Multifactorial Disease Models: Their Role in De-risking Topical Formulation Development (MedPharma)

Multifactorial Disease Models: Their Role in De-risking Topical Formulation Development

The global topical pharmaceutical market is valued at around $95 billion USD and is forecast to grow by $70 billion USD over the next 4–5 years1 . Currently, there are approximately 900 new products in development for dermatology, split between small molecules (65%) and biologics (35%)2 with a considerable focus on topicals for the treatment of conditions such as psoriasis, atopic dermatitis, and acne vulgaris.

A company’s attitude towards risk fundamentally affects their product development strategy, which can vary between the development of a simple or prototype formulation (higher risk) or a fully market-ready, commercially viable product (lower risk) to be used in initial preclinical/ clinical evaluation. Large pharma, with many potential drug candidates to prioritise, tends to be more risk-averse and so generally focuses on entering clinical evaluation with a market-ready formulation that has been developed with risk mitigation considered throughout the development process. For these large companies, an early failure is much less expensive than a failure in the clinic.

Conversely, small biotech companies that may only have a single drug candidate and are typically funded by external investment often favour the development of a prototype formulation. These small companies tend to be more risk-tolerant and prefer to address problems as they arise, so they can evaluate the drug candidate in a clinical proof of concept (PoC) study as quickly as possible. The challenge with this prototype formulation approach is that if the PoC study achieves a positive outcome then further reformulation work would be required to achieve a more patient-friendly and usable product leading to extensive bridging safety studies. In the worstcase scenario, the formulation development may have to restart to proceed to final marketing authorisation application (MAA) or new drug application (NDA). Therefore, the time and money initially saved in the early stages is often lost and/or exceeded later in the project.

Perhaps the optimal route to success sits somewhere between these two approaches where experienced formulators can leverage their extensive technical and regulatory expertise, and utilise innovative models, to mitigate the risks described above as much as possible.

Different Types of Performance Testing Performance testing is an integral part of any formulation development programme and can be used strategically throughout the development process to mitigate development risks and failures. The method that has historically been used for assessing a drug’s thermodynamic activity is in vitro drug release testing (IVRT), where a nonrate limiting synthetic membrane is used to support the semi-solid topical formulation and the drug’s release rate is measured in a receptor solution beneath the membrane. Regulatory bodies are increasingly requiring IVRT use as a quality tool in release and stability specifications and for demonstrating generic bioequivalence.

Nevertheless, IVRT methodologies can only be used to assess and compare the diffusion and release of a drug from a formulation. They provide no insight on the ability of a drug to partition in and permeate across the Stratum corneum and subsequent skin layers or any mechanistic understanding of how a formulation will affect the barrier properties of the skin.

Given the difficulties, costs and time associated with in vivo experimentation, most topical formulators will attempt to use ex vivo/in vitro methodology to optimise and compare their formulations in this regard. In vitro permeation testing (IVPT) is an established methodology recognised by regulators to assess the skin permeation (and commonly penetration) of a drug from topical formulation. It is used to understand the influence of formulation on drug absorption across and into the skin whilst also being used to optimise and compare different formulations during the early stages of development and through product development to post-marketing studies and competitor analysis.

The major limitation of any permeation and penetration model is that it shows how the drug is moving into the biological tissue but does not give any indication of how the formulation affects the skin and whether the drug is bioavailable, engages the target and can act on the desired pathway(s). Traditionally IVPT models have utilised human cadaver skin or ex vivo surgically excised healthy skin, but for many topical products these skin samples do not reflect the condition of the skin to which they will ultimately be applied. Human and Animal Studies

Clearly topical and transdermal formulations are for the treatment of patients in situ, therefore the “gold standard” in the experimental design for formulation development would be to employ human volunteers and monitor drug delivery in vivo. Practically, this is extremely difficult for most drugs (e.g., NCEs/NMEs) and would be unethical and cost-prohibitive for formulation development. Performing pharmacokinetic (PK) studies in humans for transdermal formulations, where the intent is to deliver the drug systemically with drugs of known safety, is well established. Additionally, for topical steroid formulations in vivo studies have been performed, typically using vasoactive agents with measurements of pharmacological activity, such as blanching, used to assess drug delivery. Such vasoconstrictor studies have been useful for researching dose dependencies, or the influence of thermodynamic activity on drug delivery, but are necessarily limited and do not transpose to allow predictions for delivery of other therapeutic agents.

Other in vivo techniques include skin stripping using adhesive tapes or cyanoacrylate glue, punch biopsies, suction blister techniques, various forms of microdialysis and non-invasive determinations such as confocal laser scanning microscopy, confocal Raman spectroscopy or ART-FTIR.

Animals are obviously a potential alternative to human testing. The testing of cosmetic and pharmaceutical excipients and active ingredients for possible irritation effects has remained relatively unchanged since its inception in the early 1900s. In 1944, the first U.S. Food and Drug Administration-

recognised animal testing was implemented, known as the “Draize test”. It utilised rabbits for ocular and skin irritation of cosmetics and personal care items. While these advances allowed a decrease from six test subjects to one to three rabbits per test, little else has changed.

Although the 7th amendment to the European Union Cosmetics Directive now forbids animal testing of cosmetics in Europe, irritation testing in up to two animal species (rodent and mini-pig) is still required by regulatory authorities for various toxicological studies before a new topical medicine approval (e.g., new drug application).

Furthermore, due to the time and cost associated with product failure in the clinic, a range of in vivo animal models using fish, rodents, mini-pigs, and nonhuman primates have been developed to mimic human skin diseases such as skin cancers (e.g, basal cell carcinoma), atopic dermatitis, psoriasis, skin infections (e.g., acne, MRSA, viral infections) and wounds. Many models exploit genetic engineering as many involve gene mutations. However, increasing regulatory restrictions and ethical concerns around the use of these animals in product development (mirroring existing sentiment with cosmetics), combined with the dissimilarities between animals and humans (skin thickness, density, and immune system irregularities), has led to many proponents arguing for their discontinuation over recent years.

In Vitro Alternatives Reconstituted Human Epithelium Models In the 1980s, researchers began to develop reconstituted human epithelium (RHE) skin models which have now evolved to include collagen, fibroblasts, and melanocytes. Commercially available examples include EPISKIN (L’Oreal) EpiDerm (MatTek Corporation), and ZenSkin (ZenBio). These models have quickly gained acceptability in topical product testing for various aspects of pharmacotoxicological and dermal irritation evaluation and contributed to the refinement, reduction and replacement of whole animal testing. The primary advantage of this model above ex vivo human skin is its high reproducibility, which allows for comparison of developed formulations with control formulations of known irritancy.

Most RHE models attempt to replicate “healthy” human skin, but adaptation to replicate skin disease in a controlled, reproducible, and qualifiable manner has become an increasing requirement in topical product development. Such models are generally developed in-house to screen drug candidates and/or formulations, but commercially available RHE models are becoming available. For example, RHE models for inflammatory and autoimmune diseases have been developed, including for psoriasis and atopic dermatitis where the specific pathway leading to expression of the disease state is induced by a cytokine stimulation/ inflammatory cocktail or by downregulation of filaggrin. Alternatively, a commercially available psoriasis RHE is available (e.g. MatTek), produced from normal human epidermal keratinocytes and psoriatic fibroblasts harvested from psoriatic lesions to form a multi-layered, differentiated tissue. The psoriasis tissues maintain a psoriatic phenotype, as evidenced by increased basal cell proliferation, expression of psoriasis-specific markers, and elevated release of psoriasis-specific cytokines. Morphologically, the tissue model closely parallels lesional psoriatic human tissues but lacks the inflammatory cellular components. Skin cancer models have been constructed by incorporating various tumour entities within the three-dimensional RHE matrix. The melanoma model (again from MatTek) consists of human malignant melanoma cells (A375), normal, humanderived epidermal keratinocytes (NHEK) and normal, human-derived dermal fibroblasts (NHDF) which have been cultured to form a multi-layered, differentiated epidermis with melanoma cells at various stages of CM malignancy. Structurally, the melanoma model closely mimics the progression of melanoma in vivo.

The biggest limitation of commercially available RHE models is they do not harbour the true epithelial barrier properties of human skin, potentially resulting in false positives in toxicity studies. These models also lack appendages (sweat glands, sebaceous glands, hair follicles, etc.) which may further deviate from the true performance of the formulations when applied to human skin.

Diseased Skin Models Some of the biggest recent advances in dermatology have been with the introduction of biologics for severe skin diseases. These biologics have revolutionised the management of skin disease and have also been instrumental in expanding the basic understanding of inflammatory dermatosis and new target discovery. This growing interest and understanding in the basic biology of inflammatory dermatosis has led to the development of novel pharmacological (PD) disease models using fresh human skin.

Relatively simplistic diseased skin models such as the TurChub® system have existed for some time and have been utilised to enhance early-stage topical formulation development. The modified zone of inhibition (ZOI) assay includes all the barrier functions associated with the human skin, but with the skin mounted to prevent lateral diffusion of the active formulation around the edges and with the organism growing under the skin layer. This mediumthroughput screen measures inhibition of organism growth (area of no growth) on the underlying agar. As the organism itself is used as the biomarker for permeation and antimicrobial activity, the test avoids the need for analytical methods (HPLC, UPLC, etc.) and multiple formulations containing different drugs can be screened at once.

An advance on the relatively simple modified ZOI assays is infected skin models. These rely on the ability to grow and culture the organisms on the skin, and accurately recover and quantify the viable organisms after treatment. Typical organisms used are yeasts such as C. albicans or P. ovale, dermatophytes such as T. rubrum or T. mentagrophytes, or bacteria such as P. acne, S. aureus (including MRSA), P. aeruginosa and S. epidermis. In these models, an organism most relevant and causative of the infection is artificially introduced under controlled conditions and growth is controlled. The location of the infection within the skin is also controlled, for example, superficial or on the underside of the appropriate dermal layer, such that the position of the organism closely resembles that of the clinical presentation. Additionally, where appropriate, the barrier properties of the diseased skin can be replicated. The ability of the drug or formulation to exert its antimicrobial effect is then assessed using the measurement of a biological marker in the form of ATP, a direct indicator of cell viability, PCR, or direct viable counts. This model also allows the use of living ex vivo skin to explore the effects of an infection and consequent inflammatory responses for multimodal mechanistic studies.

Both the infected skin models and the modified ZOI assays are currently limited to monocultures, meaning only one organism is included per replicate. However, advances

in analytical techniques (e.g., differential PCR techniques), are leading to more complex biofilms (comprising multiple organism types in an infection) within the tissue to be explored.

Skin research’s huge advantage is its direct access to large sections of surgical tissue. The latest advances in tissue culture have allowed scientists to keep this surgical skin alive in culture for over a month; thereby creating a living tissue explant. Recently, this has led to the development of the combination of the models described above for those diseases that comprise a multimodal mechanistic nature (e.g., rosacea, infected eczema, seborrheic dermatitis and infected wounds). This is where infected skin elicits an immune response and the therapeutic effects from a topical treatment can be assessed as a reduction in the organisms and key inflammatory cytokines, antimicrobial peptides and wound remodelling biomarkers. For example, application of live S. aureus or Ps. aeruginosa bacterial culture to wounded live human ex vivo skin tissue results in an infection causing increases in inflammatory gene expression such as the cytokines IL1α, IL1ß, TNFα and IL8; as well as antimicrobial genes DefB4, and S100A7. Such responses to viable bacterial infection can be used to monitor both anti-inflammatory activity of therapeutics based on biological activity and antimicrobial therapy as described above. Wounding of the tissue can be used to evaluate re-epithelisation capabilities of therapeutic formulations in the form of keratinocyte and fibroblast proliferation and differentiation markers (FGF, PDGF, involucrin and KRT16), as well as collagen production and extracellular matrix remodelling (Col1A2 and MMP9) along with histology to visualise re-epithelisation of the wound.

The use of human ex vivo skin culture has the advantage of the inherent physical condition of the tissue. Although as with other models, ex vivo human skin will lack cell migration from the circulatory and lymphatic system into the dermis, unlike RHE it will contain resident immune cells (lymphocytes, dendritic cells and Langerhans cells). Of these, the resident memory T cells (TRM) are known to be potent mediators against infection and autoimmune disease and in ex vivo human skin it has been shown that they secrete inflammatory cytokines.

As well as the resident immune cells, the existing cell population(s) in these explants can be stimulated with specific mixtures to elicit biological responses. This allows the exploration of different disease states, for example, the release of chemokines, antimicrobial peptides and keratinocyte differentiation biomarkers associated with clinical psoriasis can be analysed by quantitative reverse transcription polymerase chain reaction (qRTPCR).

Research has shown similar success with other inflammatory skin diseases e.g., acute (Th2 mediated) and chronic atopic dermatitis (Th1 mediated, vitiligo, and alopecia), along with wound healing, skin ageing, damage, and environmental stress, various skin infections (bacterial and fungal) and complex multi-mechanistic skin diseases, such as acne and infected dermatitis.

Final Thought While pain and itch are elusive targets to investigate by ex vivo explant culture, recent developments suggest that co-culturing neurons with ex vivo skin explants may extend the life of the skin culture for longterm assay and in order to even better understand the neuronal cross-talk in pain, itch and wound healing.

By combining an increased understanding of immunology, tissue culture, and pathway biology, these ex vivo skin PD models have allowed us to explore the possibilities of a multifactorial skin model that more closely represents the human state for the purpose of more translatable therapeutic testing, as well as de-risking the development process for topical products.

REFERENCES

1. https://www.businesswire.com/news/ home/20190816005358/en/Global-TopicalDrug-Delivery-Market-Report-2019-2024 2. BioPharm Insight, 2020

Dr. Jon Lenn

Jon Lenn has direct responsibility for MedPharm’s operations in the United States based out of Durham, North Carolina. Since joining in 2015 he has led MedPharm’s development of cutting edge performance models for assessing penetration and activity of clients’ products targeted towards key biochemical pathways. He has over 15 years’ experience in developing dermatological projects with Connetics, Stiefel and GSK and has been directly involved with the development and approval of 8 products. He received his PhD on the topical delivery of macromolecules from the University of Reading.

Marc Brown

Marc Brown co-founded MedPharm in August 1999. He has been the guiding force behind all of MedPharm’s scientific developments and intellectual property. He has been Professor of Pharmaceutics in the School of Pharmacy, University of Hertfordshire since 2006 and has visiting/honorary professorships at the Universities of Reading and King's College London. He has over 200 publications and 26 patents describing his work. His research interests lie mainly in drug delivery to the skin, nail and airways. To date, he has been involved in the pharmaceutical development of over 38 products that are now on the market in Europe, America and Japan. Prior to MedPharm he was an academic in the Pharmacy Department at KCL.