Pharma Focus Asia - Issue 42 by Ochre Media Pvt. Ltd.

ISSUE 42

2021

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PERSONALISING PRESCRIPTION A laser sharp approach for complex disease indications

Biomedical Market Leaders Learning to thrive

Brexit and Pharmacovigilance Where may pharmaceutical companies go?

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Foreword Precision Medicine Personalising health outcomes In recent years, precision medicine has emerged as a promising tool for providing patients suffering from complex diseases with comprehensive care. Whether it is traumatic cancer, cardiac disease, lung disorder, or COVID-19 for that matter, the precision medicine approach has proved to be very effective in improving patients’ health or reduction of disease symptoms. Breakthrough developments in life sciences and technological advancements have brought about a significant change in clinical diagnosis and treatment as the focus is moving towards providing precise, predictable care personalised to patients. In February, researchers at the Cleveland Clinic developed the ‘My Personal Mutanome (MPM)’ platform that promises the growth of cancer gene therapies and genome-informed drug discovery. This platform, according to the team, will pave the way for a better understanding of mutations at the human interactome network level, developing new insights in cancer genomics and treatments, possibly resulting in developing precision medicine for cancer. Interestingly, the team is also working to leverage Artificial Intelligence (AI) for drug target identification and precision medicine drug discovery for cardiovascular and other complex diseases like Alzheimer’s. A study published in the Nature Medicine in August 2020 indicated that precision medicine, aided by the application of AI, could help develop a screening tool for a subtype of autism, thus facilitating early diagnosis and intervention. All these examples indicate a paradigm shift towards use of advanced technologies for bringing precision medicine into clinical care. The path to personalised care certainly has obstacles such

as gaps in clinical research, the need to analyse complex datasets, and insufficient technologies. In order to make precision medicine more effective and integrating into everyday clinical care, organisations will require advanced technologies and relevant use cases, along with providing care givers access to analytical tools that help derive meaningful insights out of large data sets. Precision medicine as a concept has had theoretical existence for long, and operationalising the same has been a distant goal for healthcare organisations. To achieve this, it is imperative for organisations to overcome challenges related to workflow optimisation, cost minimisation, and improving both patient and physician education. This issue presents an article titled ‘Personalising Prescription’ that talks about how this approach helps with complex disease indications. In the article, the authors take us through the relevance and application of personalised prescription for oncology, orphan diseases such as Muscular Dystrophy (MD), oral diseases. The article also outlines challenges in integrating genomics and cell-based technologies into routine clinical practice and stresses on the importance of preparing next generation clinicians and researchers, and enhancing knowledge of healthcare professionals towards addressing the same.

Prasanthi Sadhu Editor

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CONTENTS 06 Innovation in Pharmacovigilance Where does Israel stand and ISOP ISRAEL role?

Irene R Fermont, Chairman, Israeli Chapter of the International Society of pharmacovigilance

10 Understanding Bio-summary Tables for ANDA Submissions Noorunnisa, Assistant Manager, Department of Medical writing, Freyr Solutions

13 Patents and Exclusivity A US perspective

PERSONALISING PRESCRIPTION

Praveen Kumar Boga, Assistant Manager, Departmentof Medical Writing, Freyr Solutions

19 Biomedical Market Leaders Learning to thrive

A laser sharp approach for complex disease indications

Brian D Smith, Principal Advisor, PragMedic

24 Brexit and Pharmacovigilance Where may pharmaceutical companies go?

COVER STORY

STRATEGY

Subhadra Dravida, Founder and CEO, Transcell Biologics Gargi Roy Goswami, Founder and Director, KROYNAS Private Limited Rajiv Gupta, Entrepreneur; Managing Partner, Lateral Consulting

Philipp Hofmann, Head, Pharmacovigilance, QPPV, Navitas Life Sciences

MANUFACTURING 41 Future of Pharmaceutical Manufacturing The role of Automation

John Young, Sales Director, APAC region, EU Automation

INFORMATION TECHNOLOGY 48 Data Integrity In pharma’s rush to end the pandemic

Ankush Lamba, Managing Director, Technology segment, FTI Consulting

SPECIAL FEATURES 50 Research Insights

CLINICAL TRIALS 28 Employing Real-World Evidence to Improve Clinical Trial Strategies

Sumeet Bakshi, Vice President, Real World Data Solutions, Certara’s Evidence, Value & Access group

Richard Tao, Associate Principal Regulatory Writer and Submission Leads Member, Synchrogenix, Certara’s regulatory science company

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Advisory Board

EDITOR Prasanthi Sadhu Alan S Louie Research Director, Life Sciences IDC Health Insights, USA

EDITORIAL TEAM Grace Jones Swetha M ART DIRECTOR M Abdul Hannan

Christopher-Paul Milne Director, Research and Research Associate Professor Tufts Center for the Study of Drug Development, US

PRODUCT MANAGER Jeff Kenney

Douglas Meyer Associate Director, Clinical Drug Supply Biogen, USA

SENIOR PRODUCT ASSOCIATES Ben Johnson David Nelson John Milton Peter Thomas Sussane Vincent

Frank Jaeger Regional Sales Manager, AbbVie, US

PRODUCT ASSOCIATE Veronica Wilson

Georg C Terstappen Head, Platform Technologies & Science China and PTS Neurosciences TA Portfolio Leader GSK's R&D Centre, Shanghai, China

CIRCULATION TEAM Sam Smith SUBSCRIPTIONS IN-CHARGE Vijay Kumar Gaddam

Kenneth I Kaitin Professor of Medicine and Director Tufts Center for the Study of Drug Development Tufts University School of Medicine, US

Laurence Flint Pediatrician and Independent Consultant Greater New York City

HEAD-OPERATIONS S V Nageswara Rao

A member of

In Association with

Confederation of Indian Industry

Neil J Campbell Chairman, CEO and Founder Celios Corporation, USA Phil Kaminsky Professor, Executive Associate Dean, College of Engineering, Ph.D. Northwestern University, Industrial Engineering and the Management Sciences, USA

Rustom Mody Senior Vice President and R&D Head Lupin Ltd., (Biotech Division), India Sanjoy Ray Director, Scientific Data & Strategy and Chief Scientific Officer, Computer Sciences Merck Sharp & Dohme, US

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STRATEGY

Innovation in pharmacovigilance Where does Israel stand and ISOP ISRAEL role? Artificial Intelligence, real-world evidence and big data are leading Pharmacovigilance toward a more integrated Public Health activity. Multidisciplinarity will be a key element for efficiency as illustrated in ISOP ISRAEL projects in hospital and community. Industry must bring its expertise to support wide campaign and projects promoting safe medication practices. Irene R Fermont, Chairman, Israeli Chapter of the International Society of pharmacovigilance

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he Israeli pharmacovigilance system was founded recently. All over the world, pharmacovigilance regulations have evolved mainly from lessons learned as a reaction to safety issues that became public health issues. This was true also for Israel which has built its regulation on the EU model, and its pharmacovigilance department six years ago, following the Eltroxin® (levothyroxin) change in the excipients; this led not only to a huge number of adverse reactions but exposed the weaknesses in pharmacovigilance organisation in Israel. The new field of Israeli pharmacovigilance has developed quite rapidly with the obligation for pharma companies, hospitals and Health Maintenance Organisations (HMOs) to designate a Qualified Person for Pharmacovigilance (QPPV) and establish a pharmacovigilance system. Training sessions have been performed throughout the country to educate healthcare professionals on the

new regulation, some of them by ISOP ISRAEL with the support of the Ministry of Health. Several programs have been created in universities in the frame of continuing education. Today, the Ministry of Health is still focused on its priorities, which are to implement the basics of pharmacovigilance in the whole Healthcare system. The last step was the issue, only two years ago, of the risk management regulation on the European model. The pharmacovigilance department is small, with active but rather junior professionals, and pharmacovigilance inspections are still not implemented. Therefore, it cannot be expected from the Ministry of Health to both strengthen this new activity, to deal with day-to-day activities and also to be the driving force of innovation. For historic reasons, the public health system is quite decentralised, leaving a certain degree of autonomy to hospitals and its four HMOs. Israeli citizens can freely choose and change one of the four HMOS which therefore are in competition to provide the best services. The benefit of this decentralisation is that it leaves space to local or regional initiative. Therefore, it is not unusual that innovations stem from a hospital, a HMO or a professional organisation. This is the case with Sheba-Tel Hashomer Hospital, which has been rated by Newsweek as one of the ten best hospitals in the world in 2019 and is leading healthcare innovation in Israel with a significant impact abroad. Sheba Hospital is strongly involved in drug safety with the implementation of an Artificial Intelligence (AI) solution, Medaware, monitoring and supporting the prescription through access to the Electronic Medical Record (eMR). The Institute for Health Improvement Trigger tool is currently screening the eMRs to detect potential Adverse Events through markers; for instance a sudden drop in haemoglobin in a patient with anticoagulant could mean an occult haemorrhage.

Similarly, ISOP ISRAEL, has endorsed a role of leadership since its creation, 6 years ago, which coincided with the setup of the new pharmacovigilance era in Israel. ISOP ISRAEL and the vision of multi-stakeholder projects.

ISOP ISRAEL, the Israeli Chapter of the ISOP (International Society of Pharmacovigilance) has the mission to foster drug safety through education and research, combining the Israeli unique assets with the new trends which are shaping pharmacovigilance of tomorrow. ISOP ISRAEL has built its strategy as follows

Multi-disciplinarity and System approach: pharmacovigilance has remained limited far too much to industry experts and to clinical pharmacologists or clinical pharmacists. It is a common understanding in other high-risk industries, such as aeronautics and nuclear power plants, that management of risks can only be efficient when applied by a multidisciplinary team and considered across the entire system. Healthcare systems are still lagging behind; and the healthcare professions continue to act in silos. The future of pharmacovigilance lies in its becoming truly multidisciplinary. The stakeholders involved in the whole treatment process are the healthcare professionals, the patients and caregivers, the hospital management, the HMOs, the industry, the authorities and the academy. Learning while doing Rather than a theoretical training, multidisciplinarity is experienced through a case study. Case study Anticoagulants were chosen as a first example of high-risk products with many interactions between disciplines and rated one of the main classes of products involved in medications errors and hospitalisation for adverse reactions. Tool box ISOP ISRAEL has gathered several tools to set up its projects:

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STRATEGY

1. EU Good pharmacovigilance Practices, Risk Management and risk minimisation 2. ISMP Safe Practice Self-Assessment for anti-thrombotic therapy. ISMP, the Institute for Safe Medication Practices has been working for 30 years with the U.S. government’s FDA to prevent medication errors. They have designed tools and alert systems targeted to industry, healthcare professionals, hospital staff, lay community and authorities. The Safe Practices SelfAssessment tools allow hospitals to identify and correct weaknesses. They illustrate how pharmacovigilance can spread beyond expert pharmacologists. 3. My eReport® , mobile application, a quick and simple way to report adverse reactions 4. BeMedWise campaign ‘Talk before you take’: an organisation dedicated to patient education on medication safety, funded by FDA 5. Going on identifying tools, the next one to be integrated will be the IHI Global Trigger Tool for measuring adverse events (Institute for Health Improvement). Pilot centres 1. ISOP ISRAEL started in advocating for its new methodology country-wide then identified hospitals ready to implement its strategy to decrease the risk of anticoagulants. Carmel Medical Center in Haifa then Maayanei Hayeshua in Bnei Braqhave been especially efficient. Setting up a multidisciplinary team, translating into Hebrew the ISMP Self-Assessment for anti-thrombotics and running it, analysing system gaps, writing and performing a CAPA(Corrective and Preventive Actions): all these steps have been implemented in less than 9 months and results have been presented in the last ISOP ISRAEL symposium, in June 2019. 2. In parallel, another innovative initiative was taking place: Superpharm, the chain of community-based pharmacies, has enrolled its pharmacists in a nationwide campaign to advise patients under anticoagulants. More than 1100

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Healthcare systems are still lagging behind; and the healthcare professions continue to act in silos. The future of pharmacovigilance lies in its becoming truly multidisciplinary.

patients have been advised within a period of 4 months. Pharmacist’s intervention includes change of dosage, follow up of the renal function and dose adjustment, follow up of INR, instructions on injection practice, detection of interactions and information on the adverse reactions. The results of this experience, first-of-itskind, were presented in ISOP ISRAEL symposium. Partnerships

This step has been crucial to build a network representing all stakeholders in Israel and abroad: • institutions: Ministry of Health, Carmel and Maayanei Hayeshua Hospitals • academy: Hebrew University of Jerusalem and King’s College London • professional and patients’ associations: clinical pharmacologists, pharmacists, physicians, • pharmaceutical companies • US organisations: ISMP (Institute for Safe Medication Safety) and BeMedWise, dedicated to patient education Each of these partners have contributed to bring its own angle to drug safety and achieve a real multidisciplinary think tank. A strong Advisory Board From the creation of the Chapter, the Advisory Board, composed of international experts, has validated the vision and the projects and actively participated in the symposium program.

Multidisciplinary and solution-oriented Symposium The research strategy was completed by the organisation of an international symposium based on the same concepts: multidisciplinary and solution-oriented. The conference, 360° of Drug Safety was held in Tel Aviv, Israel, on 3-4 June 2019 and was attended by 174 participants from all disciplines. The main topics were the prevention of medication errors, risk communication and cases studies of risk minimisation. Several initiatives have been presented and among them the results of the ISOP ISRAEL projects. ISOP ISRAEL objectives are both to provide a toolbox ready to use to participants, and to enrol more pilot centres in Israel or abroad and spread the initiative at national level. Several start ups have presented their AI and IT solutions, Sheba hospital has shared its strategy. All stakeholders have been able to interact and share their experience. The Israeli assets to build a unique pharmacovigilance system. Now that the basis of the pharmacovigilance system has been founded, the next step for Israel will be to implement the new trends which are shaping the future of pharmacovigilance. In this objective Israel can benefit from key assets: The organisation of the four Israeli HMOs can allow them to have quick and efficient dialogue with healthcare professionals and with patients. The pooling of the HMOS’ extensive and comprehensive epidaemiologic databases has started and will provide a huge amount of data [8]. Risk management activities are implemented in hospitals for more than 25 years. As a leader in digital health, Israel has facilitated the emergence of many start-ups and companies dedicated to drug safety and using the most advanced technologies of Artificial Intelligence and machine learning. In conclusion ISOP ISRAEL has the ambition to play a major role in this evolution, in providing a multidisciplinary platform for experience

STRATEGY

ISOP ISRAEL Advisory Board : Prof Herve Le Louet , president of CIOMS, Peter Pitts, President Center for Medicine in Public Interest, previously FDA Associate-Commissioner,

Prof Mati Berkovitch, Safety Advisor to Israeli Ministry of Health, Prof Ronald Litman, ISMP Medical Director, Prof Nick Sevdalis, Director Implementation Sciences and Patient Safety. References are available at www.pharmafocusasia.com

Irene Fermont co-founded the Israeli Chapter of the International Society of Pharmacovigilance (ISOP) 6 years ago, to increase drug safety awareness through research and education. Irene Fermont, who specializes in immunohematology, has dedicated her career to patient’s safety. For more than 20 years, she created and managed pharmacovigilance and Risk Management departments or companies at international level, and has been active in Israel over the past 10 years. She is also the founder and director of IFC, a strategic safety consulting firm.

AUTHOR BIO

sharing, in identifying and combining new tools and platforms and in implementing innovative projects. Israel, the Start-up nation, has the organisational, scientific, technological and cultural resources to quickly overcome the challenges and go beyond its current state to build a unique pharmacovigilance system which could serve as an example for other countries. Declarations Ethics approval and consent to participate: not applicable Consent for publication: not applicable Availability of data and material: Not applicable Competing interests: The author declares that they have no competing interests Funding: none Authors' contributions: Author wrote the final manuscript Acknowledgements Ayalah Livneh, Pascal Grin, Dr Hesh Hershman and Yehudith Wexler for ISOP ISRAEL organization

Under her direction, ISOP ISRAEL is launching its first international symposium in June 2019, multidisciplinary and solution-oriented focused on the prevention of medication errors. An innovative integrative Risk Management strategy is implemented in a pilot medical center to decrease the risk of antithrombotic therapy.

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STRATEGY

Understanding Bio-summary Tables for ANDA Submissions Bio-summary tables are mandatory requirement for Clinical Summaries submitted to USFDA as a part of ANDA. These tables provide a standard format for data representation consistent with the FDA recommendations. This paper emphasizes the importance of Bio-summary tables which are reviewed for a new generic drug product by Division of Bioequivalence (DBE). Noorunnisa, Assistant Manager, Department of Medical writing, Freyr Solutions

FDA

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io-summary tables, also known as “Division of Bioequivalence (DBE Tables)”, are one of the main prerequisites for Module 2.7, submitted to the United States Food and Drug Administration (US FDA), as a part of the eCTD dossier. The FDA mandates the submission of these tables as PDF format and in MS Word document format in the appropriate eCTD/CTD locations (Module 2.7). The main purpose of these summary tables is to provide a standard format for data to be in an Abbreviated New Drug Application (ANDA) in a concise format consistent with the current recommendations. The FDA provides specific instructions to fill in these tables. There are different types of summary tables based on the type of formulation and characteristics of the ANDA application. The different types of summary tables for which individual instructions are provided by the FDA include the following: • Bioequivalence Summary Tables for In Vitro Feeding Tube Testing • Comparative Clinical Endpoint

STRATEGY

Bioequivalence Study Summary Tables • Model Bioequivalence Data Summary Tables • Topical Dermatologic Corticosteroids In Vivo Bioequivalence Study Summary Tables and SAS Transport Formatted Tables for Dataset Submission • In Vitro Binding Bioequivalence Study Summary Tables and SAS Transport Formatted Tables for Dataset Submission • Summary Tables for the Listing and Characterisation of Impurities and Justification of Limits in Drug Substance and Drug Products (consistent with the recommendations delineated in the Guidance for Industry ANDAs: Impurities in Drug Substances and ANDAs: Impurities in Drug Products) • Model Bioequivalence Data Summary Tables: A detailed content and format information resource for generic drug applicants submitting ANDAs to the FDA • Bioequivalence Summary Tables for Aqueous Nasal Spray Products • BCS-Based Study Summary and Formulation Tables • Pharmacy Bulk Package Sterility Assurance Table • Irritation/Sensitisation/Adhesion Study Summary Tables • Bioequivalence Summary Tables for Pressurised Metered Dose Inhaler Products Bioequivalence Summary Tables

The absence a of significant difference between test and reference with regards to rate and extent at which the drug is available at the site of action when administered at the same molar dose and under similar conditions is termed as Bioequivalence (BE). Hence, reports providing data from BE studies conducted to compare the rate and extent of drug absorption in vivo for a generic and corresponding reference product, are one of the critical components of ANDA submissions. The therapeutic

MODULE 2.7.1

2.7.1.1 Background and Overview Table 1: Submission Summary Table 4: Bioanalytical Method Validation Table 6: Formulation Data Table 10: Study Information Table 11: Product Information

2.7.1.2 Summary of Results of Individual Studies Table 5: Summary of In Vitro Dissolution Studies (Comparative In Vitro Dissolution Data, Certificate of Analysis [CoA] for Test and Reference products should be included along with potency, assay, content uniformity, date of manufacture and the lot number) Table 9: Reanalysis of Study Samples Table 12: Dropout Information Table 13: Protocol Deviations Table 14: Summary of Standard Curve and QC Data for Bioequivalence Sample Analysis

2.7.1.3 Comparison and Analysis of Results Across Studies Table 2: Summary of Bioavailability Studies Table 3: Statistical Summary of the Comparative Bioavailability Data Table 16: Composition of Meal Used in Fed Bioequivalence Study (A statement of compliance to the FDA standard meal should be provided, if the standard meal is as per the CDER guidance for food effect bioavailability and fed BE studies. In case of any alternative meal used, the summary table needs to be provided, which mentions the food item(s), ingredient(s), amount (g), energy (kcal), protein (kcal), fat (kcal) and carbohydrates (kcal)).

2.7.1.4 Appendix Table 15: SOP’s Dealing with Bioanalytical Repeats of Study Samples

Module 2.7.4 (Summary of Clinical Safety) 2.7.4.1.3 Demographic and other Characteristics of Study Population Table 7: Demographic Profile of Subjects Completing the BE Study 2.7.4.2.1.1 Common Adverse Events Table 8: Incidence of Adverse Events in Individual Studies

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STRATEGY

Table 1: Submission Summary Table 2: Summary of Bioavailability Studies Table 3: Statistical Summary of the Comparative Bioavailability Data Table 4: Bioanalytical Method Validation Table 5: Summary of In Vitro Dissolution Studies Table 6: Formulation Data Table 7: Demographic Profile of Subjects Completing the Bioequivalence Study Table 8: Incidence of Adverse Events in Individual Studies Table 9: Reanalysis of Study Samples Table 10: Study Information Table 11: Product Information Table 12: Dropout Information Table 13: Protocol Deviations Table 14: Summary of Standard Curve and QC Data for Bioequivalence Sample Analysis Table 15: SOP’s Dealing with Bioanalytical Repeats of Study Samples Table 16: Composition of Meal Used in Fed Bioequivalence Study Formatting points to be followed while filling the information in the above summary tables1: Margins for the paper should be “1” for the top and bottom and “1.25” for

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the left and right sides All text should be in Times New Roman, with font size 10 Default Table Style should be used while creating the tables in Microsoft® Word (Select Menu Table-Table Auto Format-Table Normal) “Portrait” orientation should be followed for Table 1, Table 4, Table 7, Table 8 and Tables 10-16 “Landscape” orientation should be followed for Table 2, Table 3, Table 5, Table 6, Table 9. As per the checklist provided by the FDA for an ANDA application, the above tables are to be placed in Module 2.7 in the following sequence., Importance of Bio-summary Tables from Refuse to Review (RTR) Perspective

As per the RTR guidance from the FDA, it is mentioned that the FDA will RTR an ANDA, if the Study Information (Table 10) BE table is incomplete. The Study Information BE table compiles important information about study type and site locations and should be placed in Module 2.7 of the ANDA (along with the other BE summary tables). The other minor deficiencies with respect to module 2.7 and summary tables that may trigger a RTR are as follows: • Failure to provide separate PDF and Word documents of Summary tables

• Missing summary data tables in module 2.7 • Failure to provide the certificate of analysis for each strength of the RLD • Failure to provide the exact location of the long-term storage stability (LTSS) study reports and data (Table 10), along with working hyperlinks to the respective information Major deficiencies include6:

• Inadequate dissolution studies, lacking: • Minimum of 12 units • Use of the FDA-recommended test media • ½ tablet dissolution for modifiedrelease products with functional score marks • General deficiencies of in vitro dissolution (Table 5) • Not conducted on 12 units • Not conducted on all strengths (test vs. RLD) • Not conducted in all test media Conclusion

In summary, this information regarding the Bio-summary tables will help understand the standard format for data to be submitted to the Office of Generic Drugs in accordance with the current recommendations of the FDA for ANDAs. The pharmaceutical industry can take steps toward eliminating recurring problems related to summary tables by following the format and content of these tables and hence can submit the acceptable, complete, and well-organised BE submission to ANDAs without any RTRs. References are available at www.pharmafocusasia.com Noorunnisa is a PharmD Professional with significant experience in the medical writing profession. She has few international publications to her credit. Her experience involves authoring and review of clinical regulatory documents. She is a part of Freyr Solution from 5 years and currently holds the position of an Assistant Manager.

AUTHOR BIO

equivalence of an active moiety as per the Regulatory standards depends on the determination of pharmaceutical equivalence along with establishing BE. A separate division [Division of Bioequivalence (DBE)] is designated in the Office of Generic Drugs (OGD), which is involved in the review of BE studies of new ANDA applications. For ANDA BE submissions that contain the results of in vivo studies, the four major study report components are as follows: in vitro dissolution testing, bioanalytical methodology, clinical study reports and statistical analysis.2 There are a total 16 BE summary tables for a typical product, which focus on the above information in a concise manner for DBE review. They are:

STRATEGY

PATENTS AND EXCLUSIVITY A US perspective

Drug patents and drug exclusivity. How are they different from each other? Why should the applicants/sponsors be fully aware of the Regulatory patent protection and exclusivities? The draft covers a comprehensive perspective on drug patents and exclusivities in relation to the United States. Praveen Kumar Boga, Assistant Manager, Departmentof Medical Writing, Freyr Solutions

hen a pharmaceutical company first develops a new drug or any device to be used for the treatment of a disease, it is initially marketed under a brand name by which clinicians can recommend or prescribe the drug or any device for use by patients. The drug or device is covered under patent protection, which means that only the pharmaceutical company that holds the patent is allowed to manufacture, market the drug and eventually profit from it. The United States Patent and Trademark Office (USPTO) will have the right to issue a patent to a discoverer or inventor to “eliminate others from manufacturing, utilising, offering for sale, or marketing/selling the innovation to over the United States or importing the discovery into the United States” for a constrained time, in exchange for public exposure of the discovery, when the patent is granted. Generally, the term of a new patent is twenty (20) years from the date on which the application for the patent was filed with the USPTO. For any new patent, a company may submit an application from the USPTO anywhere with the

development lifeline of a drug and can cover a wide range of claims. However, so many other factors can affect the time period of a patent. The original New Drug Applications (NDAs) and supplements can be submitted on the FDA Form 3542a, prior to approval, along with patent information and if it is upon post approval, the patent information should be submitted on the FDA Form 3542. After approval of an NDA, if the patent has been issued, the applicant has thirty (30) days to file the patent to have it counted/deemed as a timely filed patent. Beyond the thirty (30) day period, the patents may be submitted, but the patent is not counted or considered a timely filed patent. If the generic application is submitted prior to the patent, an Abbreviated New Drug Application (ANDA) holder is not required to make a certification to an untimely filed patent. Patents protect the approved drug substance, drug product, or approved methods of use for the manufacturing or marketing of drugs. New Drug Application sponsors are required to submit for listing patents that protect their approved drug

substance, drug product, or approved methods of use. For submission of patent information, applicant must use the FDA provided form 3542a before approval or Form 3542 within 30 days of approval or issuance of patent (for later issuing patents). If there are no patents to list, that must be declared via a Form 3542/3542a submission. For every patent in orange book, an ANDA applicant must certify : • Patent has expired (Paragraph II Certification) • Generic manufacturer will stay off market until patent expires (Paragraph III Certification) • Generic manufacturer believes that the listed patent is either invalid or would not be infringed by the proposed generic product (Paragraph IV Certification) [if patent information has not been filed: Paragraph I Certification]. Inventors can search the USPTO's patent database to see if a patent has already been filed or granted, that is similar to your patent. Patents may be searched in the USPTO patent full text and image Database (Pat FT). The full texts of the patents issued from 1976 to

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STRATEGY

the present and the PDF images of the patents from 1790 to the present are housed by the USPTO. Exclusivity

Exclusivity is originated to promote a balance between new drug innovation and generic drug competition. It is a period when an innovator drug is protected from generic drug competition. There are different types of exclusivities for different circumstances. Types of Marketing Exclusivity in Drug Development:

Unlike a patent, marketing exclusivity is generally acquired early in drug development, runs considerably longer and is based upon intellectual property rights, rather than evidence of safety and effectiveness. When the constitutional or statutory requirements are met for a drug, the FDA would issue the approval and also the marketing exclusivity, where the exclusivity is a period of time during which no other applications can be accepted and/or approved for the same active ingredient. This means that, other manufacturers that may wish to develop alternative formulations or generic versions of the drug will not be able to have their products approved during the exclusivity period. The type of exclusivity would decide the length of the exclusivity period. Importantly, the exclusivity period is not added to patent life, so sponsors will need to be mindful of both durations and plan, accordingly. The exclusivity duration: There are a few types of marketing exclusivity, and all of them vary in duration and the statutory requirements that must be met. Some are based on product classification, others on the indication being treated on the intended patient population. The types of exclusivity include:

• Orphan Drug Exclusivity (ODE): This type of exclusivity is seven (7) years and is granted to drugs designated and approved to treat a rare disease or condition affecting fewer than 200,000 or more

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than 200,000 and no hope of recovering costs in the United States. • Biologic Exclusivity: For Biologics License Applications (BLAs), Under section 351(k)(7)(A) of the Public Health Service Act, the duration of the exclusivity is twelve (12) years. The USFDA will not accept biosimilar filings (under its 351 (K) pathway) until five (5) years after the original biologic is licensed. • New Chemical Entity (NCE) Exclusivity: In most cases, a brandname drug with a new active moiety has a five-year exclusivity. During this five-year exclusivity period, no other company can submit an Abbreviated New Drug Application (ANDA) to the FDA seeking approval of a drug product containing the NCE. • Generating Antibiotic Incentives Now (GAIN) Exclusivity: GAIN is a new law that addresses the antibacterial drug resistance by encouraging the pharmaceutical research, development and approval of new type of antibacterial and antifungal drugs. The drug products have been granted or designated by the FDA as “Qualified Infectious Disease Product” (QIDPs) and have the additional five (5) years of exclusivity. • New Clinical Investigation Exclusivity: A brand industry’s new brand-name drug with an active ingredient that has been approved before may be awarded a three-year exclusivity in certain circumstances, such as, if a new

Patents and Exclusivity protection may or may not run concurrently and may not run the same aspects of the drug product.

way of delivering the active ingredient is proposed (for example, a tablet rather than a liquid) or a different disease or condition the drug can treat is identified. To get this approval, the drug company must conduct new clinical studies in humans. • Paediatric Exclusivity (PED): A patent protection for a new drug applicant for which the sponsor has done paediatric studies (in response to a written request from the FDA) may be eligible for a six-month exclusivity, which is added on to any other exclusivities or patents for that drug (six months added to existing Patents/Exclusivity). This exclusivity is an effective tool for drug developers, delaying the FDA ANDA and 505(b) (2) approvals six months after the patent expiration. • Patent Challenge (PC): This exclusivity is for Abbreviated New Drug Applications (ANDAs) only and the exclusivity period is 180 days. • Competitive Generic Therapy (CGT) or Generic Drug Exclusivity (GDE): This exclusivity is for 180 days and is applicable for ANDAs only. • Qualified Infectious Disease Product (QDIP) Exclusivity: This exclusivity is for five years and it can be added to any existing exclusivity. An Exclusivity Board has been established by the Center for Drug Evaluation and Research (CDER) to give oversight and recommendations about exclusivity determinations made by the Center. The CDER exclusivity board manages the granting of exclusivity determinations, that means whether and what type of exclusivity will be granted. The CDER board will not review or provide any recommendations with respect to exclusivity determinations. The five year New Chemical Entity (NCE), three year new clinical trial exclusivity and biological product exclusivity will be focused by the CDER board. Difference between Drug Patents and Drug Exclusivity

Regardless of the drug product approval

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status, the patents can be issued or expired at any time – before, during or after the FDA approval process. If the drug product meets the statutory requirements of the FDAs, the drug product will be approved with an attachment of an exclusivity. Further, few drug products have both patent and exclusivity protection while others have just one or none. The patents will expire in 20 years from the date of filing, but the exclusivity is granted upon the basis of the drug product. For instance, the New Chemical Entity (NCE) gets five years of exclusivity, while orphan drugs get seven years of exclusivity.

According to the FDA, the other major difference between patent and exclusivity is patents can be issued or expired at any time irrespective of the drug approval status,, while the exclusivity is granted upon approval. The expired patent or exclusivity drug products may not be available, or it is removed from the Orange Book. Patents and Exclusivity protection may or may not run concurrently and may not run the same aspects of the drug product. Exclusivity was developed to promote a balance between new drug innovation and greater public access to drugs that result from generic drug competition.

Praveen Kumar B is a Postgraduate in Pharmacy with a significant experience in the field of Medical Writing. He has been working in medical writing profession for the last ten years. He has a few international and national publications and presentations to his credit. His experience spans across authoring and review of the various clinical and Regulatory documents. He has been a part of Freyr Solutions for a year now and currently holds the position of an Assistant Manager in the department of Medical writing.

In some countries, like India and Brazil, they have compulsory licenses, which basically allow local companies to produce and locally market drugs that have not reached a point in time when generic competition is legally allowed. Conclusion

The applicants/sponsors should fully be aware of the Regulatory patent protection and exclusivities. These exclusivities are developed to encourage the innovation in pharmaceutical research and development of new, safe and cost-effective treatment. While taking the advantage of Regulatory exclusiveness of the target country, it can help to sponsor to realise a return on investment by utilising the Regulatory exclusivities. In the way of product identification or broadening of the line of products or market extension, one needs to evaluate patent as well as Regulatory exclusivities of the target country to have profit-making products, while serving the patient population.

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Down to The Tiniest Particle

Dispersing of powders in the pharmaceutical industry The production or refinement of pharmaceutical products often involves dispersing dusty, sticky or fine powders in liquids. Conventional technology often encounters problems - such as when agglomerated powders are not completely wetted and this is precisely where modern vacuum dispersing systems come in. Denis Hunn, Process- and Application Engineering, Ystral

Example of a process plant in Ex version for the production of tablet lacquer.

Gels, creams and suspensions or coatings on tablets are among the pharmaceutical products manufactured using powders or refined powder mixtures. They usually consist of an active ingredient, the thickening or swelling agent, such as CMC and HPMC, and colourants such as iron oxide, titanium dioxide or talc.

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Manufacturing companies can have complex requirements and the topic of reliability is particularly important and so pharmaceutical processes need to be established. These processes must be reproducible and adapt to the often meticulously coordinated requirements of the production chain.

Example of a process system in Ex-design for the production of tablet coating.

Producers also have another objective: To be able to produce the widest possible range of batch sizes using a single system, meaning that the system they use must be versatile and easy to clean.

No agglomerates, air pockets or foam The system design aims to prevent the operator from coming into contact with the materials for precisely this reason. And the aim is to achieve the longest possible storage stability after production without any foam. Agglomerates or air pockets in the product have to be completely ruled out. This is a prevalent problem, because if long dissolving times or the formation of agglomerates occur during the production of a suspension, this can lead to long waiting times. We come across several problems when conventional technology is employed. Traditional agitators or dissolvers wet the powdered fillers as an agglomerate rather than singularly- This is something that cannot be avoided, as it is the principle of these systems. Powder inside these agglomerates is not

completely wetted. The inside of the agglomerates may be wetted later by capillary effect, but this is only possible on a selective basis. The way in which the powder is added is the cause of the pseudowetting. It occurs when the powder is added to the vessel from above. However, it also occurs when it is inducted into a liquid from below in a vacuum vessel by compact flow or inline by using so-called injectors. The problem is caused by the fact that the powder particles touch each other during wetting and are not dispersed.

Dispersing under high shear-effect and vacuum

The solution is provided by a system that can completely wet and optimally disperse powder particles in both liquid and viscous medium. This is exactly what ystral’s powder wetting and dispersing system ContiTDS does. The system is used to produce solvent- and water- based coating suspensions - but this is only one example of the many possible applications. A suction Advertorial www.pharmafocusasia.com

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zone. No additional transport or fluidisation air is needed for this effect because the air in the powder expands and subsequently contracts again after dispersing. The particles are individually separated and thoroughly wetted. The powder inlet is closed once the adding of powder has been completed. The system can then be continued to be used as a normal Inline Disperser or for degassing in low-viscose systems, whereby installation both on existing vessels and in complete systems is possible.

Lower temperatures required

Under vacuum, the air contained in the powder expands

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hose is used to draw in the powder or it is taken directly from the container, whereby the powder only comes into contact with the liquid in the dispersing zone. Dispersing takes place under a massive shear-effect and vacuum. The vacuum has an extraordinary effect. Under vacuum, the air contained in the powder expands by up to 98%. All the particles in the powder are in flight when they are inducted at high speed by the vacuum conveyor. When the powder is transported, the vacuum in the powder constantly increases when it moves from the point where it is added to the maximum vacuum zone and the distances between the individual particles increase accordingly. The system generates its suction effect directly in the liquid. The maximum vacuum prevails the wetting and dispersing zone and the distances between the individual particles are largest when they enter this

The system allows dispersions and emulsions to be produced with particle or droplet sizes on the nanometre scale. A higher product quality can be achieved due to the fact that the formation of agglomerates is avoided. In addition, wetting and dispersing take place at significantly lower temperatures than when conventional technology is used and this often achieves a beneficial advantage in subsequent process steps. The storage stability of the suspensions produced using the system can be increased by up to 90 %, meaning that the coating process can be carried out hours, or even days, after producing the coating. This can even be done without a separate agitator in the feed vessel, and energy consumption is also almost two-thirds lower than when using conventional technology. In pharmaceutical production, powders often have to be dispersed in liquids. Conventional technology often achieves only incomplete wetting, because the powders are introduced agglomerated. One solution is modern vacuum technology, in which the particles are separated and wetted.

Denis Hunn was born on 31.01.1990 in Freiburg. He obtained apprenticeship as a mechatronics technician in Bahlingen am Kaiserstuhl. His professional journey started as mechatronics technician at Braunform GmbH and joined ystral in 2011. He gained advanced technical college entrance qualification and studied at the DHBW Baden-Wuerttemberg Cooperative State University (industrial engineering). He is also active in the local university council of the DHBW.

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Advertorial

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Biomedical Market Leaders Learning to thrive

In a rapidly changing market, the only competitive advantage that lasts is the ability to learn. Here is how our enduring biomedical market leaders do that. Brian D Smith, Principal Advisor, PragMedic

f you have worked in the biomedical industry for more than a couple of years, you will have noticed one of its most characteristic features: change. Pharma, medtech and related businesses operate in a complex environment of two halves: the sociological and the technological. Our sociological environment, including demography, epidemiology and politics is in flux as we master the diseases of youth and enter the battle with the implications of old age and lifestyle. And our technological environment is a storm of ‘omics, systems biology, nanotechnology, and information technology. Business environments tend to exhibit “punctuated equilibrium” in which periods of slow change are interrupted with bursts of rapid transformation. There seems little doubt that our industry is in the midst of just such a spurt of evolution.

The challenge of change

Change poses a problem for biomedical companies, especially large ones. Technical complexity, regulation and culture make them slow, ponderous beasts whose proven capacity to improve lives is countered by their inability to respond to rapid market change. Anyone who has attempted

significant change in a large biomedical company will recognise this issue. This inertia accounts for the demise of many organisations, as shown by the turnover of names in the “top 50” lists of companies. Yet those lists also pose a question: if the success of many companies is fleeting and ended by market change, what about the others, those that survive for decades or centuries? What is it that those enduring companies do differently from their short-lived rivals? The ultimate advantage

The question of what causes longevity in a changing market has been investigated by many researchers, who converge onto one simple answer: organisational learning. In short, companies sustain their competitive advantages by drawing new knowledge from what is going on around them and then acting on that insight. But such a simple answer creates another set of questions: how do biomedical companies learn? What is the process by which longlasting companies learn faster and better

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THREE FORMS OF KNOWLEDGE Knowledge is more than information. It is the actionable understanding of a situation. Three kinds of knowledge enable action: Declarative knowledge, who or what something is. For example, we may or may not know the market shares in a therapy area or the organisational structure of a healthcare system Causal knowledge, what gives rise to a situation. For example, we may or may not know what causes the adoption of a new product to vary between accounts Procedural knowledge, how something happens. For example, we may or may not know how market access is achieved in a given situation. than other companies? My research has studied this for many years and, in the following paragraphs, I distil it down to a few, actionable lessons. First, admit ignorance

The journey to knowledge begins with agreeing what the firm does not know but needs to. That knowledge comes in three forms (see box 1) and learning begins with defining what kind of knowledge needs to be created. Is it declarative (e.g., What are the primary issues facing a clinical speciality?) or causal (e.g., Why do some patients not adhere to treatment?) or procedural (e.g. How do professionals choose between treatment options?). In practice, many firms collect information habitually but not reflectively, without thinking about what they are trying to learn. But, in my research, the ability to identify and admit to critical knowledge gaps is characteristic of learning organisations. To do so requires two component capabilities, 20

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first an analytical facility to spot the gap and second the cultural knack to avoid blaming. Firms in which admitting ignorance is a culturally unacceptable, blameworthy fault are very unlikely to learn or endure in a changing market. Choose your weapon

The task of creating knowledge has been usefully compared to cooking. There are many ways to cook a meal and the method you adopt depends on what ingredients you have and what it is you want to prepare. When organisations learn, their “recipe” is shaped by the kind of knowledge they want create and the data and information “ingredients” they have available. That said, there are three main ways — deduction, induction, and abduction — of turning those ingredients into either declarative, causal or procedural knowledge. Although different functions and professions tend to prefer one way of “cooking” information over the other, it is simplistic to judge them as better or worse approaches. Organisational learning is pragmatic, meaning the best approach is the one that works in a given situation. Deductive learning

When market change is clear and measurable, and the goal is to understand that change, then deductive learning is usually the best method. This involves proposing an explanation for the change and developing a hypothesis from that explanation. For example, if the market share of a premium-priced therapy is declining, one obvious explanation is that payers are switching to lower-priced options. This leads to the hypothesis that if price is the explanation, then the shares of lowerpriced equivalents should grow at the expense of high-price products. When the data supports that hypothesis, the putative explanation is upheld and becomes new, causal knowledge about how the market works. If, however, the data shows other patterns, such as shift to an equal-priced rival, then other, non-price explanations are needed and a new cycle of hypothesis testing is needed.

Deductive learning is the most obvious learning method. It is the basis of most scientific methodologies. But it is much less common in biomedical markets than you might expect. In part, this is because it is hard to isolate one variable and control for others. Just as often, however, deductive learning is feasible but managers are happier to have faith in their long-held, subjective beliefs rather than to risk them being proven false. Inductive learning

When market change is messy and hard data is unavailable, and the goal is to unravel what is happening, then inductive learning is the preferred method. Less structured than deductive learning, induction can appear structure-less when, in fact, its process is implicit. For example, prescriber behavioural issues such as adherence to patient pathways can be studied by exploring the experience of multiple prescribers. By structuring that exploration into episodes, such as when adherence persisted or failed, or into crosstherapy comparison matrices within the same group of prescribers, patterns are more likely to emerge. In this example, the research might induce that adherence is inversely proportional to experience and self-confidence, an example of new declarative knowledge. Induction can be a richer, more insightful method than deduction but it is vulnerable to cognitive biases and method design needs to guard against that. Inductive learning is the most common learning method amongst executives. But because it is pervasive and often implicit it is often not even recognised as a learning method. By recognising when they are inducing new knowledge, managers can make the process explicit, more structured and more effective. Abductive learning

Between deduction, which tests existing thinking, and induction, which draws out new ideas, lies abduction, which works well when multiple views of the world vie for acceptance. For example, why market access decisions vary between countries

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explanation or another or because firms prefer to rush to easy but unsubstantiated conclusions. Mixing it up

Inductive, deductive, and abductive learning approaches are not mutually exclusive. They can and should be applied in the same organisation at different times or to different knowledge gaps. But, powerful as they are, sometimes none of the three approaches alone is sufficient. This is the case for the most problematic knowledge gaps, those that start from very limited understanding but which are critical to commercial success. For example, what is the segmentation in this market (declarative), what causes segments’ different behaviours (causal) and how does each segment reach their decision (procedural). In such cases, a mixed-method approach is usually required, which means using more than one learning method but combining them for complementarity. In our market segmentation case, for example, we might begin with inductive methods to understand the motivations that underlie segmentation, deductive methods to find the causes of their behavioural difference and abductive methods to unravel their decision processes. The three methods can be used to build on each other in stages, known as a sequential mixed-method approach, or independently to verify or “triangulate” each other, which is known as a convergent mixed-method approach. Predictably, mixed methods approaches require more resources and demand more skill than pure, single-method approaches but they tend to produce better answers to harder questions. As in everything else, the quality and quantity of organisational learning is proportional to the effort applied to it.

top company lists for decades - pride themselves on their ability to do this. Yet, at the same time, those market changes places greater pressure on those individuals who must lead organisational learning. Buried under an avalanche of data and armed with evermore powerful information technology options, it is tempting to rely on analysis, chopping up our data in ever more arcane ways. But analysis alone never leads to learning. Any effective approach to learning relies on synthesis, the combination of different sources of information. Inductive, deductive, abductive and mixed-methods are, like cooking, different ways to combine information and whilst analytically dicing the individual ingredients ever more finely has some value it rarely creates new knowledge. The ultimate lesson from my research then is that organisational learning is essential to long-term survival in a changing market. The learning processes it requires are undoubtedly more difficult and more expensive than the datacrunching that replaces learning in some, lesser firms. But, as the saying goes, if you think knowledge is expensive, you should try ignorance.

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might be explained by rational, quantitative decision making, by variations between country’s health systems or by irrational decision processes, such as internal politics. In this situation, some facts, including decisions and explicit decision processes, are known but other facts, such as internal dissent and implicit decision mechanisms are not. This uneven mix of information ingredients favours abductive learning, which might be best described as the “best fit” method. In this example, abductive learning would start with halfformed hypotheses such as: • If market access were rational, we would expect to see… • If market access were irrational, we would expect to see… • If market access were country-dependent, we would expect to see…. With each hypothesis completed by a description of what facts would fit each explanation. These tentative hypotheses would then be compared to the findings of structured, inductive methods, such as comparisons of different cases and interviews with decision makers. The hypothesis whose prediction most closely matches the inductive observations is the most likely to be true. In this example, our research found that market access is “boundedly rational”, that is it was rational within the specific context of the country. This combination of two explanations was new insight into how market access decisions are made and is an example of procedural knowledge. Abductive learning is, in many market situations, the most powerful learning method, yielding actionable insight into important and messy situations. But it is also the method that requires most skill. Deduction requires a systematic approach and induction demands a degree of intuition and sensitivity to the data. Abduction requires a systematic approach, sensitive intuition and the ability to allow rival explanations to compete fairly. When abduction fails, it is rarely the method at fault. More often, it is because some internal political faction identifies with one

Ignorance is expensive

Our rapidly changing marketplace makes it more necessary than ever for biomedical companies to learn and to use their new knowledge to sustain their competitive advantage. The exemplar companies in our industry -those that persist on the

Brian D Smith is a world-recognised authority on the evolution of the life sciences industry. He welcomes comments and questions at brian. smith@pragmedic.com

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Bachem

Expanding into oligonucleotides Torsten Wöhr, Head of Oligonucleotides, Bachem AG, Switzerland

Our large-scale facility will come online later in 2021. We took the time to thoughtfully design an equipment train featuring some innovative engineering solutions for increased utilisation flexibility and improved process control. Then, later on our way to become a first-choice manufacturer for oligonucleotides, we hope to make our own contributions to advancing the drug class by making oligonucleotide API production more scalable and cost-effective.

The global market for oligonucleotide therapeutics is expanding rapidly. What is Bachem’s strategy in entering this competitive environment? I think it is important for our customers to understand that the decision of the Bachem leadership to enter the field of oligonucleotide manufacturing was well-prepared and ultimately taken in line with the company’s long-term growth plan. We are operative since 2019 and are gradually expanding our expert resource pool, capabilities and capacity for oligonucleotides. We have been lucky to forge strong partnerships on the customer as well as on the supplier side and have been able to hire great oligonucleotide chemists to accelerate our organisational learning. Despite having set ambitious goals we go step by step. Having said that, more and more we are shifting our focus from closing the oligo-specific technology gap to building a solid foundation for future growth. 22

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The CMO environment, in particular for oligonucleotides, is developing quickly. How do you see your chances of success and what do you think are the major hurdles? The progress in oligonucleotide-based drug development directly translates into a growing number of granted marketing authorisations. We actually might be seeing additional approvals before the end of the year. In addition, there is a strong interest in the therapeutic application of antisense technology and non-coding RNA biology, which is reflected in an ever growing number of clinical programs in operation and in a global project portfolio that is spreading across a broader range of indications. We therefore anticipate the demand for oligonucleotide custom manufacturing services to remain strong in the foreseeable future. As for challenges: mastering oligonucleotide chemistry is not trivial, and building a track record of successfully completed scale-up projects is another major hurdle for every CMO entering the market. In addition, building large-scale manufacturing facilities for oligonucleotides, meaning facilities with an output of 1-Mol per batch or more, is very expensive. At Bachem, a sizeable CAPEX budget has been granted to purchase special equipment and to build the necessary infrastructure. Finally, almost the entire equipment train is custom-built. It is important to get the design details right, ideally on the first pass. We are lucky to have a strong engineering team on site to support these critical activities.

COVID-19 exposes the weak links in the pharma supply chain. How has Coronavirus affected Bachem and your oligonucleotide development plans? Indeed, these are challenging times. COVID-19 affects all of us on a personal level, in our daily work life and social interactions. Early on in the pandemic, Bachem received essential business status from the Swiss authorities, and we remain committed to our partners and patients, who depend on Bachem’s products and an uninterrupted drug supply.

Bachem’s corona task force monitors the COVID19 pandemic closely and implements appropriate measures in a proactive way. In addition, employees are repeatedly trained in preventing infections and reminded not to become complacent in the process. So far the virus has not impacted Bachem’s ability to produce, and so our oligonucleotide program is still on track. Fingers crossed that we continue navigating this situation successfully and can make a contribution in battling COVID-19.

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What are the major technical challenges for the production of oligonucleotide therapeutics? Where can Bachem benefit from their expertise in peptide synthesis? Similar to peptides, the manufacture of oligonucleotides requires expert knowledge in solidphase synthesis and protecting group chemistry. Downstream processing typically includes purification by chromatography and isolation by ultra/diafiltration techniques, precipitation and finally lyophilisation. The manufacture of peptide APIs follows the same basic principle. And it is the core technology Bachem has developed over decades. Still, there are important differences between peptides and oligonucleotides. The synthesis in flow-through columns consumes large volumes of solvents and reagents for which our facility infrastructure will be appropriately expanded. Furthermore, oligonucleotides are negatively charged and highly water soluble, requiring the handling of aqueous solutions throughout the entire downstream process. And let’s not forget that oligonucleotide APIs, especially double stranded entities, are considerably larger then peptides and pose challenges also from an analytical point of view. Overall, it is fair to say that our peptide manufacturing background and our analytical capabilities are certainly very helpful in our quest to build a successful oligonucleotide business.

Torsten Wöhr joined Bachem‘s Sales and Business Development team in 2017 to lead the company’s business with commercial products. In 2019 he accepted his current role to start up Bachem’s oligonucleotide program offering CMC development and manufacturing services for oligonucleotide drug substances. Torsten has more than 20 years of experience in customer-facing roles for chemical contract manufacturing (pharma/biotech) and life sciences companies. He studied biochemistry/molecular biology at the Swiss Federal Institute of Technology in Zürich (ETHZ) and obtained his doctorate in bio-organic chemistry from the University of Lausanne. He also holds a post-graduate degree in Industrial Engineering and General Management from ETHZ.

Bachem is a leading, innovation-driven company specialising in the development and manufacture of peptides and oligonucleotides. With 50 years of experience and expertise Bachem provides products for research, clinical development and commercial application to pharmaceutical and biotechnology companies worldwide and offers a comprehensive range of services. www.bachem.com Advertorial www.pharmafocusasia.com

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Brexit and Pharmacovigilance

Where may pharmaceutical companies go? W The United Kingdom decided to leave the European Union; this ‘Brexit’ impacts pharmaceutical companies and their Pharmacovigilance systems in the UK and EU. This article describes the consequences for Marketing Authorisations, for instance ICSRs, aggregate reports, signal / risk management, the QPPV and the PSMF. Pharmaceutical companies need to be prepared for the results of the ongoing negotiations between the political stakeholders, which will detail future processes and needed changes for the pharmaceutical companies. Philipp Hofmann, Head, Pharmacovigilance, QPPV, Navitas Life Sciences

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hat may happen to sick people should medicinal products suddenly not be available owing to Brexit? Some 45 million patient packs go to the European Union (EU) from the United Kingdom (UK) every month, with a further 37 million patient packs moving from the EU to the UK. On 23 June 2016, the UK, together with the British Overseas Territory of Gibraltar, voted with a 51.9 per cent majority to leave the EU. Subsequently, the UK government initiated the official EU withdrawal process by triggering Article 50 of the Lisbon treaty. The

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UK left the EU on 31 January 2020 with an agreed transitional period until 31 December 2020. Philipp Hofmann, MD, Head of Pharmacovigilance and QPPV, Navitas Life Sciences (a TAKE Solutions Enterprise) explores the potential impact of Brexit to Pharmacovigilance (PV) in both the UK and the EU. Hard Brexit was not to be

The ghost of a hard Brexit, one in which “no deal” between the UK and EU could be reached, luckily disappeared. According to a study in the Lancet 2019, a “no deal” Brexit would have an adverse impact on health in the UK under every scenario, and that “no deal” would have the worst impact. In total, a “no deal” Brexit could have cost the UK GBP 90 billion . The transition period, published on 19 March 2018, gave pharmaceutical companies time to prepare for Brexit, to continue with the current PV system to ensure patients’ safety .

The current situation: Soft Brexit

As of December 2020, the negotiators in the UK and the EU reached an agreement on the regulation. Many details still need to be agreed upon in specialist working groups to cover all aspects of daily life. So far it seems clear that the Medicines and Healthcare products Regulatory Agency (MHRA) will not continue to be part of the European PV system, including the PV Risk Assessment Committee (PRAC) or EudraVigilance. This will have a tremendous impact for both the involved Health Authorities and pharmaceutical companies. This approach will require double efforts in PV in order to satisfy the needs of Health Authorities in the UK and the EU, and could leave room for certain conjectures. Consequences for Marketing Authorisations

With this in mind, just what are the consequences for Marketing Authorisations (MA), and how can

supply with Centrally Authorised Products (CAP) be ensured in the UK and in the EU? After 31 December 2020, a pharmaceutical company solely located in the UK will no longer be able to hold an EU MA. On the other hand, an EU pharmaceutical company can continue to hold a UK MA for a period of time after 31 December 2020, but they will have had to establish a contact person based in the UK. The MHRA has issued a businesscontinuity plan for more than 370 CAPs to rapporteurs and co-rapporteurs, which are registered within the EU. All MAs need to be converted to national licenses. The plan is that CAPs will automatically be converted to UK MAs on the day of Brexit. Mutual recognition and decentralised MAs will be unaffected since they already hold national UK MAs. Additionally, MAs, which are only held in the UK, will need to be transferred to an entity or affiliate which is located within the EU. Submission of Individual Case Study Reports (ICSR) and Aggregate Reports

Post Brexit on 31 December 2020, the UK requires submission of all UK ICSRs (serious and non-serious) and serious ICSRs from other countries via the new MHRA Gateway and/or ICSR Submissions portal which have been developed. Obligatory registrations should have been made prior to Brexit. The MHRA may, at some point, issue a new list of UK-specific reference dates for Periodic Safety Update Reports (PSURs) potentially resulting in the submission of PSURs at different times in the UK and the EU. In the meantime, the European Union Reference Dates (EURD) should be followed and, from 01 January 2021, PSURs should be submitted in parallel to the UK and EU. The Authorities may require joint signatories for both the UK and the EU.

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Owing to a separate deal known as the Northern Ireland protocol, Northern Ireland’s ICSRs, Risk Management Plans (RMPs), and PSURs for products being marketed in the Northern Ireland region will remain aligned with EU requirements, while the rest of the UK will follow national procedures. For products placed on the market in Northern Ireland signals should be additionally reported to the European Medicines Agency (EMA).

After 31 December 2020, a pharmaceutical company solely located in the UK is no longer able to hold an EU MA. On the other hand, an EU pharmaceutical company can continue to hold a UK MA for a period of time after 31 December 2020, but they will have had to establish a contact person based in the UK.

Signals and safety management

Risk Management Plans (RMPs)

The MHRA will continue to accept EU versions of the RMP, but where the UK has made a specific request for information to be included; this may need to be provided in a UK specific annex. For centrally approved products the current approved version of the RMP should be included in the initiating sequence as part of the conversion process. PV System Master File (PSMF)

According to EU legislation , the PSMF must be housed within a member state of the European Economic Area. Post Brexit, the Marketing Authorisation Holder (MAH) will need to change the location of the PSMF to a Member State within the European Economic Area (EEA). Typically, the PSMF is electronically accessible from any location in the world. The MHRA is, therefore, unlikely to ask for a separate UK PSMF. However, there may need to be UK-specific annexes. MHRA says the

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format and content requirements for the UK PSMF are “equivalent to that of the EU PSMF.” On the other hand, the MHRA says that each UK PSMF must have a unique identification number. EU QPPV and UK QPPV

According to Article 8 of Directive 2001/83/EC and Article 74 of Directive 2001/82/EC, the Qualified Person responsible for PV (QPPV) must reside and carry out their tasks in a Member State of the EEA. The QPPV will, therefore, need to change his/her place of residence and carry out his/her tasks in the EEA, or a new QPPV residing and carrying out his/her tasks in the Union (EEA) will need to be appointed. Based on information by the MHRA, “the role and responsibilities of the UK QPPV are equivalent to that of the EU/EEA QPPV,” but require that the QPPV be based in the UK.

AUTHOR BIO

In addition to the current practices for the EMA, the MHRA requests notifying of signals arising from any data source/ emerging safety issues within three working days if they have not already been notified to them. After Brexit, this also includes standalone signal notifications submitted to the EMA as well as signals raised by the EMA. The MHRA will carry out assessment of signals and issue UK decisions for both signals identified by the MHRA and those highlighted internationally.

Comparable to the EU QPPV for products in the EEA, the UK QPPV will be responsible for establishing and maintaining a PV system for all UK authorized products. This includes oversight of what is happening at all times and visibility of the global safety database. However, the MHRA also notes that it will allow MAHs an exemption to the requirement that the QPPV be based in the UK for a short period of time in order to transition. Pharmaceutical companies need to prepare a plan after the transition period. This will most likely reference having in place an EU QPPV as well as a UK QPPV to have direct points of contacts in place for the Authorities. What are the most likely next steps?

The UK and the EU will continue to independently negotiate on the detailed terms of Brexit to ensure the continuous provision of medicinal products and medical devices. There are still some areas in PV post Brexit, which will be defined in specialised working groups by the authorities. Pharmaceutical companies are advised to be ready for upcoming changes, which will be published in due course on an independent system for PV, PSMF and QPPV. The outcome of the detailed discussions between the involved parties is so far unclear. Our QPPV office will continue to closely monitor and keep abreast of any further developments. We will ensure that, as an end-to-end PV Service partner, our QPPV services remain uninterrupted. References are available at www.pharmafocusaia.com

Upon gaining his MD with the Philipp’s University of Marburg, Germany, Philipp Hofmann started his career as a medical writer in the pharmaceutical industry, responsible for Annual Safety Reports. This very first position was soon followed by a continuous professional development from Medical Manager Vigilance, Safety Officer, Deputy EU QPPV, Director Global Safety to Head of Pharmacovigilance and EU QPPV prior to joining Navitas Life Sciences. Philipp, with his specialization in Pharmaceutical Medicine, is now heading the pharmacovigilance department of Navitas Life Sciences and the QPPV office.

STRATEGY

FPS Reactors and Process Vessels Charging Isolators

Tailor made solutions for your needs Stefano Butti, Sales Director, FPS, Italy

AUTHOR BIO

FPS is an Italian company specialised in the design and manufacture of containment & isolation systems and micronisation solutions for the handling and production of active and sterile pharmaceutical ingredients; it is mainly addressed to pharmaceutical, chemical and cosmetic companies all over the world. With almost 100 employees, more than 1,300 systems in operation worldwide for handling pharmaceutical substances, FPS presents itself on the market as an international company, extremely flexible and able to adapt to different customer's needs. During the past years the necessity to have a safe loading system for process vessels became more and more actual due to the increased activity of product to be handled and also to the updated requirement from REACH regulation. Based on this FPS has designed a full range of containment systems suitable to load process vessels in a fast, safe and ergonomic way. Always starting from the process evaluation and from a risk assessment we have realised more than 100 systems for this specific topic. They are almost all different one from the other due to the specific requirement and need of our customer. The project definition always starts from on-site supervision to check installation areas and discuss with the end user about specific needs. In function of possible physical constraint first evaluation is about the possibility to use gravity discharge or to evaluate alternative systems like Vacuum Transport System (VTS).

First of all, a preliminary design is realised and once the real project starts an ergonomic study on a 1:1 scale mock-up is performed to check real activities to be performed. Once this critical step is completed the final manufacturing of the system is started. FPS propose different configurations: • Single chamber mobile reactor loading system for small quantities of product handling, up to OEB4 category (CPT down to 1mg/m3). • Single chamber reactor charging system for larger quantity of product handling, up to OEB4 category (CPT down to 1mg/m3). • Double chamber reactor charging system for larger quantity of product handling, up to OEB5/OEB6 category (CPT down to 0.1 and 0.01 mg/m3). • Double chamber reactor charging system for larger quantity of product handling, up to OEB5 category (CPT down to 0.1mg/m3). For more information contact: sales@fps-pharma.com

Stefano Butti has studied Mechanical Enineering at university of Milan and graduated in 2000. ISPE member since 2002, he participated as speaker to different congress and seminar on Containment and Micronisation topic both for HPAPI and Sterile application as well as published different articles in technical newspapers. He Joined FPS company in 2008 starting as Technical Sales Manager and he is now head of the Sales group for the company Containment and Micronisation system provided worldwide.

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CLINICAL TRIALS

EMPLOYING REAL-WORLD EVIDENCE TO IMPROVE CLINICAL TRIAL STRATEGIES T

here are many advantages to studying a drug’s performance under everyday conditions that cannot be matched in clinical trials, which are essentially controlled experiments. A traditional, Randomised Controlled Trial (RCT), which has a specific design with established inclusion and exclusion criteria, cannot anticipate all the real-world situations that can occur when external factors come into play. Those factors can include behavioural patterns, other therapeutic interventions, and health system effects, such as whether a drug is reimbursed in that country and the treatment protocols in that region or hospital. External factors can be global, local, or specific to the individual patient.

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CLINICAL TRIALS

Employing real-world evidence allows for the study of many aspects of diseases, such as natural history, patient populations, and outcomes, under everyday conditions. Therefore, real-world evidence has many applications in clinical research, ranging from optimising clinical trial design and population/outcome selection to reducing the burden of regulatory commitments. Sumeet Bakshi, Vice President, Real World Data Solutions, Certara’s Evidence, Value & Access group Richard Tao, Associate Principal Regulatory Writer and Submission Leads Member, Synchrogenix, Certara’s regulatory science company

Under clinical trial conditions, a patient with diabetes who is prescribed an anti-diabetic drug must adhere to the study protocol and their outcome is measured based on that protocol. If the patient misses several doses, their data may need to be removed (or censored) from the final analysis dataset. Poor medication adherence is not typically factored into a clinical trial. However, in the real world, people frequently forget a dose of medicine, take the wrong dose, or take it at the wrong time. Medication adherence is just one real-world example; there are many others that need to be considered when trying to truly understand patient outcomes. For example, inclusion and exclusion criteria for a clinical trial prevent people with specific comorbidities or prior treatments from participating in the trial. Many of these people are likely to receive the intervention in real life despite not qualifying for the RCT. A real-world study generally includes a wider variety of people and circumstances and better reflects everyday situations. However, the experimental design of traditional RCTs allows for easier isolation of the treatment effect of one therapy compared with another as it is less subject to the bias which can present challenges in analysis of Real-world Data (RWD). Thus, RWD are usually employed in conjunction with and in complement to RCTs rather than as a replacement for them.

Challenges Using Real-World Data

Despite their obvious advantages, the adoption of real-world studies involves challenges such as gaining access to and managing the heterogeneity and messiness of the data. The Real-world Evidence (RWE) currently being generated is predominantly from structured data, i.e., electronic medical records, Electronic Health Records (EHRs), and healthcare claims. These data sources, although large and complex, form the tip of the iceberg, as many other data sources, such as free-text entries, paper records, telephonic and video consultations, images, or video camera footage are now available and could form valuable RWE input. Increasing Adoption of RWD in Rare Diseases/Populations

Rare disease research is an area that can benefit greatly from using RWD. Many rare diseases are not well studied, their natural history is unknown, and the outcomes that should be targeted are unclear. Traditional RCTs include treatment and control arms in which participants receive the current standard of care or placebo. Of course, in the case of rare and orphan disease populations, a sufficiently powered traditional RCT may be very difficult to carry out due to recruitment challenges and the potential absence of a current standard of care. As a result,

single-arm trials are increasingly being relied upon as pharmaceutical companies focus on small populations in therapeutic areas of extremely high unmet medical need. This phenomenon is not only limited to rare and orphan disease populations, such as Batten disease and Lennox-Gastaut Syndrome, but also includes smaller underserved segments of more common diseases, such as HER2negative hormone-positive breast cancer or non-muscle invasive bladder cancer, where there is no current effective treatment available. Similar scenarios occur with some types of advanced cancers where the disease is both rare and life threatening, making it impractical and unethical to recruit a control population for a clinical trial. In these instances where it is not possible to establish a control group, regulators and payers are increasingly accepting single-arm trials, but they prefer to see some comparative data as part of the marketing authorisation submission. With single-arm trials, RWD can be used to generate an external comparator arm; a practical approach that also saves time and lowers costs. Creating an External Comparator Arm

Regulators are often interested in the use of so-called natural history studies to offer pure external comparators, especially in circumstances where there are no approved treatments or accepted standards of care. However, the term natural history is really a misnomer because all patients receive at least some kind of intervention in the real world. Even in cases where there is no standard of care, doctors always try to alleviate patients’ symptoms with some type of treatment. Consider Batten disease, which refers to a group of rare, fatal, inherited nervous system disorders that affects about 50 children in the UK. These children have about 20 to 25 seizures a day and reduction in seizure frequency is the desired outcome. Although there is no approved treatment, doctors do prescribe different

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CLINICAL TRIALS

types of anti-epileptics. Therefore, there cannot be a true natural history study offering a pure non-treated comparison. In such cases, the control group would be an arm that provides information about the treatments used and outcomes for these patients, without the interventional drug in question. In addition to the use of natural history studies and historical controls, external control arms can leverage data synthesised from other clinical trials that are not part of the same protocol. A synthetic arm is designed by selecting patients from placebo groups in past clinical trials, matching them to participants in the current trial, and then studying the outcomes. Techniques (such as matching techniques) are often used to adjust outcomes for valid comparisons similar to those used in classical realworld studies. New Trends in the Use of RWD in Clinical Trials

The development of COVID-19 vaccines and drugs provides some good examples of evolving RWD trends because they were created in response to a new disease where there was limited pre-existing clinical data that could be used for RCTs. Much of the COVID-19 disease knowledge and epidemiology was established using RWD and integrated into clinical trials. RWD is also being employed with clinical data to support prescribing decisions for patients with COVID-19. For example, the World Health Organization (WHO) Solidarity Trial for COVID-19 treatments included RWD in its drug comparisons. It compared four types of existing antiviral or antiinflammatory drugs – remdesivir, hydroxychloroquine, lopinavir/ritonavir, and interferon beta – without a traditional control group. It was not a double-blind study, but a direct comparison study. The results were published by the WHO, and decisions were made by regulatory decision makers and policymakers about which drugs were useful for COVID-19 treatment and which were

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RWD can offer regulators and other decision-makers additional insights into the effectiveness of treatments in the ultimate setting in which they will be used by broadening the population for whom the evidence is assembled and by offering insights into how a drug is likely to perform under nonideal conditions.

not recommended. The Solidarity Trial included local hospital standard-of-care procedures, which refer to real-world situations. In addition to the changes in development and research pathways for COVID-19-related treatments, many non-COVID-19-related clinical trials are currently adversely impacted and progressing slowly because sites are busy caring for COVID-19 patients, physicians are not able to give trials the attention they need, and it is very difficult to recruit patients. As a result, researchers are looking for operational models that are less site dependent and which can leverage RWD. That impetus has further strengthened the trend toward democratising patient data, reinforcing patients’ ownership of their data. As a practical example, by obtaining consent from patients to access their EHRs, insurance claims, and administrative or social demographic data, the burden of collecting such information through the standard trial case report form (a duplicative effort) can be avoided. Blockchain technology is becoming one of the key

tools that allows all those data to be brought together in a validated and secure environment, thereby reducing patient recruitment requirements, data collection needs, and dependence on personnel at the sites going forward. Artificial Intelligence (AI) is also being used for real-world studies. Its earliest and most common uses were in epidemic modelling. AI is now being employed to screen patients and identify those at risk for a particular disease, predict clinical outcomes, and determine optimal drug doses for specific patient groups. The Asian regulatory context

In some Asian countries, pharmaceutical companies are using RWE to obtain marketing approval for traditional herbal medicines without the need for RCTs. As these herbal medicines have already been on market for many years, China’s National Medical Products Administration (NMPA) encourages companies to collect pertinent patient data and submit them for approval under its real-world study regulations. NMPA released its “Guidelines for Real-World Evidence to Support Drug Development and Review (Interim)” in January 2020 and they were joined by the Center for Drug Evaluation’s “Technical Guidelines for Real-World Research Supporting Child Drug Development and Evaluation (Trial)” in November 2020. Furthermore, when NMPA approved an Allergan glaucoma treatment product in March 2020, it became the first medical device approved in China using RWE. As the product was already marketed for glaucoma treatment in the US, Allergan could compare RWD from patients in China with the US clinical trial results to determine if there were any ethnic differences between the two patient populations. As no differences were detected, an additional double-blind RCT was not required in China. Using this approach, it took less than one year for Allergan to secure NMPA approval of its glaucoma

CLINICAL TRIALS

Looking Ahead

While post-marketing studies will likely remain the primary use for RWE for the next 10 years, the COVID pandemic could be a watershed that elicits real change. It has injected a sense of urgency and promoted a shift from the traditional way of doing things to a more innovative and proactive approach using RWE to access more data and accelerate timelines. The drive is toward getting better and broader data and not just clinical trial data. Regulatory agencies were already becoming more accepting of the use of RWD in clinical trials, but COVID19 will likely accelerate their adoption. The US Food and Drug Administration (FDA) introduced draft guidelines for “Submitting Documents Using RealWorld Data and Real-World Evidence

to FDA for Drugs and Biologics” in May 2019 and it plans to issue additional guidance in 2021. In the interim, the FDA-funded RCT-DUPLICATE project has conducted 10 non-interventional, RWE studies designed to emulate RCTs and evaluate cardiovascular outcomes of anti-diabetic or anti-platelet medications. Initial results from the study, which is being conducted by Brigham and Women's Hospital and Harvard Medical School in close collaboration with the FDA and Aetion, were published in December 2020. The researchers selected three activecontrolled and seven placebo-controlled RCTs for replication using patient claims data from US commercial and Medicare payers. Nine of the 10 RWE studies achieved at least two of the three agreement metrics. Six of the nine studies also achieved ‘regulatory agreement,’ i.e., interpretation of the results would have resulted in similar regulatory decisions. The results did highlight one significant challenge – as placebos are not administered in everyday clinical practice, they cannot be observed in RWD. Pharmaceutical companies are also looking to incorporate RWD into decision making earlier in the drug-development process. This change is affecting some pharmaceutical companies’ organisational structures, which, in the past, were very clearly demarcated in terms of pre-launch and post-launch activities.

AUTHOR BIO

treatment system. There are, however, challenges in comparing clinical data between countries. One difficulty is that most clinical trials are conducted from a western market perspective. For example, the comparator chosen in a clinical trial is often from the US, UK, or a European market. Some diseases are classified differently in the Asian and European markets, presenting significant challenges for clinical trials. For example, certain tumours are defined differently in Asia and Europe depending on the prevalence of tumour sub-types. Another issue is that treatment pathways in some Asian countries may be very different from those in western countries due to drug availability and pricing, physician preferences, and other factors. The contexts can also vary greatly, not just in terms of disease definition, but also social demographics and the availability of traditional medicine methods. As a result, it can be challenging to overlay evidence from one market on the other. RWD can be leveraged to identify and address these differences as part of designing the clinical development program.

RWD used to be the domain of the postlaunch team and often in the context of market access. Increasingly, the market access staff are joining drug development programs very early, much earlier than they did even as recently as two or three years ago, and bringing their expertise with RWD to address challenges across the development life cycle. Conclusion

Use of RWD has the potential to help improve development program designs by enabling researchers to test hypotheses and define appropriate clinical trial endpoints for efficacy and safety. The insights gained can help avoid unnecessary clinical trials and improve the probability of success of a development program. RWD can offer regulators and other decision-makers additional insights into the effectiveness of treatments in the ultimate setting in which they will be used by broadening the population for whom the evidence is assembled and by offering insights into how a drug is likely to perform under non-ideal conditions. These data may be less structured and may require more handling and manipulation expertise to make them usable in conjunction with clinical trials, but they can provide a valuable and often more meaningful picture of a product’s potential. References are available at www.pharmafocusasia.com

Sumeet Bakshi is Vice President, Real World Data Solutions in Certara’s Evidence, Value & Access group. Sumeet qualified as a physician at the University of Mumbai in India and holds an MBA from the Saïd Business School at Oxford University in the UK.

Richard Tao is Associate Principal Regulatory Writer and Submission Leads Member at Synchrogenix, Certara’s regulatory science company. Richard qualified as a medical doctor and public health research scientist from Nanjing Medical University in China and received an MS from Jiangsu Institute of Parasitic Diseases and postdoctoral training at the University of Massachusetts and Harvard School of Public Health.

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CLINICAL TRIALS

PERSONALISING PRESCRIPTION A laser sharp approach for complex disease indications Personalising Prescription is a sub-set of the Personalised Medicine concept. Both the concepts were always in practise while the gene and cellular technological advances have made them sound fancier. There are common, complex diseases like cancers; the diseases for which there are no single underlying target like psychiatric conditions; the diseases that are rare like Orphan categories; the oral diseases that are at the helm of integrated treatment approaches. The personalisation happens both at the disease and patient level while the factors to be considered are genetic, environmental and personal variables for prescription. Subhadra Dravida, Founder and CEO, Transcell Biologics Gargi Roy Goswami, Founder and Director, KROYNAS Private Limited Rajiv Gupta, Entrepreneur; Managing Partner, Lateral Consulting

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he finale of the 20th century brought fresh hopes of a revolution in medicine based on advancing knowledge of the human genome decoded. The Human Genome Project was possible due to swift advances in genetic technologies that made possible the parallel testing of many Single Nucleotide Polymorphisms (SNP) in a cost effective manner. The beginning of these technological advances led to a Science

CLINICAL TRIALS

Personalised Prescription in Clinical Practice of Psychiatry

To apply personalised prescription in clinical practice requires a thorough understanding of the pharmacokinetic and pharmacodynamic principles of psychiatric drugs and does not depend on new developments in pharmacogenomic or other biomarker testing. It appears to require only that sophisticated clinicians understand that genetic, environmental or personal variables influence pharmacokinetic and pharmacodynamic response; the therapeutic window of the drug to be taken into consideration. Blood levels, called therapeutic drug monitoring, have been used by psychiatrists to personalise dosing for lithium, tricyclic antidepressants and some antipsychotics including clozapine in the past. Risperidone prescription is the best known example with genetic, environmental and personal variations reviewed in clinical practice. Personalised Prescription for Orphan diseases

journal editorial comment in 1997 that defined personalised prescription as tailoring drugs to a patient's genetic makeup and predicted that personalised prescription would become a reality in clinical practice. The discovery of induced Pluripotent Stem Cells (iPSC) has revolutionised some of the concepts in personalising and precision components of Medicine The concepts of personalised or individualised medicine and prescription are not new to the medical jargon. However, genetic advances have made discussing 'personalised medicine' and 'personalised prescription' in genetic terms more appealing and practical while exploiting mainly genetic differences between patients. Physicians have traditionally practiced personalised medicine in their efforts to decide the best treatment for each of their patients and was based on subjective physician preferences and not on scientific knowledge. Personalised medicine

is known as a global concept that may include personalised surgery, personalised rehabilitation, personalised nutrition and personalised prescription. A personalised prescription includes not only the use of new tests, that may or may not be pharmacogenetic tests, but also the concerns of all scientific information valid for prescribing medication. For a comprehensive view of personalised prescription, clinicians are expected to consider genetic, environmental, and personal variables when prescribing any medication. Known important genetic variables in specific drug response can be explored using pharmacogenetics; environmental variables such as co-medication, supplements, foods, beverages, and smoking etc for some drugs; and personal factors such as age, gender or medical illnesses (renal or hepatic insufficiency) as crucial personal variables in the response to some other drugs.

For those with a rare disease and for which there is no targeted drug available, almost any medicine is personalised. Whatever drugs or treatments they take are to alleviate the symptoms with better or combination of drugs. In some cases genome sequencing, iPSC technology incorporated in personalising prescription are next generation in nature with scope to offer cures for patient segments who are neglected by the pharma research. Muscular Dystrophy (MD) is a neuromuscular degenerative disorder, is one such Orphan disease category that afflicts individuals of different age, race, and gender. MD is a group of rare hereditary muscle diseases characterised by progressive skeletal muscle weakness. There is no traditional small molecule targeted drug based treatment available to the Physicians to prescribe till date. Using some of the drug test endpoints like cell viability, apoptosis, creatine kinase secretion and dystrophin levels on patient’s sourced iPSC platforms in the labs, the drug’s suitability can be ascertained for

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CLINICAL TRIALS

the particular patient. Another area of therapy that is considered in personalising is how the physiology of muscle pathology will interact with these therapies and a precision approach with tailored physical activity is likely to benefit individual patients. Personalised Prescription In and For Oncology

The Genetic Connection and Use of Stem Cells in "Everyday" Medical Applications There is significant ongoing effort by a large cross section of the medical and research fraternity, across the globe, to work with Cryopreserved Adult Healthy Stem Cells to develop highly Personalised Prescriptions, specifically tailored for, both, lifestyle enhancements and specific ailments/diseases, even the chronic ones for each individual. The individual's Whole Exome/Genome Map is becoming an essential detail to better plan for Personalised Prescriptions. The increasing success of biobanks for preserving the Umbilical Cord Blood of the newborn is an example of awareness among the modern day parents of the need for their children to have access to their own cell and related genetic information for targeted health solutions. The same set of ‘aware’ parents are among the initial target audience which understands the need for having access to healthy stem cells for Personalised Prescriptions based on their own genetic map and hence, they are opting for not only the Whole Exome/ Genome Mapping but also going ahead to Cryopreserve their Healthy Stem Cells for any future requirement for themselves in combating cancers or anyone else from their genetic family tree. Genetic Mapping can definitely enable deeper understanding of the state of health, chances of potential diseases and inherent immunity for that individual. Personalised Prescriptions, based on Genome Mapping, can suggest a host of preventive therapies to mitigate or minimise the occurrence of debilitating diseases. There is a distinct lowering of age limit at which chronic diseases have

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started to impact the general population. A range of diseases resulting from stressful lifestyle, pollution and general degradation of living standards are agnostic of age, geography, race and even social class and levels. Stem cells are integral to Personalised Prescriptions for quite a few of such diseases, such as Autologous and Allogeneic procedures for HSCT - Hematopoietic Stem Cell Treatment for cancers impacting the blood cells. The recent advancements enable Precise Cell Selection from the patient's own Peripheral Blood Stem Cells (PBSC) or Bone Marrow (BMT) and even Umbilical Cord Blood (UCBSC) for an autologous procedure, which has a much higher probability of the body accepting the infusion of healthy stem cell selection and minimised occurrence of GVHD Graft vs Host Disease, post-transplant. In fact, there is a preference for cryostorage of a patient's healthy stem cells much earlier in the treatment regime, before chemotherapy or any other such procedure which may render the patient's own healthy stem cell unfit for use at a later date. An Autologous procedure also reduces the need to find a donor and definitely reduces the total cost and time for the treatment. In the event, Allogeneic procedure is necessitated, Personalised approach would prefer opting for PBSC of the Donor instead of bone marrow as a source, along with procedures to derive the necessary cell selection to reduce occurrence of GVHD post transplantation.

of disease pathways, genomic interactions, and novel biomarkers of oral conditions continues to increase. Below are a few examples to get a glimpse of how research is unravelling newer molecular targets that are promising personalised oral health care in the near future.

Personalised Prescription for Oral diseases

Acute and Chronic Orofacial Pain

Most oro-dental pathologies such as dental caries, periodontal diseases, oral and pharyngeal cancers, chronic orofacial pain, etc. and craniofacial disorders, such as cleft lip/cleft palate, arise from a complex interaction of genetic, biological, behavioural, and environmental factors. Using high-throughput ‘omics’ approaches to assess disease susceptibility, prevent disease, and holistic treatment is slowly becoming a reality as our understanding

Head and Neck Squamous Cell Carcinoma

Head and Neck Squamous Cell Carcinoma (HNSCC) is a disease with complex gene alterations. These alterations result either in shutting down or amplification of regulatory signals within a cell that accelerates cellular growth giving rise to tumours. Current treatment options for HNSCC include surgery and cytotoxic therapies. Most of these treatment strategies result in drastic reduction of the quality of life of the patient. Better understanding of the biological heterogeneity of head and neck cancer will help customise treatment and optimise outcomes for this malignancy. Cancer management has long focused on care based on tumour stage, subtype, and histology. Knowledge obtained with the help of genomic technologies offers a scope for a more refined tumour classification based on signalling pathways that can be targeted more precisely. Molecularly targeted therapies developed and tested for use HNSCC are namely include EGFR -directed drugs like cetuximab and EGFR - tyrosine kinase inhibitors namely gefitinib, lapatinib, erlotinib is being used for targeted therapies for HNSCC. Individual genetic variation in Cytochrome P 450 superfamily of enzymes is known to be involved in the metabolism and bioactivation of most of the drugs. People with a certain allelic variation of CYPD26 gene are unable to convert codeine to morphine. So these groups of people experience insufficient analgesia but they are able to withstand many of the adverse side effects associated with opioids. On the other hand, morphine intoxication is

CLINICAL TRIALS

Oral Infectious Diseases

Genomic approaches have revealed several interesting information about genomes of oral pathogens involved in the progression of common oral infectious diseases such as dental caries and periodontal disease. A noteworthy example is the Human Oral Microbiome Database (HOMD) which is an assembly of almost 1000 predominant microorganisms that inhabit oral tissues. This has opened up a new arena of early intervention tactics to the development of novel strategies for oral polymicrobial disease diagnosis, prevention and treatment. For instance, a new bacterial species, Scardovia wiggsiae was identified through HOMD project. This bacteria is a potential pathogenic indicator for early childhood caries risk. Thus, information about this microorganism and similar findings about additional pathogens drive the decision of a success-

ful treatment strategy that may call for a change in the diet pattern as well as microbiota. Several Genome Wide Association Studies (GWAS) findings from dental caries investigations have identified specific caries susceptibility loci and related information. An interesting example is about the TAS2R38 and TAS1R2 genes that mediate taste sensation. Some individuals with variations in these two genes have been shown to be more predisposed to eat cariogenic food choices that automatically makes them potential candidates for dental caries. Dental signature of every individual is unique and while dentistry has also many oral treatments which are generic and applicable for the vast majority of the population, the advancements in the field have made the need for Personalised Prescriptions move up from being a value add to being inherent to the field of dentistry itself. One such example is the use of Stem Cells Extract (SCE) to enable regenerative recovery after a Tooth Implant procedure. A typical tooth implant is a Bio-inert external item, however, the use of SCE makes the same Tooth Implant to become Bio-Active offering regenerative capabilities, reducing the sense of pain and aiding recovery in much less time. Stem Cell Extract is fast on its way to become an integral part of every Personalised

AUTHOR BIO

observed in individuals with multiple copies of CYP2D6 because of extremely rapid metabolism of codeine. Interindividual differences in response to anesthetics such as isofurane, halothane and fentanyl are produced due to variants in CYP2E1 and OPRM1 genes. Thus, dentists would be able to customise safer and more effective peri-operative and post-operative pain management by identifying and monitoring such individual genetic variation. Another important area of concern of chronic Orofacial pain management is TMD or temporo mandibular joint disorders. Identification of genetic variations in an individual’s pain perception can offer clues about susceptibility to TMD. This information will be beneficial to segregate individuals most susceptible to developing chronic TMD and early treatment strategies can be designed for them. One example of such an approach is the Orofacial Pain: Prospective Evaluation and Risk Assessment (OPPERA) study which is the most comprehensive analysis till date.

Prescription for Tooth Implant and it has the potential to be included in any dental surgical procedure because of its regenerative properties. Challenges of Practising Personalised Prescription in Traditional Medicine

The integration of genomics and cellbased technologies into routine clinical practice comes with its own challenges. Few of the barriers which calls for a thought to find strategies to overcome are: • Skepticism by providers and payers about the usefulness and credibility of information • Added cost to both caregivers and patients • Lack of insurers • Lack of integration of data sets with patient history data • Lack of knowledge, resistance, risk aversion at the Practitioner’s end • Lack of public awareness. To overcome present and future challenges, the authors feel that the first step should be to engage in a dialogue focused on preparing next generation clinicians, researchers and educators; upgrading the knowledge base of current health professionals through interdisciplinary training and skill sets; adopt, practise and build credibility in the space.

Subhadra Dravida is the Founder and CEO of Transcell Biologics (www.transcellbio.science) that is into translating adult stem cell technology prowess into real time applications

Gargi Roy Goswami is the Founder and Director of KROYNAS Private Limited, India (www. genedent.com) focused on education and training to support translation of research into clinical applications in the domain of Dental Genetics and Saliva Diagnostics.

Rajiv Gupta is an Entrepreneur; Managing Partner at Lateral Consulting.

www.pharmafocusasia.com

Bioreactor Automation Enhances Productivity with Biologics Driven by real-time sensing

Bioreactor performance for mammalian cell culture has been automated and improved with better soft-sensor inline analytics and model predictive process control. With simple customisation of CHO cell models using a novel software tool, precise and stable glucose feed-regulation specific to user cell lines and media requirements is enabled. This fully-integrated hardware and software system provides unprecedented custom automation and reproducibility for biologic product performance in CHO cell culture at the lab-scale. Hiroaki Yamanaka, Yasuhito Murato, Paul E Cizdziel Members, Life Innovation Business Headquarters Division, Yokogawa Electric Corporation

In the quest for improved quality and productivity in drug manufacturing, the industry is moving toward increasing use of bioreactor systems with realtime integrated monitoring and advanced analytics that have the potential to enable automation, drive performance and improve data-rich quality control. However, there exist multiple options in sensors and technologies for monitoring important cell culture variables or Critical Process Parameters (CPP). Furthermore, cell culture vessel configurations can be disposable Single-use Bioreactors (SUBs), glass or even stainless-steel. They can be stirredtank in design, rocking platform bags or perfusion configurations. The sensors and monitoring technologies selected for these configurations need to be suitably designed and compatible with the bioreactor architecture. When properly selected, the Process Analytical Technologies (PAT) provide not only analytical insights into ongoing bioprocesses, but can be leveraged for real-time control and automation; especially in fed-batch or continuous culture. Automation is a key trend driving improvements in manufacturing; especially for production of biologics in the biopharmaceutical industry. One big

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challenge is the integration of systems for effective automation. For example, recent uses of in-line sensors in mammalian cell culture can be used to monitor biomass production, key media nutrients, or metabolism indicators. Spectrophotometric techniques such as Near-infrared (NIR) and RAMAN technology have been employed to quantitate and correlate spectroscopic data with precise chemical signatures. As a result, the monitoring of glucose concentrations and lactate in real-time are becoming more common, reducing the need for off-line analysis and enabling the automated delivery of media supplements as required.

PAT Integration & Data Alignment

Comparing the capability of RAMAN and FT-NIR for spectroscopic applications in cell culture is frequently a debate among researchers. The ability to spectroscopically identify and differentiate cell culture media components is primarily affected by considerations of bandwidth, sensitivity, interference and software. And the latter (software), is quite capable of having a sizable impact on the prior three mentioned parameters as described in the examples below.

Figure 1 – Refinement of the Cell Culture Calibration Model in Three Steps Illustrated in this figure is the three-step protocol for customising a calibration curve specific to a user cell line, media and process. Beginning with a baseline calibration curve (provided), it generally takes three cell culture runs on the BR1000 bioreactor to accurately align PAT sensor data with off-line reference values.

For instance, in bioreactor culture, two issues frequently cited and considered limitations of FT-NIR included baseline data variation due to cell density dependent optical scattering and interference by water in glucose detection. We have convincingly addressed the first issue largely by digital noise suppression software improvements. Whereas the second issue of signal interference by water or other molecules overlapping with glucose bond absorption profiles was reduced by the proper selection of, and integration of multiple wavelength micro-scan data to provide a more specific footprint of reliable molecule-specific bond signatures. Both of these NIR limitations were thus overcome by innovative data use and novel software approaches. Consequently, the determination of glucose concentration in cell culture media with FT-NIR can now be robustly correlated with actual reference concentrations or off-line cell culture glucose measurements using bench top chemical analyzers. The derivation of cell models and the proper selection of micro-scan data for a particular cell line and cell culture medium are fairly complex tasks requiring significant resource time and effort to achieve the proper calibration. And although a universal cell model for any expression system would seem to be ideal, it is not an effective viable option. To this end, we have settled on a precondition and a calibration approach to simplify the alignment of NIR data with user-specific cells and media. The precondition is to focus on a specific cell type so that deviations from the standard model are minimised.

And the calibration approach is one that leverages a baseline calibration curve to provide a suitable starting point for subsequent user data-derived refinements. By limiting the developed FT-NIR application to specifically CHO cells, we were able to derive an effective lap-top software tool that uses a ‘datadriven’ model refinement algorithm. The tool enables the custom calibration of FT-NIR data to any CHO-cell based user expression system by smartly adjusting spectroscopic micro-scan data. It has been our experience that starting with the pre-set baseline calibration algorithm, and performing only three consecutive bioreactor runs, enough user data can be gathered to construct a precise custom cell model. By using this overall approach and the final cell model, NIR spectroscopic monitoring can provide highly accurate glucose and lactate concentration sensing in real-time specific to the user cell line and culture medium. For some purposes, simple monitoring of CPP such as glucose & lactate may provide significant value for production and quality control. However, it can also be coupled with intelligent bioreactor software systems to enable automated feeding of glucose for maintaining constant concentrations or dynamic control. More will be discussed about this in a later section. In-line biomass measurement is an important PAT to understand the culture growth phase and cell viability in bioreactors in real-time. Sometimes referred to as bio-capacitance, electrical impedance

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Figure 2 – Precision Control of Glucose and Alignment with Off-line Analytical Data Use of the refined calibration curve with Model Predictive Control (MPC) software results in strong alignment of glucose concentrations in fed-batch culture. DG44 CHO cells were seeded at 5 x 105 cells/ml in FortiCHOTM (ThermoFisher) medium containing 5 gm/L glucose. Cells were cultured for 14 days with a set-point concentration of 2 gm/L glucose. The delivery of glucose feed solution (450 gm/L) was automated via peristaltic pump action.

sensors monitor cumulative charge polarisation across intact plasma membranes to estimate total live cell biomass. This technology has been widely adopted in biopharma as a popular method to calculate Viable Cell Density (VCD) in real-time. Like many users, we had encountered issues with adopting this technology, including reliability issues at higher cell densities and interference from some media components. However, we have innovated around these issues in several ways. Firstly, a thorough investigation of the cell physical size and growth dynamics (in suspension) during the cell culture timeline was completed to properly relate biomass data to proper cell counts. This is critical information that was collected by using a wellknown immortalised strain of CHO (Chinese hamster ovary) cells. Secondly, electrical impedance data was selected at multiple frequencies to overcome some types of interference from media components or other factors. And thirdly, we derived growthphase-specific data conversion algorithms that recognise transitions, and switch or compensate (as appropriate) in the cell culture timeline. For instance, we have found that calculation of VCD using bio-capacitance data from early and exponential growth phase culture can be used to derive an algorithm that provides high reliability specific to those earlier growth phases. However, this algorithm becomes a poor predictor of actual VCD in stationary or later stage cultures. This fact is likely due to changes in cell morphology that are reflected in later stage culture perhaps due to physiological shifts and changing protein expression profiles. It has been shown that in recombinant cell lines, the biologic expression of monoclonal antibodies is generally

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induced to higher levels when cultures achieve peak cell densities. In bioreactor culture, as CHO cells approach peak cell densities, glycolytic pathways transition to more oxidative phosphorylation metabolism, cell sizes may change and membrane composition also likely changes. These events appear to result in a deviation from the calibration parameters applied for determination of VCD in early stages of culture. Hence, an entirely different data correlation model seems to be appropriate once cultures reach the peak VCD. By integrating and switching from early stage to late stage models at the appropriate time, the generation of accurate VCD data from bio-capacitance measurements can be achieved with appropriate CHO cell models. In our hands, using this approach, bio-capacitance can provide high accuracy in calculation of VCD data throughout the entire bioreactor run, including up to 100 x 106 cells per ml in cell culture.

Model Predictive Control

As mentioned earlier, the employment of in-line PAT for real-time Process Control (PC) is an automation approach that has potential benefits for biotherapeutic drug manufacturing. A key example is to automate feed-control of nutrients in fed-batch culture. Spectroscopic technology for monitoring nutrient concentrations is limited in ways discussed earlier. However, it is universally agreed that glucose concentration is one CPP that has a direct impact on manufacturing performance. Hence, production cell lines are usually well characterised regarding glucose sensitivities and optimal concentration requirements for growth in bioreactor cell culture.

Glucose concentrations have been shown to affect the metabolic state and growth rate of cells in culture, in addition to the expression yields, and post-translational modification of recombinantly produced biologic drugs. To control glucose concentration in bioreactors in real-time with PAT, both accurate detection and feeding strategy are important for process control. PC considerations are obviously not unique to biopharmaceutical production, but rather have been widely studied and employed in many other industries such as chemical plants and oil refineries since the 1980’s. Early strategies, especially in the chemical industry, employed Proportional-IntegralDerivative (PID) control, largely dependent on feedback mechanisms. These PID-based methods work primarily via input sensing and rapidly reiterative feedback adjustment cycles. However, in bioprocess, in-line sensor data collection is slower and living systems take longer time periods to adjust and equilibrate to environmental or process changes. Hence the effectiveness of new prediction-based approaches can exceed the traditional reactive feedback manner. In bioprocess, we believe that PID-methods will increasingly be supplanted with more sophisticated feed-forward predictive control strategies requiring defined constraints factored into decision outcomes. We have developed an intelligent CHO cell Model Predictive Control (MPC) algorithm for glucose-feed control that accommodates multiple constraints including VCD, growth phase, future-state, current concentration, feed-volume dilution factors, and selfcorrection based on differences in measured versus predicted data values. PAT-driven use of this dataadaptive MPC for automated delivery of glucose by peristaltic pump action, has been shown to provide precision control of glucose in fed-batch bioreactor culture, even in low glucose concentration ranges such as 1 gram/liter. With flexible programming features the dynamic regulation of glucose within a single bioreactor run can be implemented, enabling growth phase-specific concentration control, among other unique applications. The effects of this strategy for bioproduction have largely been unexplored mostly due to prior limitations in precision control of glucose concentrations during cell culture.

Summary

The integration and calibration of all these technologies and software requires expertise in cell culture, model building, spectroscopy, programming and data

Figure 3 – A fully integrated bioreactor system with PAT sensing and precision glucose control for CHO cell culture Shown is the BR1000 Advanced Control Bioreactor System from Yokogawa Electric Corporation. Capable of handling 1 to 5 liter stir tank bioreactor vessels, it uses near-infrared & bio-capacitance in-line spectroscopic sensors with model predictive control software for automated delivery of glucose with precision control. In addition to glucose and pH adjustment alkali, up to four other reagents can be automatically delivered by peristaltic pump action. The BR1000 is the only fully-integrated PAT sensing, automated glucose delivery bioreactor system for mammalian cell culture.

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integration. Hence, it is less common for resourceconstrained organisations to explore sophisticated in-line bioreactor automation and process control technologies for biopharmaceutical production. Piecemeal assembly of the many components usually from many independent suppliers and software integrations are a challenging and time-consuming task. In this article we have attempted to explain how we have addressed and solved many hurdles by creating a novel and fully-integrated bioreactor system that we call the BR1000, with unique software for operation and calibration. To maintain strong utility and precision in PAT sensor data but still allow customised use for CHO cell culture, the BR1000 bioreactor system contains programming with a significant degree of CHO cell application bias. Use of other cell lines or significantly divergent media compositions would likely not perform optimally due to these built-in biases. However, for applications with CHO-cell culture, the system we have assembled is likely the most accurate and efficient glucose control, lab-scale technology platform for recombinant monoclonal antibody process development. Currently we are working on next generation glucose sensing PAT control systems that provide similar process control benefits to CHO cell culture and automation for pilot and manufacturing scale bioproduction.

REFERENCES

E. Tamburini, M.G. Marchetti and P. Pedrini. “Monitoring key parameters in bioprocess using near-infrared technology”. Sensors (2014), Vol. 14, p.18941-18959. T. Yardley, D. Kell, et al. “On-Line,RealTime Measurements ofCellular Biomass using DielectricSpectroscopy”. Biotechnology andGenetic Engineering Reviews, Vol. 17,2000, p.3-35. B. Moore, A. Sanford and A. Zhang. “Case Study: The characterization and implementation of dielectric spectroscopy (biocapacitance) for process control in commercial GMP CHO manufacturing process”. Biotechnology Progress (2019), 35e2782. https:// doi.org/10.1002/btpr.2782 Danny Chee Furng Wong, Kathy Tin Kam Wonq, Lin Tang Goh, Chew Kiat Heng, and Miranda Gek Sim Yap. “Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures”. Biotechnology and Bioengineering (2005), Vol. 89, No. 2, p.64-177. Y.-S. Tsao, A.G. Cardosos, R.G. Condon, M. Voloch, P. Lio, J.C. Lagos, B.G. Kearns, Z. Lui.

“Monitoring Chinese hamster ovary cells culture by the analysis of glucose and lactate metabolism”. Journal of Biotechnology (2005), Vol. 118, Issue 3, p.316-327. M. Buchsteiner, L-E Quek, P. Gray and L. K. Nielsen. “Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect”. Biotechnology and Bioengineering (2018), Vol.115, Issue 9, p.23152327. N. Templeton, J. Dean, P. Reddy and J.D. Young. “Peak antibody production is associated with increased oxidative metabolism in an industrially relevant fed-batch CHO cell culture”. Biotechnology and Bioengineering (2013), Vol.110, Issue 7, p.20132024. T. Namatame and P. Cizdziel. “Making antibody medicines manufacturing a reality”. Pharmaceutical Manufacturing (2019), September Issue, p.30-36. AUTHOR BIO

Hiroaki Yamanaka is currently employed as a bioengineer at the Life Innovation Business HQ of Yokogawa Electric Corporation (Tokyo) working on bioreactor process control technologies and applications. A Ph.D. graduate of Kyoto University in Life Science.

Yasuhito Murato is the manager for bioprocess international sales at Yokogawa (Tokyo) corporate headquarters. Having prior international business experience with Novozymes and an advanced science degree in cell biology, Yasu is also an expert in Yokogawa process instrumentation.

Paul Cizdziel has nearly three decades of experience in market-leading global life-science research supply companies including ThermoFisher, Merck Millipore, and REPROCELL in executive positions of technology management, business development, marketing and sales. He has also held scientific positions at the NIEHS (NC, USA) and the Yokohama RIKEN Institute.

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MANUFACTURING

Future of Pharmaceutical Manufacturing

The role of Automation The breakthrough in the search for a coronavirus vaccine highlights the speed with which the pharmaceutical industry can mobilise scientific ingenuity in the service of humankind’s core goals. Yet, industry leaders know that such speed is the exception to the norm. Here, John Young, APAC director at automation parts supplier EU Automation, looks at four areas where greater automation could have a significant impact in helping speed the process of bringing a new drug to market in the years ahead. John Young, Sales Director, APAC region, EU Automation

he Asia-Pacific pharmaceutical sector is set to grow at a compound annual growth rate (CAGR) of 7.1 per cent in the period to 2027. Manufacturers wanting to take advantage of these opportunities should stay informed about the latest trends in automation technology. Unfortunately, many key decision-makers fear their companies will struggle to keep pace with technological innovation in this highly regulated sector. Here are four key areas where greater levels of automation could have a positive impact on the industry in the coming years: (Ultra) High throughput screening

In drug discovery, the chances of discovering a compound that produces the desired impact on the target is very low, so scientists typically check hundreds of thousands of potential compounds.

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MANUFACTURING

Automating QC

This screening process, due to the high volume, is known as high throughput screening (HTS). Automation plays a key role in HTS. Robotics and other forms of automation technology are a key part of any HTS system. Robots will transport assay plates from station to station and specialised automation analysis will often be used to run experiments on the wells. For example, measuring reflectivity to show evidence of protein binding. Manufacturers are considering greater levels of automation to speed up the process and free up skilled workers for other tasks. When 100,000 or more compounds are screened in a single day, the process is sometimes known as ultra-high throughput screening. Naturally, screening on this scale involves significant automation, such as multiple robotic arms operating as colony pickers. Robotic liquid handling

An effective way of automating the

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screening and experimenting referred to above is through investing in robotic liquid handling devices. The simplest version dispenses a fixed volume of liquid from a motorised pipette or syringe. Adding greater levels of automation, such as a Cartesian coordinate robot, allows for the position of the pipette to be altered. The latest systems are highly precise, even when dispensing liquids on the nanolitre scale. Robotic liquid handling has been used to manage precise quantities of coronavirus, for instance, saving time by speeding up repetitive work, reducing the risk of error and lessening the exposure of human beings to the virus. Robotic liquid handling systems are becoming increasingly versatile. An automated workstation can combine multiple operations into a single footprint, helping save floorspace in the laboratory. They can also be customised with different add-on modules, from centrifuges to colony pickers.

Quality Control (QC) is another key area where pharmaceuticals manufacturing may be missing out by not investing early in automated technologies. QC is especially important in any industry, but in pharmaceutical manufacturing, the stakes could not be higher. By reducing the need for human intervention, automation reduces human error. Repetitive tasks can be automated to free up time and resources. These include, for example, colony counts, incubation transfers and data entry. Concerning the latter, many QC labs at the forefront of technological change are increasingly paperless. Data transcription is being automated and advanced data analytics software is capturing realtime insights. Automated laboratories might also use predictive maintenance technologies to help schedule infrequent tasks like planned equipment maintenance. By pairing predictive maintenance technology with a reliable equipment supplier like EU Automation, laboratories can reduce the potential for costly downtime. Automation will not replace the need for qualified QC managers and technicians, but it will enable them to perform their jobs more effectively. Many of the technologies that could achieve this are already available, but QC leaders often struggle to make a convincing business case to secure investment in digitising and automation their laboratories. Personalised medicine

The idea that medicine should be tailored to the specific needs of the patient is an idea as old as the Hippocratic Oath, but the scientific breakthroughs of recent years have led to excited talk of ‘personalised medicine’. Also known as precision medicine or stratified medicine, the concept promises to revolutionise the world of healthcare delivery. The successful mapping of the human genome has played a key role in opening the possibilities of personalised medicine. Specifically, sequencing an individual’s

MANUFACTURING

John Young has vast experience of working in a B2B environment and has worked with EU Automation’s customers all over the world. John helps supply manufacturers with the parts they need to keep their facilities running, whether it is hard to find obsolete parts or the latest in automation technology.

AUTHOR BIO

DNA or RNA can reveal genetic mutations that increase an individual’s risk level and susceptibility to certain diseases. In terms of drug delivery, analysing an individual’s DNA, RNA or other biomarkers can help identify which drugs will be most effective for them or identify if they are more at risk of developing certain side effects, an area called pharmacogenomics. In short, it means abandoning the traditional trial and error strategy used by most doctors and replacing it with a method that is tailored to a unique individual. This approach may play an important role in securing approval for drugs, helping reduce the excessive costs of bringing a drug to the market. This is because it will allow scientists to identify who will benefit most from a clinical trial. Furthermore, the threshold of efficacy for passing through the clinical trial stage could be lowered, because personalised genomes can be used to single out individuals who might benefit most from the drug. Automation will be essential in fulfilling the revolutionary promise of personalised medicine. Many of the technologies associated with personalised medicine will

rely on the generation of vast quantities of data and an appropriate digital infrastructure. As things currently stand, the capacity to gather the data is ahead of our capacity to analyse it. Processing and analysing the data will require artificial intelligence and deep learning algorithms. However, the scale and pace of data generation will continue to grow as digitally enabled devices, including wearable devices and implantable sensors, become more common and accessible. Automation plays a role in many stages of drug discovery and development. Although the speedy development of a coronavirus vaccine will remain the exception to the norm, strategic investments in automation will allow the pharmaceutical industry to speed up the process of bringing drugs to market in the coming years.

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Functional Respiratory Imaging An innovation at just the right time

Every patient deserves to receive the treatment they need. In pulmonology, however, where it is becoming increasingly clear that diseases such as asthma and COPD have many phenotypes, it is difficult to prescribe individualised treat-ments. The gold standard for diagnosis, spirometry, is unable to differentiate between pulmonary disease phenotypes, but Functional Respiratory Imaging does have that ability. It is a technique that makes better follow-up of diagnostics and treatment selection possible. Wilfried De Backer, Executive Chair, Bod Fluidda Jan De Backer, Ceo, Fluidda

‘COVID-19 has made the demand for innovative diagnostic techniques greater than ever.“We are having difficulty bringing the pandemic under control, partly because the tools we use to detect and treat the disease are not sufficiently responsive to the situation. Hence there is a substantial need for innovation in the respiratory field”, says Dr Jan De Backer, CEO of Fluidda. De Backer developed Functional Respiratory Imaging (FRI) in collaboration with his father, Dr Wilfried De Backer, Emeritus Professor of Respiratory Medicine at the University of Antwerp. FRI shows the anatomy of the airways and blood vessels in the lungs and the flow of air through them at millimetre-level resolution.

QUANTITATIVE TECHNIQUE A radiologist will examine a CT scan of the lungs purely by eye and then describe it, with the result that only larger abnormalities and abnormal patterns are detected. ‘It is very difficult even for a practised eye to see small abnormalities or create a three-dimen-sional reconstruction of the lungs in the mind’s eye. With our technique we can do that’, continues Jan De Backer. ‘FRI is a quantitative technique that produces a 3D map of all the clinically relevant structures in the lungs, such as the alveoli, blood vessels and lung volume,

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using images from a standard CT scanner‘, says Jan De Backer. FRI even shows small abnormalities. The technique developed by the De Backers is able to show diffuse disease-related changes at very high resolution and monitor the changes over time. FRI thus enhances a radiologist‘s performance, and outperforms spirometry, which at present is still the gold standard for analysing lung function. FRI analysis starts with two CT scans, the first after deep inspiration and the second after normal expiration. The patient uses a mouthpiece that records his or her breathing to enable the scans to be timed correctly. After that, the work of the hospital and the patient is done. The two scans are sent to Fluidda in the Belgian village of Kontich, only walking distance from the university hospital in Antwerp. Jan De Backer: ‘Our systems are based on machine learning and artificial intelligence. Every CT scan that we analyse improves our algorithms. We analyse and model the images using computational fluid dynamics, a flow simulation system that has its origins in the aerospace industry. That yields a three-dimensional image of the lungs, showing the flow of air and revealing areas of increased air resistance or places where inhaled particles have been deposited.

ONE MILLIMETRE Jan De Backer gives an example of just how sensitive the technique is. ‘Pulmonary hypertension can be due to constriction of the smallest blood vessels in the lungs. This causes the blood to accumulate in the feeder blood vessels, which expand slightly. We can see the expansion even in blood vessels with a diameter of only one millimetre. That makes FRI a very patient-friendly option for diagnosing and monitoring pulmonary hypertension, which at present is done using invasive pressure measurement in the pulmonary artery.’ Using FRI, scientists have discovered that thrombosis in small blood vessels plays a major role in severe COVID-19. The finding made using FRI has now been confirmed by autopsies (see the interview with Muhunthan Thillai). Like the field of Computational Fluid Dynamics (CFD), Jan de Backer also comes from a background in aerospace. De Backer first encountered CFD while studying aerospace engineering at Delft University of Technology in the Netherlands. It was his father, Wilfried De Backer, who then hit on the idea of using the technique to model the flow of air through the lungs. The big challenge for the Fluidda researchers was to adapt the computer technology to use lung

Several completed and current phase 1-4 clinical trials of drugs for pulmonary disease owe their success (or potential future success) to the Fluidda technique. data – after all, a lung is rather different from the smooth surface of an aircraft. Jan De Backer: ‘A lung – especially a diseased lung – has an irregular structure. You need to set the right boundary conditions to map flow in the lungs using computational fluid dynamics. We managed to obtain those boundary conditions directly from the CT scans, so that each measurement is individual.’ Wilfried De Backer adds: ‘FRI yields an accurate, personal representation of the anatomy of the airways and blood vessels in the lungs and the flow of air through them, so it is not based on a general model.’

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BRONCHOLAB

FRI yields an accurate, personal representation of the anatomy of the lungs and the flow of air and blood in them, so it is not based on a general model. GOLD STANDARD The motivation for developing FRI was EUROSCOPE (European Respiratory Society Study on Chronic Obstructive Pulmonary Disease), a study conducted in the 1990s to explore the effects of inhaled corticosteroids on COPD. Wilfried De Backer: ‘In order to reach a worthwhile conclusion, we had to monitor 1,200 patients over three years. Although the study produced significant results, it gave rise to lengthy discussions among respiratory physicians on the value of inhaled corticosteroids for treating COPD. The EUROSCOPE study set me thinking about an alternative to spirometry. My son Jan then told me about computational fluid dynamics, and I thought we could obtain the requisite information from CT scans. We started developing FRI in 2005, and thanks to huge progress in computer technology and artificial intelligence we’ve managed to develop a technique that works well.’ At present, GPs and respiratory physicians diagnose lung diseases based on symptoms, blood tests, and usually spirometry. Jan De Backer: ‘The treatment for most pulmonary diseases is the same for a substantial majority of patients, who are given medication via an inhaler, for instance. Spirometry is then used to check whether their lung function has improved. Spirometry is the gold standard, but it only gives a general idea of lung function, whereas FRI gives a detailed, personalised picture.’ Pulmonologist Wilfried De Backer adds: ‘Take people with end-stage COPD, for example: they all have an FEV1 of 25 per cent, but they‘re not all the same. If you treat a patient based on that one FEV1 value, the treatment is not really targeted, as you have no information on the pathophysiology in the lungs. FRI provides pathophysiological information on such things as blood vessels, pulmonary hypertension and the sites of resistance in the distal alveoli.’

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The FDA cleared Fluidda's device Broncholab, which is a diagnostic support tool. Jan De Backer: ‘We expect to receive European approval in the first quarter of 2021. It is not Fluidda’s aim to test all patients using FRI immediately. Most patients respond well to the treatment they receive from their GP or respiratory physician, and for them spirometry is adequate to analyse their lung function. We are focusing initially on patients whose treatment options have currently been exhausted: the question is whether the respiratory physician can find out, using FRI, whether there are reasons for the failure of their treatment. FRI also lends itself to predicting clinical outcomes. In COVID-19 trials , for instance, we find that vascular problems in the lungs detected using FRI are predictive of a poorer clinical outcome. The physician can then opt for more aggressive treatment, based on the FRI information.’ Wilfried De Backer goes on: ‘With FRI we can see sooner whether treatment goals are being met. At present it can take a year for treatment using biologics to produce an improvement in the FEV1 in patients with pulmonary disease. With FRI we can see after only one or two months whether there are improvements in geometry and/or function that spirometry does not detect. This has the advantage that we can review expensive treatments quickly and discontinue them if necessary, thus avoiding unnecessary adverse effects on patients and saving on the high cost of biologics.’

COST SAVINGS As for the cost savings from FRI, Jan De Backer says: ‘Cardiac catheterisation to diagnose pulmonary hypertension costs between five and twenty thousand dollars in the USA. An FRI scan costs only a few hundred dollars. COPD exacerbations in the USA cost over ten billion dollars a year. We know that a substantial proportion of those people are struggling with a treatable but undiagnosed vascular problem in the lungs. With FRI it can be diagnosed. If the treatment prevents 10% of exacerbations, that already yields a billion dollars in savings.’ FRI is proving its value not only in patient care but also in pharma-ceutical research. Wilfried De Backer’s personal experience again comes into play: ‘As a member of the European Medicines Agency, I noticed that a lot of products were failing to be approved because there was insufficient evidence of their efficacy based on spirometry, while there was indirect evident that they worked. There

AUTHOR BIO Wilfried De Backer has built his academic career at the University of Antwerp where he finished his affiliation as full professor of Respiratory Medicine in 2017. He obtained his PhD in 1988 with a thesis on the Chemical Regulation of Breathing in men. Starting from this basis, he mainly studied the pathogenesis of sleep related breathing disorders. The focus of his research has always been on pathophysiology of respiratory disease. In the same spirit he encouraged the development of a new functional respiratory imaging technique FRI, that helps to understand the functional behaviour of the upper and lower airways. Pheno-typing of patients with FRI belongs to his ongoing clinical research topics.

was huge frustration about the fact that we were missing a lot of innovations because there were no good quantitative endpoints for clinical trials. There was an intensive search going on for markers of that kind, for example biomarkers in the blood, but those markers suffer from huge variability. If you want to reach a clinically relevant conclusion about a particular patient, such variable markers will not do. FRI solves the problem, as we show the individual patient‘s anatomy as it really is. We have never seen any significant variability unrelated to the disease or the effect of medication in our studies. So FRI provides stable parameters for clinical trials.’

CLINICAL RESEARCH A recent example of the success of FRI in clinical research is from the Dutch respiratory physician Maarten van den Berge (see the interview with him). Wilfried De Backer: ‘The contrast between Van den Berge‘s study and EUROSCOPE could not be greater. Whereas EUROSCOPE needed three years and 1,200 patients to show a significant difference, Van den Berge achieved that in one month with just 23 patients.’ Jan De Backer: ‘The study looked at the difference between LABA/LAMA and LABA/LAMA plus inhaled corticosteroids for people with COPD. Thanks to FRI, Dr Van den Berge was able to show convincingly that adding inhaled corticosteroids works significantly better than treating them solely with LABA/LAMA. He also showed where in the lungs the drugs act and what differences there are between patients in which the treatment does and does not work well. Spirometry did not yield any significant results in that study, given the small number of patients involved.’

FRI was behind the development of smallparticle inhalers that send the particles deeper into the lungs. Jan De Backer: ‘Starting in 2005, we carried out ten to fifteen studies resulting in the approval of drugs using those small particles.’ Several completed and current Phase 1-4 clinical trials of drugs for pulmonary disease thus owe their success (or potential future success) to the Fluidda technique. Researchers are increasingly embracing Functional Respiratory Imaging for clinical trials into new drugs for pulmonary disease. Broncholab is currently being used as a routine diagnostic support of patients with pulmonary diseases in American hospitals, and Europe will probably follow in 2021. As soon as doctors start making widespread use of FRI, the days of non-specific diagnosis using spirometry will be over. FRI is able to map each patient‘s disease at millimetre-level resolution and monitor the effects of treatment, thus making the prospect of a new scientific and diagnostic gold standard a real possibility. ‘It’s all coming together now’, concludes Jan De Backer. In recent years the radiation exposure from CT scans has decreased substantially, treatments have become more and more expensive and the possibilities of data analysis have improved to unprece-dented levels. Furthermore, a pandemic is showing the world that we need innovations in pulmonary medicine. The introduction of FRI as a diagnostic technique has come at just the right time.

Jan De Backer graduated from Delft University of Technology, The Netherlands as aerospace engineer. He obtained an MSc degree in aerodynamics and specialised in applied biomedical computational fluid dynamics leading to a PhD from the University of Antwerp, Belgium. He is an alumnus of the MBA programs at London Business School, London and Columbia Business School, New York. De Backer has received several awards for his innovative research in the field of airway modeling in respiratory and sleep medicine. His work has been published in international journals. De Backer founded FLUIDDA in 2005 and he has held the position of Chief Executive Officer since 2007.

AUTHOR BIO

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INFORMATION TECHNOLOGY

DATA INTEGRITY In pharma’s rush to end the pandemic The industry has worked through 2020 to quickly develop a COVID-19 vaccine under pressure. Soon, regulatory activity will shift back to its standard focus, and pharma companies may realise that data integrity standards may have slipped. This article will discuss the importance of data integrity and best practices to follow. Ankush Lamba, Managing Director, Technology segment, FTI Consulting

oday, we are in the midst of one of the most challenging periods of modern times. The coronavirus pandemic has engulfed the world, with more than 47.5 million infected and 1.2 million lost livesand those numbers are rising every day. The life sciences and pharmaceutical industry has been at the centre of

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this storm, working around the clock to evaluate, create, trial and bring to market a wide range of drugs as plausible lines of prevention and treatment for COVID-19. However, these efforts are haunted by a shortage of resources, restrictions on importing API, social distancing at facilities, disturbed supply chains, and

tremendous pressure to quickly manufacture and distribute products. Despite these arduous circumstances, it remains critical for pharma companies to maintain quality and compliance and follow regulatory guidelines. Doing so requires pertinent measures to ensure adherence to Current Good Manufacturing Practice (CGMP) guidelines, and data integrity to meet the requirements of regulators including the U.S. Food and Drug Administration (FDA), the Medicines and Healthcare Products Regulatory Agency (MHRA), and others, in case of an inspection or inquiry. Regulatory Focus

With normal routines altered in a profound way, what is likely to catch the attention of regulators are the measures taken in regard to the health of the employees, safety protocols and hygiene processes at research and manufacturing facilities. In preparation for these, companies should conduct a rigorous review of policies and procedures to ensure adequate documentation and reporting. Another area of inspection regulators are likely to focus on is data integrity. Lapses in reliability and accuracy of data — which are essential for ensuring the ‘safety, efficacy and quality of drugs’ — will be closely scrutinised as part of regulatory efforts to protect public health. Electronic data of the analytical equipment and batch records of the products manufactured during this phase are prime examples of data regulators may examine during an inspection or inquiry. According to regulatory guidelines, data integrity is: “…the completeness, consistency, and accuracy of data, which should be attributable, legible, contemporaneously recorded, original or a true copy, and accurate.” (FDA) “…the degree to which data are complete, consistent, accurate, trustworthy and reliable,” and, “Risk-based system design and controls should enable the detection of errors, lapses and omissions of results and data during the data life cycle. Controls

INFORMATION TECHNOLOGY

As per the FDA Warning Letter of 2018, the regulations most frequently cited are: 21 CFR reference

Title of CFR Section

211.194

Laboratory Records, Review of All Data

211.188

Batch Production and Control Record

211.192

Production Record Review, Deviations and Investigations

211.68

Automatic, Mechanical and electronic Equipment

211.165(a and b)

Testing and Release for Distribution

Data integrity enforcement action

Poor data integrity practices in the manufacturing and testing of pharmaceuticals products have resulted in a steady rise in the number of enforcement actions, such as warning letters, import alerts and consent decrees. Criticality of data integrity is substantiated by a global report that states that roughly 50 per cent of all Center for Drug Evaluation and Research (CDER) inspection observations (form 483) issued between 2014 and 2018 cite data integrity violations. These violations are even more prevalent in warning letters, with 79 per cent of global drug warning letters during this period citing data integrity issues. Further, the underlying similarity in the recent warning letters issued by the FDA is to engage a consultant to audit a company’s operations and assist in meeting FDA requirements for the data integrity remediation activity. Examples from warning letters include:

“Your quality system does not adequately ensure the accuracy and integrity of data to support the safety, effectiveness, and quality of the drugs you manufacture… We strongly recommend that you retain a qualified consultant to assist in your remediation. In response to this letter, provide the following…A comprehensive investigation into the extent of the inaccuracies in data records and reporting.”

“We acknowledge that you are using a consultant to audit your operation and assist in meeting FDA requirements. We strongly recommend that you retain a qualified consultant to assist in your remediation…An assessment of the extent of data integrity deficiencies at your facility. Identify omissions, alterations, deletions, record destruction, non-contemporaneous record completion, and other deficiencies. Describe all parts of your facility’s operations in which you discovered data integrity lapses…A comprehensive retrospective evaluation of the nature of the manufacturing data integrity deficiencies. We recommend that a qualified third party with specific expertise in the area where potential breaches were identified should evaluate all data integrity lapses. ” Conclusion

With the increase in the issuance of warning letters and forms 483 over the years, pharmaceutical companies have become increasingly cognisant of the importance of data integrity to ensure the safety, efficacy and quality of their products. Under the pressures of the

AUTHOR BIO

may include procedural controls, organizational controls and functional controls.” (WHO)

COVID-19 pandemic in particular, company leadership should focus on taking proactive measures to manage data integrity risk and ensure compliance with the CGMP regulations before the next FDA inspection happens. These efforts may include review and assessment of procedures as per the guidance of CGMP for responding to COVID-19 infection in employees, to prevent or mitigate potential adverse effects on the safety and quality of drugs from an infected or potentially infected employee engaged in drug manufacturing. Companies should also utilise advanced digital tools and technologies that can proactively identify data integrity lapses in electronic data of certain equipment (e.g. Chromatography Data Systems (CDS), NON-CDS, LIMS, etc.) and highlight on a real-time basis, so management can enable the company to take necessary actions. Companies that have already received a form 483 and warning letter should consider implementing a remediation plan for the violations identified. This would involve a comprehensive investigation that includes identification of the causes of the lapse, ongoing monitoring and preventive steps for potential recurrence. Since data integrity is a critical component of the basis for maintaining quality and the confidence of regulators, pharmaceutical companies should seek the expertise of a third-party expert to audit company operations and assist in meeting FDA requirements. References are available at www.pharmafocusasia.com

Ankush Lamba is a Managing Director in FTI Consulting’s Technology segment in Mumbai. He brings more than 12 years of experience in fraud data analytics and digital forensics. He has worked on high profile data integrity investigations for pharma companies involving regulators including the USFDA.

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Research Insights The Evaluation of Drug Delivery Nanocarrier Development and Pharmacological Briefing for MetabolicAssociated Fatty Liver Disease (MAFLD): An Update Reem Abou Assi, Discipline of Pharmaceutical Technology, College of Pharmacy, Al-Kitab University Ibrahim M Abdulbaqi, Academic Staff Member, College of Pharmacy, Al-Kitab University Chan Siok Yee*, Academic Staff Member, School of Pharmaceutical Sciences, USM *Author to whom correspondence should be addressed. Dedicated to the memory of my father and the physician in the field who has passed recently, Ahmad Abou Assi. Academic Editor: Dimitris Tsiourvas Pharmaceuticals 2021, 14(3), 215; https://doi.org/10.3390/ph14030215 Received: 22 December 2020 / Revised: 22 January 2021 / Accepted: 27 January 2021 / Published: 4 March 2021 (This article belongs to the Section Pharmaceutical Technology) Keywords: non-alcoholic fatty liver disease (NAFLD); metabolic fatty liver disease (MAFLD); insulin resistance; obesity; nanoformulations; nanotechnology; nanocarrier; nanosystem

Abstract: Current research indicates that the next silent epidemic will be linked to chronic liver diseases, specifically non-alcoholic fatty liver disease (NAFLD), which was renamed as metabolic-associated fatty liver disease (MAFLD) in 2020. Globally, MAFLD mortality is on the rise. The etiology of MAFLD is multifactorial and still incompletely understood, but includes the accumulation of intrahepatic lipids, alterations in energy metabolism, insulin resistance, and inflammatory processes. The available MAFLD treatment, therefore, relies on improving the patient’s lifestyle and multidisciplinary pharmacotherapeutic options, whereas the option of surgery is useless without managing the comorbidities of the MAFLD. Nanotechnology is an emerging approach addressing MAFLD, where nanoformulations are suggested to improve the safety and physicochemical properties of conventional drugs/ herbal medicines, physical, chemical, and physiological stability, and liver-targeting properties. A wide variety of liver nanosystems were constructed and delivered to the liver, only those that addressed the MAFLD were discussed in this review in terms of the nanocarrier classes, particle size, shape, zeta potential and offered dissolution rate(s), the suitable preparation method(s), excipients (with synergistic effects), and the suitable drug/compound for loading. The advantages and challenges of each nanocarrier and the focus on potential promising perspectives in the production of MAFLD nanomedicine were also highlighted.

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1. From NAFLD to MAFLD—What Is Going on?

Non-alcoholic fatty liver disease (NAFLD) is defined by the presence of excessive fat in the liver, which is identified either by imaging or liver biopsy. It is the term used to describe various histologic anomalies, ranging from benign steatosis to non-alcoholic steatohepatitis, in people who consume little to no alcohol. NAFLD has a substantial potential to progress to cirrhosis, hepatocellular carcinoma, end-stage liver disease, liver-related death, and recurrence after transplantation. In 2013, scientists released an alarm about the future of liver diseases in which NAFLD is turning to become an epidemic. Given the drastic and growing prevalence of NAFLDs, which affect more than one-quarter of the world’s population, the severe hepatic and extra-hepatic sequelae, and the inadequacy of treatment options, thus, re-naming and re-defining the disease are welcome. The term “fatty liver” was first described in 1836, while the name “NAFLD” was first used in 1980 and for 40 years until 2020 when it was re-evaluated for a more precise nomenclature indicating the existence of metabolic dysfunction, rather than the absence of other conditions, such as alcohol intake, as a safe limit of alcohol consumption could not be set. As a result, the well-known NAFLD nomenclature was replaced by metabolic-associated fatty liver disease (MAFLD), to open the door to the development and implementation of a set of “positive” criteria for defining the condition rather than depending on a “non” or “negative” definition.

MAFLD is expected to overburden the annual economy dependent on Medicare recipients with an increase of USD 188 billion in corporate costs alone. Approximately the same costs are split into four European countries, including Germany, France, Italy, and the United Kingdom, with the highest costs for patients aged between 45 and 65, with no apparent gender preference as some research is suggesting that MAFLD is more common in females while others were also highlighting that it is more common in males , this conflict could be due to the limited data about MAFLD in females rather than males, as will be noticed in the illustrated in vivo models later in this article. Interestingly, MAFLD impacts children’s health in an asymptomatic profile that is incidentally diagnosed at a mean age of 11–13 years. Overall, MAFLD mortality is escalating with an estimated rise between 65% and 100% from 2019 to 2030 in the Asia-Pacific region alone, as well as the US, China, and France, which is combined with an alarming MAFLD predicted prevalence escalation to occur in Germany, Italy and the UK by 2030. Clinically, MAFLD is considered to be the hepatic manifestation of multiple metabolic syndromes, which is present when three or more of the following conditions are met, including obesity, elevated glucose, blood pressure, triglyceride, as well as high-density lipoprotein cholesterol. To recapitulate MAFLD pathogenesis’s complexity, “multiple hit theory” was recently established, where multiple synergistically acting factors are involved in the disease incidence and progression. These factors include genetic predisposition, dietary factors, altered gut microbiota and insulin-resistance-induced alteration in production and secretion of adipokines, mitochondrial dysfunction, and endoplasmic reticulum stress. These factors contribute to the development of steatosis and steatohepatitis and reflect different disease patterns among MAFLD patients.

2. Available MAFLD Treatments and Their Efficacy

MAFLD’s etiology is multifactorial and still incompletely understood, but involves accumulation of intrahepatic lipids, alterations of energy metabolism, insulin resistance, and inflammatory processes. Consequently, the available MAFLD therapy relies on changing the patient’s lifestyle and multidisciplinary pharmacotherapeutic options. 2.1. Non-Pharmacological Approach In the absence of approved single pharmacological therapies for MAFLD, the current European Association for the Study of the Liver, European Association for the Study of Diabetes, and European Association for the Study of Obesity Clinical Practice Guidelines for the management of MAFLD recommend lifestyle modification as the strategy of choice for prevention and improvement. Diet calorie reduction is the first therapeutic technique in MAFLD; the diet should minimize both saturated fat intake to <7% of the total calories and trans-fat intake, and maintain dietary cholesterol intake at <200 mg/day, and total fat at 25%

to 35% of the total calories in order to achieve moderate weight loss (7 to 10%). In addition, increased exercise, such as 150 to 200 min/week of moderate-intensity aerobic physical activity is reported to enhance liver histology, insulin resistance, and life quality. Therefore, this should form the basis of any treatment strategy. This approach may be challenging to maintain over the long-term, even with the implementation of cognitive-behavioral therapy; thus, the pharmacological intervention will be the other choice in such cases. 2.2. Pharmacological Approach 2.2.1. Anti-Obesity Drugs The addition of pharmacotherapy resulted in more significant weight loss and weight loss maintenance than lifestyle modifications alone. Currently, there are nine pharmacological interventions approved by the US Food and Drug Administration (USFDA) that can be used for weight loss, including phentermine, diethylpropion, benzphetamine, phendimetrazine, orlistat, phentermine/topiramate extended release, lorcaserin, bupropion/naltrexone, and liraglutide. In MAFLD, anti-obesity medications did not directly affect the liver independent of the effect on weight loss. For now, orlistat and sibutramine are mainly the available options for the long-term prescription. However, there are still insufficient safety data regarding the long-term outcomes of anti-obesity therapy. The choice to initiate obesity drug therapy should be controlled, first by avoiding contraindications and drug–drug interactions, and second, by choosing a therapy with a mechanism of action that targets the patient’s dietary behavior; close monitoring is vital for assessing efficacy, tolerability, and switching to an alternative option if needed. 2.2.2. Hypoglycemic Agents Since MAFLD and type 2 diabetes mellitus share pathophysiological characteristics, such as insulin resistance, hypoglycemic agents have used mainly metformin and thiazolidinedione. In the case of metformin administration (0.5 to 3 g/day), a meta-analysis reported its use to be essential for improving liver function and body composition in non-diabetic MAFLD patients. However, some increase in insulin sensitivity disappears after three months of metformin use in MAFLD patients, with no assessed direct impact of metformin on the MAFLD activity score; side effects include gastrointestinal tract (GIT) discomfort and general weakness with records of interactions when used with various medications. Although the use of thiazolidinediones in MAFLD is reported at a much lower dose (4 to 45 mg/day) compared to metformin and is a promising choice in the treatment of MAFLD manifestations due to its beneficial impact on insulin resistance and hepatocyte fatty acid metabolism, the side effects (lower extremity edema, and weight gain) and the need for long-term use establish a disadvantage. Scientists recommend further investigations on the use of recent

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hypoglycemic agents, including glucagon-like peptide 1 receptor agonists, dipeptidyl peptidase 4 inhibitors, and sodium/glucose co-transporter 2 inhibitors and their impact on MAFLD—in particular, the oral versus parenteral route of administration profile in humans. 2.2.3. Lipid-Lowering Agents Statins, fibrates, and omega-3 polyunsaturated fatty acids are commonly used to manage dyslipidemia. Due to their potential antioxidant properties and favorable effect on adiponectin levels, they are suggested to benefit MAFLD patients. Statins function as anti-MAFLD by controlling metabolic risk factors with dose-related hepatotoxicity; higher lipophilic statins show a greater hepatic excretion rate, while lower lipophilic statins exhibit more pronounced active renal excretion. Food intake affects the bioavailability of statins, as most of them have low bioavailability, except for Pitavastatin (>80%), while several meta-analyses of major statins have consistently recorded modestly increased risk of incident diabetes (9–13%) in MAFLD patients. In addition, as per a pilot study, other options such as fibrates are considered safe and improve metabolic syndrome, glucose, and liver tests; although their effects on liver histology are minimal, fibrates are suggested to be useful for the prevention and management of MAFLD. Other lipidlowering agents, such as ezetimibe, are reported to attenuate hepatic steatosis and may benefit MAFLD biochemical markers (i.e., fatty acids). The majority of the results in such regard are from animal studies, which not are always compatible with human physiology, as authors point out. Indeed, the study on humans suggested the possibility of the animal results heterogenicity, where later ezetimibe was reported to improved hepatic fibrosis in 80 patients’ results, but increased hepatic long-chain fatty acids. 2.2.4. Cytoprotective and Antioxidant Agents Inflammation and oxidative stress are believed to play a role in the pathogenesis of MAFLD, where anti-inflammatory and potent antioxidants own a crucial role in anti-MAFLD therapy, such as bile acids (ursodeoxycholic acid). In such regard, phytochemicals and micronutrients are also used, including flavonoids, polyphenols, carotenoids, and phenolic compounds, as well as vitamin E, and silymarin. They act as attractive therapeutic agents for short-term treatment until the full profile of safety and effectiveness is further explored with their impact specifically in diabetic and non-diabetic MAFLD cases. 2.3. Surgical Intervention With a causal link established between MAFLD and obesity-related metabolic syndrome, bariatric surgery interventions promote weight loss and would be expected to improve MAFLD; the surgery is a debated option for MAFLD treatment where, despite it having the potential for inducing excellent weight loss and possibly improving the symptoms of metabolic syndrome, type 2 diabetes mellites, and potentially reversing the pathological liver changes in MAFLD patients, it could also, reportedly, be a secondary outcome of the bariatric surgery, while other

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patients developed new or worsened features of MAFLD, including the development de novo, worsening of fibrosis, MAFLD activity score, MAFLD histology, and change in liver volume. Performing this costly surgery might add a few years to the patient’s life, but it does not reduce the overall healthcare costs in the long-term. On the other hand, liver transplantation cures cirrhosis but does not treat MAFLD’s underlying metabolic disease. Thus, strategies to control comorbidities in patients with MAFLD before transplant are needed to decrease waitlist mortality, and the recurrence of MAFLD after liver transplant.

3. Nanomedicine Alternatives for MAFLD Smart Drug Delivery

Other currently emerging approaches to MAFLD therapy include developing new targeting drugs that are already under phase 2 and phase 3 trials, or improving the safety and physicochemical properties of conventional drugs and herbal medicines, as well as physical, chemical, and physiological stability, along with liver-targeting properties using a wide range of nanoformulation techniques, as illustrated in Figure 1. The United States National Nanotechnology Initiative described nanotechnology as science, engineering, and nanoscale technology, approximately 1 to 100 nanometers (nm) [74], however, several formulations were reported under the nanosystem umbrella for the merit of providing a particle size scale below 1000 nm. Compared to the research size conducted on MAFLD alone, a minimal number of nanoformulations directly address this disease by evaluating the designed nanosystems on the in vitro, ex vivo and in vivo models. Searching the PubMed database alone showed around 122,877 articles covering “nano” topics, out of which only 31 were in the MAFLD field, which started to trend in 2018. Using the searching keyword Nano-MAFLD or Nano-NAFLD yielded the same article number, while searching NAFLD or MAFLD alone yielded 25,471 and 83 studies, respectively (October 2020), as illustrated in Figure 2.

Figure 1. The main challenges that nanoformulations are overcoming to enhance drug/phytochemical delivery.

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Figure 2. The published article number from the PubMed search (October 2020) vs. the years, using the keywords of A: NAFLD, B; Nano, C: MAFLD, D: NAFLD nano, E: MAFLD nano with a peak in research since 2018 (expressed within the orange line). NAFLD, non-alcoholic fatty liver disease; MAFLD, metabolic fatty liver disease.

4. Opportunities and Limitations of Nanosystems for MAFLD Therapy 4.1. Nanoparticles 4.1.1. Polymeric Nanoparticles Polymer nanoparticles (P-NPs) are the most widely used nanocarriers in the pharmaceutical sector to develop controlled, sustained and burst drug delivery systems with release profiles directly related to the constructed polymeric shell. P-NPs can be either nanospheres or nanocapsules, with particle size (PS) ranging from 1 to 1000 nm and two fundamental preparation techniques, including “top-down” and “bottom-up” methods. They can be used to mask the taste and to protect drug/compounds from environmental and gastric degradation, offering enhanced bioavailability, increased stability of any volatile materials with non-immunogenicity, and a low to nontoxicity nature. Challenges of this nanosystem design are merely summed up by the proper selection of the polymer. Within the P-NP design for MAFLD therapy, polymer use is mainly limited to chitosan, and poly lactic-glycolic acid (PLGA) (Figure 3), with promising results of chitosan alone as an excipient owning synergetic anti-MAFLD effects. Other polymers with anti-MAFLD therapy that are not used in P-NP formulation to address the disease include accase-catalyzed catechin polymers, pyrrole-based polymers, Hericium Erinaceus exo-polymers, beta-cyclodextrin, and colestipol. These polymers were reported to have a lowering influence of cholesterol, low-density lipoproteins (LDL)cholesterol fraction, hyperlipidemia, cholesterol and

lipid levels, respectively. Additionally, there is a need to investigate glucose-lowering polymer synergetic effects, such as cellulose, which positively impacts diabetes mellitus type 2, and inulin, which was reported to reduced serum insulin levels but simultaneously increased body weight in diabetic rats. Thus, preliminary studies are recommended when exploring glucose-polymers as excipients for nanoparticles, such as dextrans, icodextrin, and amylose, to ensure that there are no short-term or long-term consequences on MAFLD patients, including the blood glucose levels. Chitosan is a golden approach for MAFLD polymeric drug delivery, which is not fully explored yet. It is highly available as a natural cationic aminopolysaccharide polymer of marine origin, prepared from crustaceans’ shells, and classified as generally recognized as safe (GRAS) by USFDA; thus, it owns ideal biological properties. It also exhibits hepato-protective effect, regulatory impact of carbohydrates and lipid metabolism, ameliorating insulin resistance, and decreasing the prevalence of MAFLD, along with the decrease in aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels in the in vivo studies. In addition, it owns a lipid-lowering capacity, through promoting weight loss, lowering LDL, and boosting highdensity lipoprotein (HDL), with a suggested mechanism linked to the polymer’s positive charge, which attracts the negatively charged fatty acids and bile acids binding them to the indigestible chitosan fiber. Interestingly, this unique polymer can deliver genes and target the liver, which nominates it to be the polymer of choice for smart anti-MAFLD nanomedicine delivery. In MAFLD patients, a decline in nicotinamide adenine dinucleotide (NAD1) biosynthesis is observed; thus, a high dose of nicotinamide (NAD1 precursor) with a dose of the relatively unstable antioxidant, ascorbic acid, was usually administrated. Accordingly, chitosan nanoparticles were prepared by the ionotropic gelation technique to overcome the drawbacks of high dose intake and instability challenges. On the in vivo level (using male albino rats), these NPs showed an insulin-resistant status amelioration, and reduction in ALT, AST, as well as liver tissue total cholesterol, triglycerides, and 8-hydroxy-2-deoxyguanosine (8-OHdG) levels. The NPs also decreased oxidative and nitrosative stresses along with a significant increase in the hepatocellular energy upon the oral intake of only 10 and 20 mg/kg of loaded nicotinamide and ascorbic acid in NPs in comparison to their conventional forms oral dose of 100 mg/kg. Both chitosan nanoparticles were suggested to be cytocompatible, with encapsulation efficiency (EE%) of 75% and 85.5%, respectively, PS less than 200 nm and a positive zeta potential (ZP) (22.5 to 29.8 mV). Authors further suggest that the positive charge of these NPs, and the use of chitosan, would offer superior stability and a controlled-release manner, respectively, of the loaded materials. Poly lactic-glycolic acid (PLGA) is another polymer known to be a biodegradable, highly biocompatible polymer from

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Figure 3. Chemical structures of the most used polymers in engineering polymer-based nanoparticles for MAFLD therapy.

the family of USFDA-approved polymers, with the ability to deliver active ingredients, proteins, and macromolecules, and promote their transportation across biological barriers. Mostly it is used for the intravenous route of administration (IV) with an accelerating potential for oral drug delivery during the last decade. To the best of our knowledge, there is no study about the synergetic impact of PLGA alone on one or more of MAFLD manifestations. In a first-time report, PLGA-based curcumin NPs were evaluated clinically in a double-blind, randomized, placebocontrolled study on 84 overweight/obese MAFLD patients. Curcumin is considered a folk medicine in MAFLD therapy primarily because of its antioxidant and anti-inflammatory properties. Treating patients between the age of 25 and 50 with supplementation of curcumin PLGA-NPs at 40 mg capsules/day after meals for three months leads to improved glucose, lipids, inflammation, and nesfatin, hepatic transaminases, and fatty liver degree indices. These curcumin PLGA-NPs were initially patented turmeric-based colloidal dispersion technology named THERACURMIN, with a limited pharmaceutical characterization of PS 0.19 µm and stability of 28 days in comparison to 1 h for pure curcumin powder. The THERACURMIN was found to have the ability to raise the plasma concentration of curcumin by 39.8 to 81.7 times at 1–2 h after oral administration, compared with pure curcumin powder, yielding a higher bioavailability of curcumin by 40 folds. Further trials of curcumin loaded in other nanocarriers as anti-MAFLD prospective remedies are encouraged to provide a better clinical and pharmacological understanding through expanding the studied variables, mainly ethnicity, age, group number and the preparation recipe with full characterization and evaluation. Another phytomedicine, resveratrol, from the polyphenol group, has been identified as a potential new pharmacological approach in the treatment of MAFLD; however, its low solubility, could govern its effects that may be improved through encapsulating it with freezedried PLGA-NPs by the evaporation method. This has also improved its stability (up to 6 months under 4 °C) and bioactivity. The resulting resveratrol PLGA-NPs had PS of 176.1 nm with Đ of 0.152 and ZP of −22.6 mV, while the EE% and loading percentage were 97.25% and 14.9%, respectively. The NPs offered a sustained release profile of the resveratrol with a much lower release in the acidic medium than the alkaline one. Resveratrol PLGA-NPs was more effective in alleviating lipogenesis, promoting lipolysis, and reducing hepatocellular proliferation compared to the free resveratrol when studied on the fat-emulsion-treated

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human hepatic cells (HepG2), which were incubated with oleic acid for 24 h to induce steatosis and build a closer cell culture model of MAFLD. In this work, authors recommended further in vivo studies on such NPs to correlate in vitro and in vivo results. On the nano-molecular level, a novel polymer, lactosylated poly(2-(dimethylamino) ethyl methacrylate, was used to construct NPs and load it with “miR-146b mimic”, which plays a vital role in the regulation of metabolic gene expression and MAFLD therapy through its ability to modulate hepatic lipid homeostasis. The resultant NPs had PS, Đ, and a positive ZP of 150 to 350 nm, 1.21, and 10.3 mV, respectively. The loaded formulation was cytocompatible and efficiently capable of directly targeting the hepatocyte and has been taken up by HepG2 and mouse hepatocyte cell lines (AML12). In addition, these NPs showed the ability to significantly suppress the formation of lipid droplets on the in vivo level, offering a specific liver delivery, with a significant inhibition to the lipid accumulation when injected via female C57BL/6 mice tail vein in a single dose of 1 mg/kg. 4.1.2. Metal-Oxide-Based Nanoparticles The metallic NPs (M-NPs) consist of a metallic core with a standard PS of 10–200 nm. They can comprise pure metallic substances (e.g., gold, silver, copper, platinum, and palladium) or metallic oxides (e.g., zinc oxide, titanium dioxide, cerium oxide, iron oxide), while their comparatively small PS is responsible for their high surface area to material ratio, leading to disproportionately more considerable exposure to the body. Size and shape are the two major factors responsible for M-NPs’ properties robustness, any variation in them is significantly reflected in the M-NPs’ color, melting temperature, and electrical conductivity, even if the two NPs are made of the same pure metal. They are known for their strong plasma absorption offering controlled release pattern, in addition to their applications in biomedical sciences including biological system imaging and disease diagnosis, with the possibility of large-scale production. M-NPs can be synthesized in different shapes, including spheres, rods, and tubes; indeed, their instability, challenging synthesis with the presence of impurities, and toxicity are matters of controversy. In particular, M-NPs’ toxicity is of concern, as the liver is the first point of contact for NPs entering the circulatory system; an example of such toxicity histological observation is illustrated in Figure 4. In this regard, NPs that can escape pre-systemic elimination could eventually accumulate in the liver post-entry, leading to profound interactions with hepatic cells and other non-parenchymal cells. Given the fact that M-NPs are notoriously resistant to degradation, this could lead to long-term persistence and subsequent hepatic toxicity. In contrast, recent studies are reporting these NP benefits in healing the liver from critical situations, including the titanium dioxide (TiO2) and silicon dioxide (SiO2) NPs through inhibiting the cellular hepatic fibrosis, with no data on the possibilities of owning anti-MAFLD activities.

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Figure 4. Histopathology of the liver after consuming 100 µL gold nanoparticles by different groups of Swiss albino female mice via intraperitoneal injection at different periods and particle size (PS). (1) Control mouse liver section showing normal hepatic architecture, (2) marked steatosis, and the abundance of micro and macro vesicles after one-day consumption at 5 nm, (3) and (4) look healthy with normal hepatocytes after one-day consumption at 20 nm and 50 nm, respectively, (5) cytoplasmic degeneration and some aggregation of inflammatory cells after seven days consumption at 5 nm, (6) looks healthy with mild activation of Kupffer cells after seven days consumption at 20 nm, (7) multi-necrotic foci filled with hemorrhage and also the presence of infiltrative cells after seven days consumption at 50 nm, (8) necrotic foci filled with edema and surrounded by inflammatory cells after one and seven days consumption at 5 nm, (9) and (10) look healthy with bi-nucleated cells and the activation of Kupffer cells after one and seven days consumption at 20 nm and 50 nm, respectively. Adapted from reference.

Interestingly, the spherical cerium dioxide (CeO2) NPs with PS of 4–20 nm paves the way in anti-MAFLD therapy through enabling the reduction in hepatocyte lipid droplets size and content, the hepatic concentration of triglyceride, and cholesterol ester-derived fatty acids, as well as the expression of several genes involved in cytokine, adipokine, and chemokine signaling pathways. These nanoparticles’ original formulator suggested their hepatoprotective and therapeutic value in chronic liver disease. Later, they were reported to be up-taken by HepG2 cells line that were intentionally treated with oleic and palmitic acids to induce the hepatosteatotic condition. These NPs have also reduced oxidative stress, improved cell viability, and reduced fatty acid content in steatotic conditions by inducing specific changes in fatty acid metabolism, suggesting their potential for treating MAFLD. 4.1.3. Nanographene Oxide Particles Graphene was first reported for drug delivery in 2008, with an earlier established employment in the fields of photonics and electronics, while in nanomedicine it is considered the future boon of biotechnological kits and drug delivery for theranostics, high throughput biosensors and bioassay, smart scaffolds for tissue regeneration, and ultra-high sensitive biomarkers. These various applications could be due to their wide PS range in both nano- and

microscales. Nanographenes (NGs) offer a controlled and slow-release profile, while their synthesis using the “bottom-up” method is considered more appropriate than the “top-down” one in terms of the produced uniformity. New synthesis methods that can avoid NGs’ scalability and introduce industrial-level amounts are current challenges. This inexpensive carbon nanomaterial can exist with a positive and negative charge, which is mainly used to remove heavy metals. Graphene-based materials include pristine graphene, graphene oxide, reduced graphene oxide, graphene quantum dots, graphene nanoribbons, graphene nanoplatelets, and three-dimensional graphene foam. NGs can offer high electrical conductivity and surface-to-volume ratio resulting in significant altitude of the drug/compoundloading capacity and high mammalian cell internalization, particularly by the nanographene oxide particles (Figure 5) that are up-taken into the cell cytoplasm. They are also known for their aqueous, colloidal stability, and the delivery of insoluble drugs/compounds . Challenges such as NGs agglomeration in solution, due to van der Waals interactions, might impact the loaded drug/compound pharmacological behavior and limit cell specificity (in case of chemotherapy or radiotherapy and cancer cells); in addition, unexplored toxicity issues must be of further research concerns. In MAFLD, NGs’ synthesis was very limited to developing sensors as essential indicators for clinical diagnosis, and prognosis judgment, including environmentfriendly immunosensor of leptin using porous graphene-

Figure 5. Scanning electron microscopy images of (a) 5.34% silver nanoparticleembedded graphene oxide (Ag/rGO), (b) 7.49% Ag/rGO, (c) 6.85% zinc oxide nanoparticle-embedded graphene oxide (ZnO/rGO), (d) 16.45% ZnO/rGO, (e) 3.47% silver nanoparticle, and 34.91% zinc oxide nanoparticle-embedded graphene oxide (Ag/ZnO/rGO), and (f) 7.08% silver nanoparticle and 15.28% zinc oxide nanoparticleembedded graphene oxide (Ag/ZnO/rGO). Adapted from reference.

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Figure 6. Structure of conventional liposome encapsulating hydrophilic and hydrophobic drugs. Adapted from the reference.

functionalized black phosphorus and gold nanoparticles successfully. The sensitive and selective biosensor was also reported for the fibroblast growth factor 21 as an important MAFLD biomarker, while there was no record on anti-MAFLD NGs as drug delivery systems or the synergetic impact of NGs alone on one or more MAFLD manifestation. 4.2. Lipid-Based Formulations 4.2.1. Liposomes This nanocarrier is one of the first nanoformulations described in the early 1960s, with PS range of 0.025 μm to 2.5 μm. Liposomes are non-toxic, flexible, biocompatible, completely biodegradable, and non-immunogenic nanocarriers that are suitable for systemic and non-systemic administrations. They offer significant advantages for hydrophilic and hydrophobic drug/compound delivery, including increment in efficacy stability, reduced toxicity, side effects, and targeting potentials. However, they are generally offering a short half-life due to oxidation and hydrolysis (particularly the negatively charged liposomes), with possible drug/compound leakage, and high cost of production. Parameters such as liposomes PS and bilayers’ number are critical as they control the circulation half-life and drug encapsulation percentage, respectively. Thus, considerations are mainly given to its lipid composition, bilayer components, surface charge, size, and preparation method, which will significantly contribute to producing liposomes that can meet the set selection criteria. The structural evolution of liposomes is illustrated in Figure 6. Liposomes are prepared by passive or active loading techniques that include various mechanical dispersion methods. In liver diseases, liposomes are promising nanocarriers for liver targeting as anticancer agent carriers; this could be due to the fact that the liver exhibits the largest capacity for liposomal uptake, followed by the spleen among the other organs in the reticuloendothelial system. In MAFLD therapeutics, liposome characteristics were mainly employed to enhance anti-MAFLD phytoand conventional medicine properties. For instance, fenofibrate, an anti-dyslipidemia drug with prophylactic and/or inhibitory activity against inflammation, oxidation, and apoptosis, was loaded in liposomes. Fenofibrate is

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bio-pharmaceutically classified as a Class II drug, which means that, despite its high permeability, the drug’s poor solubility restricts its clinical outcomes. Fenofibrate liposomes were prepared using a dry-film dispersing method with PS, Đ, and ZP of 122.1 nm, 0.293, and −2.92 mV, respectively. The EE% and drug-loading percentage were 96.6% and 7.44%, respectively, while the transmission electron microscope (TEM) images confirmed the spherical shape of the developed liposomes and the reported PS analysis by dynamic light scattering. The resulting liposomes offered a sustained in vitro release profile of fenofibrate, with a raised peak plasma concentration of 34.9-fold in comparison to the pure fenofibrate. Pharmacologically, fenofibrate liposomes (20mg/kg/day) reduced hepatic lipid accumulation significantly almost to the level of the control group of male C57BL/6 wild-type mice upon oral administration. Interestingly, unlike the pure fenofibrate, fenofibrate liposomes were able to remarkably reduce hepatic triglyceride content by 62.4% due to the increased oral absorption, which might offer a prophylactic effect against MAFLD. Likewise, the flavonoid naringenin, which owns suggested anti-MAFLD activity due to its potent antiinflammatory properties, was also successfully loaded (25 mg) in liposome by thin-film rehydration method, producing a liposome with PS 98 nm, EE%, and drugloading percentage of 96.66% and 8.43%, respectively, with most of the naringenin encapsulated in the formulation’s lipid bilayer. The sustained release of the naringenin-loaded liposomes was significantly higher (81.74%) than the crude naringenin (24.35%), and the pharmacokinetic results on male C57BL/6J mice reveal the increase in maximum serum concentration (Cmax) by 6.7-fold. This naringenin liposome at 25 mg reduced the ALT, AST levels, and liver lipid accumulation in MAFLD induced by methionine cholinedeficient diet compared to 100 mg of crude naringenin for the same reduction impact. 4.2.2. Hybrid Lipid–Polymer Nanoparticles Hybridization is defined as a material involving two or more types of chemical bonds formed by “hybridization” of two or more monolithic materials. The principle of hybridbased NPs is emerging with some advanced nanosystems that were first reported in 2012 and 2014. The hybrid lipid– polymer nanoparticles (HLPNPs) are a novel generation core-shell nanostructure, notionally derived from both polymeric NPs and liposomes, where a polymer core remains enveloped by a lipid layer, as illustrated in Figure 7. This two-in-one nanosystem exists in both liquid or solid status, offering a drug control release profile, and owning a spherical shape and an outer surface that can be decorated in multifarious ways for active targeting of different approaches, including the smart delivery of DNA and RNA. HLPNPs have two main methods of preparation, the Two-Step method, which usually yields PS between 200 nm and 400 nm and involves the preparation of polymeric NPs and lipid vesicles separately, and the One-Step method, where the separate preparation of the polymeric NPs

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Figure 7. The structure of hybrid lipid–polymer nanoparticles. Adapted from the reference.

and lipid vesicles is not a prerequisite. Other methods of preparation are still evolving, such as the use of bath sonication approach with the One-Step method (PS < 100 nm), or the micro-vortex platform (PS ~30 nm to 170 nm), in addition to the nanoprecipitation technique (PS ~60 to 190 nm). Thus, the selection of the right technique influences various parameters, such as size, dispersity, and shape. The involved polymers in this NP synthesis are either previously reported in other nanosystems including Polystyrene, Maltodextrin, PLGA, Poly(ethylene glycol) monomethyl ether-Polylactic acid (mPEG-PLA), or Polylactic acid (PLA) alone, or a specifically functionalized polymer such as PFBT (poly[(9,9-dioctylfluorenyl-2,7-diyl)-alt-co-(1,4-benzo-(2,1′,3)thiadiazole)]), and poly-beta amino esters (PBAEs). Factors such as the core-shell nature, the polymer–oil ratio and the compatibility between encapsulated drug and dispersed oil are critically influencing the hydrodynamic characteristics and drug entrapment capacity of the hybrid matrix. HLPNP accumulation in the liver is still controversial, for instance, solutol-HLPNPs showed low drug accumulation, in contrast, vitamin E or D-ɑ-tocopheryl polyethylene glycol succinate (TPGS) HLPNPs demonstrated high drug accumulation, which may be attributed to variations in solubility between the loaded materials and/or the existence of an inverse correlation between increased blood retention of HLPNPs and their accumulation in the liver. In MAFLD nanomedicine discovery, recent research studied the value of developing silymarin-HLPNPs for better hepatoprotective efficacies upon enhancing silymarin low oral bioavailability (0.73%), poor aqueous solubility, and low membrane permeability. Silymarin was loaded into a previously developed HLPNP with PLGA alone, and compared with the modified silymarin-HLPNPs with PLGA and chitosan using a modified nanoprecipitation technique. It was noticed that the loaded HLPNPs with PLGA increased PS and Đ from 125.8 nm to 286.5 nm, and 0.142 to 0.226, respectively, upon hybridization with chitosan, while the ZP charge changed from negative (−43.1 mV) to positive (45.3 mV) due to chitosan presence. The

X-ray diffraction confirmed silymarin dispersion within the developed nanoformulations. As for the EE%, there were no significant changes upon hybridization (from 97.39 to 97.05%). The hybridization with chitosan had enhanced silymarin cellular uptake by human epithelial colorectal adenocarcinoma cells (Caco-2) and HepG2 cells and boosted its lipid-lowering effect and the triglyceride content in HepG2 cells in comparison to PLGA hybridization alone. Both formulations offered a burst release drug delivery, and the relative bioavailability study on healthy male Wistar rats with an oral dose of 20 mg/kg showed that silymarinHLPNP with PLGA and chitosan offered a bioavailability of 1.23-fold and 14.38-fold higher than that of silymarinHLPNP with PLGA alone, and pure silymarin suspension, respectively. The hepatoprotective and antihyperlipidemic effects of silymarin-HLPNP with PLGA and chitosan in MAFLD conditions were suggested through reducing the AST and ALT serum levels significantly in PNPLA3 I148M transgenic male and female mice with notably less macrovesicular steatosis compared to the group treated with pure silymarin suspension. 4.2.3. Solid Lipid Nanocarriers Solid lipid nanocarriers (SLNs) are colloidal drug delivery systems that were developed in the late 1980s. They are best described as a combination of liposomes and niosomes containing phospholipids and surfactant molecules, with a PS range from 40 to 1000 nm; they are derived from oil-in-water (O/W) emulsions by replacing liquid lipids with a lipid matrix that is solid at room and body temperatures. The lipid core typically consists of fatty acids (e.g., stearic acid), monoglycerides (e.g., glycerol monostearate), diglycerides (e.g., glycerol behenate), triglycerides (e.g., tripalmitin, tristearin, trilaurin), waxes (e.g., cetyl palmitate), or steroids (e.g., cholesterol), and is stabilized by appropriate surfactants. Similar to most of the lipid-based formulations, SLN successfully delivered phytomedicines, proteins, and peptides, as well as a wide

Figure 8. Classifications of (a) solid lipid nanoparticles (SLNs); (b) nanostructured lipid carriers (NLCs). Adapted from reference.

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variety of genes and drugs. SLNs are classified into three types I, II, and III. As illustrated in Figure 8, type I is a homogeneous matrix model where the drug is dispersed in the lipid core with controlled release properties, while in type II a drug-free lipid core is formed, and a solid exterior shell with both lipid and drug is formed. Unlike type II, type III is adequate in achieving a prolonged drug release. SLN has a wide range of advantages over the other nanosystems, precisely polymeric NPs and liposomes in terms of safety and owning the options of excluding organic solvents from excipients when desired, respectively. In addition, SLN offers excellent reproducibility and feasible large-scale production upon using the cost-effective high-pressure homogenization method, with a relative enhancement in loaded materials’ physicochemical stability compared to their pure form, along with biodegradable and cytocompatible natures. This nanosystem offers significant loading capacity with site-specific targeting capabilities and a controlled release profile. An initial study suggests that SLN parenteral administration can bypass the gastrointestinal route if the drug is pH sensitive, which may eventually result in high concentrations of drugs in the liver, but further investigations on SLN allocation and its benefit to liver and MAFLD is recommended. Main critical challenges should be taken into consideration when choosing the SLN preparation process, as each method produces SLNs with different morphological characteristics, and has its own challenges, including the possibility of drug expulsion from nano-vehicles and unsuitability to encapsulate hydrophilic materials, machine(s) unavailability in each laboratory, instability due to relatively high dispersion and metal contamination, the use of organic solvents, and limitation related to material solubility in carbon dioxide upon using hot homogenization technique, cold homogenization technique, ultrasonication or highspeed homogenization, solvent emulsification/evaporation method, and supercritical fluid extraction of emulsion method, respectively. In MAFLD therapy, SLN was reported in the enhancement of berberine solubility (very slightly soluble in water), which is a major component of Coptis chinensis. It is believed to have potential anti-MAFLD activities through multi mechanisms, including mediating insulin resistance, regulating the AMP-activated protein kinase pathway, and modifying the gut microenvironment. SLNs were prepared using the authors’ previously patented method that yielded SLNs with PS, Đ, and ZP of 76.8 nm, 0.402, and 7.87 mV, respectively. However, under TEM, while the particles were reported to have spherical shapes, with PS ranging from 50 to 150 nm, the XRD and NMR measurements ensured that berberine was dispersed and wrapped in the lipid carrier. Pharmacokinetically, and upon administrating berberine in pure and loaded SLNs forms at 50 mg/kg to different groups of male Sprague Dawley male rats, Cmax was increased by 3 folds, indicating that SLNs could minimize fluctuations in drug concentrations, promote absorption, and possess a slow-release character. Berberine-SLNs at 100 mg/kg dosage showed more potent hypoglycemic effects

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than the equivalent dose of berberine alone in db/db male mice, especially in improving glucose tolerance and insulin sensitivity. In addition, it has uniquely suppressed the gain of body weight and offered hepatoprotective effects by lowering the ALT serum level and hepatic triglyceride content. The authors urged for preclinical studies of berberine-SLNs with further chemical and physical characterization as well as safety assessments to ensure the availability of the berberine-SLNs in the market. 4.2.4. Self-Emulsifying Drug Delivery System and NanoStructured Lipid Carrier These two lipid-based formulations’ application impact on MAFLD therapy was also limited to enhancing drug/ compound physicochemical properties. This is due to their superior abilities in overcoming the leading factors behind insufficient oral bioavailability of lipophilic materials, including drugs efflux through P-glycoprotein and their first pass elimination due to metabolism through cytochrome P450. Self-emulsifying drug delivery systems (SEDDs) (Figure 9) have sparked profound interest from pharmaceutical researchers and industries through mainly antibiotic and antiviral SEDDs products in the market; this micro/ nanosystem offers droplet sizes of 5 µm to 200 nm and <100 nm as well. It can enhance the oral delivery of various therapeutic agents with different physicochemical properties, with either initial in vitro burst release, followed by a gradualrelease phase or a sustained release profile. SEDDs exist as liquid and solid formulations, where the solid form is suggested to provide better stability, reproducibility, and patient compliance, in addition to ease of process control, and various pharmaceutical dosage forms production (powders, capsules, tablets, or pellets) with the possibility of developing a controlled in vitro release attitude by mixing these dosage forms with suitable polymers or coating with polymeric films [193,195]. They are mainly composed of oil, surfactant, and cosurfactant phases mixed using the aqueous titration

Figure 9. The typical self-emulsifying drug delivery system (SEDD) structure after dispersion in aqueous phase. Adapted from reference [200].

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method, and the self-emulsification area is selected from the constructed pseudo-ternary diagram. The use of GRAS-type excipients with the least possible surfactant and cosurfactant (or co-solvent) ratios should be taken into consideration to assure that the final formulation is cytocompatible. As with other lipid-based formulations, the particle size relies on the excipients’ hydrophile–lipophile balance (HLB) value, where it is usually found that the higher the HLB value is, the lower the PS and Đ are, while the ZP charge is usually negative due to the presence of anionic surfactants and fatty acids unless specific lipids that grant the positive charge to the developed system are specifically employed, such as cationic surfactants, namely, tertiary amine surfactant, oleylamine, and the quaternary ammonium surfactants or polymers (e.g., Eudragit RS or RL). Incipient reports suggested that SEDDs could increase materials’ liver uptake, with further studies recommended on the cytocompatibility and mechanism behind such increment. On the same line, nanostructured lipid carrier (NLC) is the enhanced version of SLN, as it outweighs the drawbacks of SLN. This includes loading capability issues by conceiving a less organized solid lipid matrix via blending a fluid lipid with the solid lipid, which provides less drug expulsion during storage. NLC’s usual diameter ranges from 10 to 1000 nm, but most sizes between 50 and 300 nm are recommended for an easier crossing of barriers, increased uptake in cells and rapid action. Whereas NLCs usually own a controlled release profile, NLCs with size above 300 nm suggested providing a sustained drug delivery. The particle size of this formulation is affected by several factors, including compositions’ properties, manufacturing process, processing temperature, pressure and number of cycles during highpressure homogenization, sterilization and lyophilization. They are mainly prepared by three different methods: the high-energy (homogenizer, sonicator, or microwave), the low-energy (microemulsion and double emulsion), and the organic solvent-based preparation. They are primarily composed of lipids (solids and liquids at room temperature), surfactants, and co-surfactants, and other ingredients such as organic salts and ionic polymers. NLC offers significant chemical stability of the incorporated materials and effortless production possibilities on a large scale granting more affordable nanosystems than the polymeric NPs. The NLC classes are based on the structure of the formed crystals based on the lipid content, as explained in Figure 7. NLC type I offers an imperfect crystal model due to the presence of sufficient mixture amounts of liquid lipids and solid lipids, where the drug can place itself; NLC type II, known for its amorphous model, is linked to the use of special lipids that do not recrystallize after homogenization and cooling, which in turn will minimize drug expulsion. NLC type III, or the multiple models, shows a phase separation due to its composition of small oil nanoparticles that are inside the solid lipid matrix; this type is usually produced upon mixing solid lipids with higher amounts of oils in a ratio where the solubility of the oil in the solid lipid is exceeded. In addition, NLC is also able to load lipophilic and hydrophilic materials, with no need to use organic

solvents, offering control, and targeted drug release profile; however, as with most of the lipid-based formulations, inappropriate lipid/surfactant selection can lead to issues of stability and cytocompatibility. As reported in SLN, NLC parenteral administration is recommended in liver delivery, as it demonstrated excellent liver tumor targetability on the in vivo level. In the MAFLD drug discovery, silibinin’s oral bioavailability (flavonolignans) was enhanced with better efficacy to treat obesity-induced MAFLD upon loading it in different lipid-based nanocarriers, including SEDDs and NLCs. The characterizations of SEDDs and NLCs showed that they had PS, Đ, and ZP of 83.89 nm and 25.49 nm, 0.31 and 0.23, and −8.55 mV and −34.27 mV, respectively. However, upon examining these formulations under TEM, the PSs were spherical but larger than the values by the Zetasizer (150 and 300 nm, respectively). The authors suggested that this is due to the additional preparations that the samples went under for TEM screening. Furthermore, silibinin’s loading capacity inside SEDDs and NLCs was significantly high, with 89.21% and 93.55%, respectively. Interestingly, SEDDs showed greater intensity and higher uptake in GIT than silibinin aqueous suspension and NLCs, probably due to SEDDs prolonged residence time in GIT. On the in vivo level, these lipid nanocarriers oral administration to male Sprague Dawley rats at a dose of 0.32 mg/kg could reduce the pathological signs of liver steatosis when compared to Roux-en-Y gastric bypass surgical option and upon treating MAFLD caused by obesity. Recent successful application of NLC in MAFLD therapeutics was also reported using naringenin, a natural dihydroflavone that widely exists in citrus plants, with previously reported intense anti-inflammatory activity. Naringenin at 100 mg/kg/day attenuates MAFLD in male C57BL/6 wild-type mice via down-regulating the NLRP3/ NF-κB signalling pathway. The naringenin-loaded NLC was prepared by emulsion evaporation and solidification techniques to overcome its crude form poor water solubility (0.072 mg/mL), susceptibility to oxidation, and low oral bioavailability. Upon characterization, the PS, ZP, EE%, and drug loading were 162.9 nm, −6.4 mV, 94.5%, and 22.5%, respectively, with good stability at 4 °C for 7 days. Naringenin-NLC cellular permeability was significantly higher than the naringenin alone on the Madin-Darby Canine Kidney (MDCK) cell line model, suggesting that naringenin could be a substrate of P-glycoprotein. The same enhancement profile was reported on the absorption in either ileum or jejunum of SD bio-breeding male rats. This could be why crude naringenin and naringenin-NLC had the same pharmacokinetic profile but at 100 mg/kg/day and 12.5 mg/kg/day, respectively. Interestingly, in MAFLD-induced mice, only a significant reduction in triglyceride levels was reported upon the oral Recent successful application of NLC in MAFLD therapeutics was also reported using naringenin, a natural dihydroflavone that widely exists in citrus plants, with previously reported intense anti-inflammatory activity. Naringenin at 100 mg/kg/day attenuates MAFLD in male C57BL/6 wild-type mice via down-regulating the NLRP3/

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NF-κB signalling pathway. The naringenin-loaded NLC was prepared by emulsion evaporation and solidification techniques to overcome its crude form poor water solubility (0.072 mg/mL), susceptibility to oxidation, and low oral bioavailability. Upon characterization, the PS, ZP, EE%, and drug loading were 162.9 nm, −6.4 mV, 94.5%, and 22.5%, respectively, with good stability at 4 °C for 7 days. Naringenin-NLC cellular permeability was significantly higher than the naringenin alone on the Madin-Darby Canine Kidney (MDCK) cell line model, suggesting that naringenin could be a substrate of P-glycoprotein. The same enhancement profile was reported on the absorption in either ileum or jejunum of SD bio-breeding male rats. This could be why crude naringenin and naringenin-NLC had the same pharmacokinetic profile but at 100 mg/kg/day and 12.5 mg/kg/day, respectively. Interestingly, in MAFLD-induced mice, only a significant reduction in triglyceride levels was reported upon the oral intake of naringenin-NLC (12.5 mg/ kg/day) in comparison to naringenin (100 mg/kg/day). Such a reduction pattern was not seen in ALT and AST levels, despite the fact that naringenin-NLC (12.5 mg/kg/ day) recorded the highest quantification concentrations in the liver compared to crude naringenin at 50 and 100 mg/kg/day. 4.2.5. Nanoemulsion Nanoemulsions (NEs) are characterized as colloidal systems with droplet sizes ranging from ~50–500 nm, low viscosity, transparency and spherical shape. They are designed to enhance a variety of compounds’ functional and physicochemical properties and shelf life, as well as cosmetical, nutritional, and pharmacological values. NEs can be delivered through the conventional and novel routes of administration, including transmucosal and transdermal routes. This nanosystem offers a sustained release drug delivery, while the small droplet size promotes the material burst release pattern from NEs. NEs’ release profile is directly proportional to its droplet size, smaller droplet size, and larger interfacial area, which promotes rapid drug release. It consists of the main three phases, the oil, the aqueous phase and the emulsifier phase, as explained in Figure 10. The oil phase could be made of triacylglycerols, diacylgycerols, monoacylglycerols, and free fatty acids, non-polar essential oils, mineral oils, lipid substitutes, waxes, and oil-soluble vitamins. Oil phase selection significantly influences the formulation’s stability and characteristics. The production of homogenous NEs relies on the preparation method using mainly either high or low energy methods (e.g., phase inversion temperature, phase inversion composition, ultrasonication, high-pressure homogenization and microfluidization, etc.); such selection is fundamentally reflected on the NEs’ loading, encapsulation efficiency, PS and Đ. Usually, NEs are confused with microemulsions due to significant similarities between the two systems in terms of their physical appearance, components, and preparation techniques. However, NEs are kinetically stable and thermodynamically metastable, while microemulsions are thermodynamically stable. 60

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NEs and NLC surpassed SLN in terms of bioaccessibility and small droplet size output (<100 nm), while NEs offer higher release rates for oral or topical routes, with equal abilities of NEs and NLC to provide protection and stability against UV radiation damages. NEs were also found to be superior to SLN and polymeric nanosuspension when enhancing drug’s solubility and dissolution rate. Despite the various advantages of NEs, the safety concerns associated primarily with the use of synthetic emulsifiers are a crucial problem to be tackled. Most of the synthetic emulsifiers can trigger toxic symptoms with the prolonged administration, including the potential binding of anionic emulsifiers to proteins, enzymes, and phospholipid membranes in the human body, resulting in various adverse alterations, such as dysfunction of enzymes, modification of protein structure, and phospholipid in the membrane cell. Accordingly, replacing the synthetic emulsifiers with natural substitute(s) is one of the on-demand novelties in the NEs’ construction. Initial studies show that NEs’ intravenous administration provided drug delivery to various organs, including the liver, with promising targeting effects, while other studies indicate a preferential liver uptake to the oral NEs. In MAFLD cases, the fat-soluble vitamin D (cholecalciferol) deficiency was observed. It is known for its anti-inflammatory, antioxidant and immune-modulating activity; however, the intake of conventional vitamin D is associated with variable oral bioavailability, poor water solubility and sensitivity to environmental factors, such as light, oxygen, and heat. The vitamin D NEs were prepared using the high-intensity ultrasonication method to overcome these physicochemical drawbacks through employing purposely designed pea protein natural amphiphilic surfactant resulting in droplet size of 88.90 nm, and EE% of 93.2% with a significant UV light stability for 180 min (remaining 74.22% of vitamin D in NEs) in comparison to the pure vitamin D (remaining 8.71%) . The authors proposed that the light stability could be due to the presence of the aromatic side chains and double bonds in pea proteins which might absorb UV light. The digestion test for this oral

Figure 10. The structure of nanoemulsion droplet, consisting of lecithin as an emulsifier dissolved in oil phase, and Tween 80 as another emulsifier dissolved in aqueous phase, while the active material is curcumin. Adapted from reference [222].

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NE showed good recovery of vitamin D after three hours (62.9%). As an anti-MAFLD, this NE demonstrated superiority to the conventional vitamin D in terms of reducing elevated liver enzymes, improved lipid profile, enhanced fatty acid oxidation and attenuates liver inflammation and fibrosis in high-fat diet male albino rats, suggesting a remarkable hepato-protectivity effect of the developed NEs. 4.2.6. Micelles Micelles are self-assembling colloidal dispersions with a hydrophobic core and hydrophilic shell that have a PS ranging from 10 to 100 nm. For the hydrophilic materials and proteins, the reverse micelles are the more suitable carriers, while the unimolecular micelles that are composed of block copolymers (i.e., core(laur)-polyethylene glycol) offer a much more thermodynamic stable micelle form, as explained in Figure 11. These polymers have several hydrophilic and hydrophobic regions in one molecule, which enables the selfassembly of one molecule into a micelle. Micelles are known for their high drug content and kinetic stability, and usually they are administrated through topical, and ophthalmic routes, but they could also be taken orally and intravenously. Micelles are mainly composed of the amphiphilic molecule (surfactants and copolymers), and they are favorable for their simple preparation procedure that offers increased drug solubility, reduced toxicity, increment in circulation time, enhanced tissue penetration and targetability, particularly upon using pH-sensitive polymers. Care must be given when designing this nanocarrier in terms of assuring drug system stability, excipients-drug/compound compatibility, particularly for hydrophilic ones, and the suitability of the short-sustained release profile offered by micelles to the aim of the study [243]. The most controlling experimental factors upon designing micelles are the degree of swelling of the corona, excipient’s concentration, temperature, pH, ionic strength, and sample preparation. Polymeric micelles will have additional factors impacting the micelles characteristics, mainly PS, including the polymer/copolymer molecular weight, the relative proportion of hydrophilic and hydrophobic chains, and the quantity of solvent trapped inside the micellar core. Micelles have surpassed other nanosystems for liver targeting therapy in terms of ideal distribution and relatively proper staying duration in the site upon employing smart excipients such as arabinogalactan polymer. Polymeric micelles exhibited a high capacity to incorporate various bioactive molecules such as antisense oligonucleotides, plasmid DNA, proteins, small interfering ribonucleic acids (siRNAs), messenger RNAs. Micelles could offer positive liver-targeting, mainly for anticancer agents], with further research needed in the anti-MAFLD future. On the nano-molecular level, loading GDC-0449 and miR-29b1 mimics in micelles significantly reduced the collagen deposition in the liver, which in turn improved the liver fibrosis cases, suggesting that these micelles represent a promising candidate in treating MAFLD. The GDC-0449 and miR-29b1 mimics are a hedgehog ligand inhibitor that plays a vital role in treating liver fibrosis by

Figure 11. Schematic representation for the micellization of diblock copolymers and drug encapsulation in polymeric micelle. CMC: critical micelle concentration. Adopted from reference.

inhibiting several pro-fibrotic genes. The measurements of PS, Đ and ZP were 80 nm, 0.2 and −0.5 mV, respectively, while the TEM image showed micelles’ spherical shape and well-dispersed particles, with one-week stability upon placing the micelles on the bench, in addition to 24 h stability in the serum. Moreover, the cellular uptake study indicated that micelles could transfect miRNA efficiently in the immortalized rat liver stellate cell line (HSC-T6), even in the presence of serum, which indicates the in vivo applicability of these micelles over many of the commercially available transfection reagents. 4.2.7. Nanocrystals Nanocrystals (NCs) are currently considered as a promising drug delivery system (PS < 1 μm) with more than twenty approved formulations in the market including those that can treat MAFLD accompanied manifestations of hypercholesterolemia, diabetes, and inflammation. NCs’ structure has various shapes, including dot, sphere, cube, rod, triangle, and hexagon; an example of it, expressed under TEM, is in Figure 12. The advantages of NCs are focused on overcoming erratic absorption of poorly soluble drugs by significantly improving drug/component solubilization and bioavailability ratios which have a positive effect on their pharmacokinetics and therapeutic applications, as well as surface/cell membrane adhesion. This nanosystem excels above the previously reported nanocarriers in terms of drug-loading capacity, and delivery efficiency to cells or tissues, which in turn lead to much more desirable pharmacological activities as compared to nanocapsules, liposomes SLN, NLC [258], and NEs. NCs are primarily administered via oral, parenteral, pulmonary, ocular and topical routes, using different methods of preparations depending on the desired final NC specifications where the combination technique is used to produce NCs with PS < 100 nm. Furthermore, the bottom-up method is for laboratory-scale use, high-pressure homogenization for PS > 100 nm and media milling for less contamination and pharmaceutical industry scale production [263]. NCs with an amorphous crystalline substructure have an increased dissolution rate and are better suited for the delivery of highly hydrophobic rather than hydrophilic drugs, with the ability to protect drugs from environmental conditions and to provide a controlled release profile. Critical steps to study during NC formulation include the freeze-drying process and dependence on critical microcell concentrations for improved stability and drug-loading capacity. At the cellular level, NC charge plays a crucial role in their efficiency,

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Figure 12. Transmission electron microscopy image of a typical nanocrystal sample loaded with titanium dioxide. Adapted from the reference.

where positive NCs can further interact with negatively charged organelles and DNA, leading to higher cell uptake and cytotoxicity compared to negatively charged particles. However, negatively charged NCs also exhibit significant uptake through clathrin- and caveolae-mediated uptake mechanisms. Although NCs were effective in improving the drug delivery and pharmacological outcomes of cardiovascular, ophthalmic, viral, helminthic, bacterial, inflammatory and cancer diseases, NCs impact on liver disease is not well explored; it is proposed that oral and injected NCs might offer liver targeted drug delivery options. As for directly addressing MAFLD, the anti-obesity and first time reported anti-inflammatory nanocrystalline cerium dioxide (CeO2) were suggested as part of MAFLD therapy based on CeO2 antioxidant auto-regenerative ability that was proven on the in vivo level. These effects are linked to the significant ability of the NCs to reduce lipid peroxidation in the liver tissue, and to reversibly switch between Ce3+ and Ce4+ presented on its surface, resulting in the formation of oxygen defects in the crystal lattice that act as “reactive sites” or “hot spots” for free radical scavenging. In addition, CeO2NCs reported reducing steatosis, lobular inflammation and pro-inflammatory cytokines (IL-1β, IL-12Bp40) in rat serum. Interestingly, CeO2-NCs also restored the level of antiinflammatory cytokines (IL-4, IL-10, TGF-β) to the control values. Regarding the CeO2-NC formulation, details were limited to its PS of 4.9 nm with a ZP of-20 mV, while the X-ray diffraction is single-phase and corresponds to the cubic cerium dioxide. 5. Future Perspectives In liver drug delivery, nanoformulations including liposomes, polymeric nanoparticles, and polymeric micelles are the primarily successful nanocarriers mainly in targeting hepatocytes selectively, enhancing loaded materials safety, stability, and physicochemical properties, such as solubility, which is limiting their pharmacological activities based on the drug’s biopharmaceutical classification]. As 62

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can be seen from our previous illustrations, additional detailed answers are needed to address the issues of the nanomedicine impact on MAFLD therapy from a variety of different perspectives, such as the logic behind the selection of a particular nanosystem, excipients, their ratios and preparation method(s). Selecting the route of administration is an additional issue that is critically linked to the type of nanocarrier, the efficiency and the nature of the loaded drug/compound. First and foremost, researchers should consider some key factors while designing each formulation. For example, lipid nanoparticles are known to promote lymphatic transport and can bypass the liver and avoid hepatic first-pass effect, which may lead to a conflict between in vitro and in vivo outcomes when using such nanocarriers. Although many studies report boosted in vitro dissolution rate and in vivo bioavailability of different drugs/ compounds upon loading in lipid-based nanoparticles, further investigation is needed into the mechanism behind which physiological enhancement in the MAFLD-patient liver has occurred, even though the drug/compounds may be bypassing the liver. Another critical question to be answered is nanomedicine’s ability to overcome the absence of a single anti-MAFLD therapy. In addition, a variety of nanocarriers should be considered to examine their unreported effects on MAFLD for therapeutic and diagnostic purposes, such as nanospheres, nanocapsules, nanosuspensions, niosomes, dendrimers, nanogels, carbon nanotubes, polymerized nanographene oxide particles, nanodiamonds, quantum dots, exosomes, polymers and nanoparticle-mediated targeted drug delivery systems, using specific ligands. Future formulators are advised to use the concept of quality-by-design (QbD) formulation, as conventional pharmaceutical development processes are based on quality through testing, and are out of use. It is essential to characterize the developed formulations through a boarder range of assays to clarify excipient– excipient, drug–excipient compatibility and long-term physicochemical stability. This includes fourier-transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, differential scanning calorimetry, X-ray diffraction, and scanning electron microscopy. The effect of the nanosystem charge (positive and negative) on the safe and efficient delivery of anti-MAFLD liver drugs, as well as the mechanism of metabolism, excretion rate, and route, should be extensively studied. Unfortunately, when examining nanoformulations at the in vivo level, gender is an overlooked factor; future research should involve both female and male animals to compare their responses to the formulation with a toxicity profile. Furthermore, many phytochemicals were recognized for their anti-MAFLD therapeutics as illustrated in Table 1, and yet, when their nanosystems were developed (if any), they were never evaluated for the anti-MAFLD activities. Future studies can therefore focus on assessing such already developed nanosystems using the proper in vitro, ex vivo and in vivo MAFLD models. Silybin-nanosuspension and micelles that improved the low oral absorption and

RESEARCH INSIGHTS

bioavailability of silybin are examples of such instances., as well as Schisandra extract loaded in liposome-encapsulated with β-cyclodextrin [281]. Natural polyphenol compounds are a major current trend in the development of MAFLD therapy and have a strong presence worldwide in many traditional diets, mainly Mediterranean diets, and common Chinese dietary herbs [289]. Studies are limited on such phytomedicinal compounds having an impact on MAFLD when loaded into nanosystems, mainly flavonoids and phenolic acids that account for about 60% and 30% of the polyphenols, respectively [290]. In terms of folk medicine, curcumin was one of the most investigated compounds for liver hepato-protectivity and MAFLD treatment potentials. However, these investigations still did not cover most of the famous nanosystems. For instance, curcumin-loaded polymerized graphene oxide nanocarrier was reported to improve its sustained release behavior and bioavailability with a cytocompatible profile [291], while curcumin liposomes were reported to improve the oxidative stress/ antioxidant balance and alleviate inflammation in experimental acetaminophen-induced hepatotoxicity. Studying the impact of such nanosystems on MAFLD might grant answers to many questions concerning its generic drugs (anti-obesity, hypoglycemic, lipid-lowering, cytoprotective and antioxidant agents), and phytomedicinal therapies. The use of synergistic excipients in the treatment of MAFLD was restricted to mostly chitosan polymers,

so more research should consider investigating and/or using other synergistic excipients such as oils, surfactants, co-surfactants, co-solvents. Ginger and garlic oils are promising to begin with as they have been successfully used in the construction of different nanoemulsions for their anti-obesity, anti-hyperlipid activity, along with their hepatoprotective ability to minimize serum total cholesterol, low-density lipoproteins, very low-density lipoproteins, triglycerides, and atherogenic indices. In addition, the resultant and improved serum high-density lipoprotein cholesterol levels suggest that animals are on the way to recovery from MAFLD. However, the ability of such smart excipients to produce nanosystems meeting the required selection criteria is a priority too. Another overlooked consideration in MAFLD nanomedicine therapy is the fate of nanocarriers and related safety issues following administration through various routes, with reports of inflammation-related DNA damage in the liver associated with exposure to nanoparticles, in addition to a persistent rise in body weight and elevated blood glucose levels due to manganese oxide nanoparticle subcutaneous injection at 100 μg/kg, along with a drop in low-density lipoprotein levels. On the same line, and as the liver is suggested to be the major organ for nanoparticle deposition after their absorption, a single oral dose of silver nanoparticles induced acute liver inflammation in healthy male mice; this emphasizes an increased threat

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RESEARCH INSIGHTS

to the susceptible overweight population. Therefore, the sequence of safe, effective and stable novel nanoformulation should not be neglected. 6. Concluding Remarks NAFLD has been re-identified and renamed to a precise name, MAFLD. The disease is escalating all over the world, burdening the health’s of millions due to its multiple manifestations of intrahepatic lipids, alterations of energy metabolism, insulin resistance, and inflammatory processes that contribute to the lack of a specific and effective single treatment. On the other hand, the proper selection and design of the nanocarrier, and it’s in vitro, ex vivo and in vivo evaluation models could provide a clearer vision about the possible solutions to the available conventional anti-MAFLD drugs and traditional herbal remedies in order to protect them from the harsh physiological or environmental conditions. The relation between enhanced materials’ solubility, dissolution rate, bioavailability, and MAFLD nanomedicine discovery is in need of further investigation, mainly when using lipid-based nanocarriers, where the possibility of drug bypassing the liver is high. In this regard, targeted drug delivery might be more promising in MAFLD therapy as exploring such

Author Contributions Conceptualization, R.A.A., I.M.A. and C.S.Y.; methodology, R.A.A. and I.M.A.; formal analysis, R.A.A. and I.M.A.; investigation, R.A.A., I.M.A. and C.S.Y.; resources, R.A.A. and C.S.Y.; original draft preparation, R.A.A., I.M.A.; writing—review and editing, R.A.A. and I.M.A.; visualization, R.A.A., I.M.A. and C.S.Y.; supervision, C.S.Y.; project administration, C.S.Y. All authors have read and agreed to the published version of the manuscript. Funding Universiti Sains Malaysia, grant number 203/PFARMASI6711686, gratefully supports this work. Acknowledgments Authors Reem Abou Assi and Ibrahim M. Abdulbaqi are the recipients of the Universiti Sains Malaysia USM Fellowship Award (P-FD0009/20(R)) and (P-FD0030/19 (R)), respectively, from the Institute of Postgraduate Studies (IPS), Universiti Sains Malaysia (USM), Malaysia. Conflicts of Interest The authors declare no conflict of interest. References are available at https://www.mdpi.com/1424-8247/14/3/215/htm

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Pharma Focus Asia - Issue 42

Articles inside

Research Insights

Future of Pharmaceutical Manufacturing The role of Automation

Data Integrity In pharma’s rush to end the pandemic

Employing Real-World Evidence to Improve Clinical Trial Strategies

Brexit and Pharmacovigilance Where may pharmaceutical companies go?

Biomedical Market Leaders Learning to thrive

Patents and Exclusivity A US perspective

Understanding Bio-summary Tables for ANDA Submissions