Bridging study to evaluate the effects of lodonal hiv

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A Bridging study to Evaluate the Effects of LODONAL™ as an Immune-System Regulating Agent in Subjects in which Immune System is Compromised: LODONAL™ IN THE TREATMENT OF SUBJECTS WITH HUMAN IMMUNODEFICIENCY VIRUS (HIV) Dr. Abayomi Oni1, Mr. John Babafemi Aiyegbo RN1, Dr. Femi Aina1, Dr. Olanrewaju Ibigbami1, Mr.Damilola Tugbobo1, Noreen Griffin3, Dr. Jill Smith2, Dr. Nicholas P. Plotnikoff3, Professor Fengping Shan4, Dr. Gloria B. Herndon3,5,6, Dr. Estelle-Marie Heussen5, Dr. Richards Afonja6 1State Specialist Hospital Asubiaro, Osogbo, Osun State, Nigeria

2Georgetown University, College of Medicine, Washington DC, USA 3Immune Therapeutics, Inc, Orlando, Florida, USA

4Department of Immunology, China Medical University,

No.77, Puhe Road, Shenyang, China Washington DC, USA 6American Hospital and Resorts [AHAR] Pharma, Lekki Lagos, Nigeria 5GB Pharma Holdings,

On June 11, 2014, approval was received from the National Agency for Food and Drug Administration and Control [NAFDAC] for AHAR Pharma to conduct a 90-Day bridging clinical trial to evaluate the effects of Low Dose Naltrexone (LodonalTM) as an Immune-System Regulating Agent in Subjects in which Immune System is Compromised and to demonstrate the efficacy and safety of LodonalTM in the local population. Conditional to NAFDAC approval of LodonalTM as an over the counter ‘immune booster’, to be marketed and sold in Nigeria, are results that demonstrate a positive trend leading to an increase or halting the reduction of CD4 count. Therefore, the primary objective of this clinical trial was to confirm that LodonalTM has a beneficial effect on the immune system of HIV+ patients in Nigeria and to also prove its safety. This was determined by assessing mean changes in CD4 levels from baseline [Day-1] to Days 30, 60 and 90. This was a Phase 3, single center, open labeled, randomized, bridging study. A total of one hundred and fifty [150] patients of both gender between the ages of 18-60 were recruited.

Results showed that LodonalTM has a positive effect in increasing CD4% in comparison to the Placebo group. The mean % change between Day 1 and Days 30, 60, 90 was 44% for the Treatment group and 11% for the Placebo group, which was further reflected in the difference between Day-1 and Day 90 mean absolute CD4 count increase of 168 (cell/mm3) for the Treatment group compared to 50 (cell/mm3) in the Placebo group. This increase was sustained over the 90-Day period.

Considering the robust results of the study it is recommended that given Lodonal’sTM’ tolerability and efficacy it should be considered as a first line treatment because of its medical impact, significant increase in quality of life and subsequent economic impact in developing countries. LodonalTM price of about $1/day could drastically reduce first line treatment costs so that financial resources may be diverted to reaching more patients and disease management.

Aims: Primary objective of this clinical trial is to confirm that LodonalTM has beneficial effects on the immune system of patients in Nigeria with compromised immune system. This was determined by stabilization or increase in the CD4 count and or CD4% and stabilization of T cells (lymphocyte) count from baseline to Day 30, 60 and 90.

Secondary objectives include: 1. To confirm stabilization or increase of haemoglobin levels from baseline to Day 30,60 and 90 2. Evaluation of subjects’ weight from baseline to Day 30, 60 and 90 3. To monitor interferon alpha levels from baseline to Day 30,60 and 90 4. To evaluate the safety of LodonalTM in adult participants with compromised immune system using standard measure of safety (physical examination, vital signs, clinical laboratory parameters (chemistry and hematology) and adverse event(s) reporting).

Key Words: HIV, LDN, CD4 Count, Low Dose Naltrexone, LodonalTM, immune-enhancement, ART, Immune Therapy, immune balance.

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1.0 Introduction Immune Therapeutics, American Hospitals and Resorts Pharma [AHAR Pharma], Lekki, Lagos, State Specialist Hospital, Asubiaro, Osogbo and GB Pharma Holdings collaboratively engaged in a 90-Day Bridging trial within a population of HIV+ patients. The study was conducted at one of Nigeria’s leading HIV/AIDS clinics in order to assess the benefits and safety of Low Dose Naltrexone [LDN, marketed as LodonalTM] in HIV+ patients. Naltrexone is an opioid antagonist that was approved by FDA in 1984 [50mg] for the primary use in the management of alcohol and opioid dependence. However, there is accumulating evidence to suggest that in its low dose form [3.0-4.5mg] can promote health by supporting immune modulation, which reduces various oncogenic inflammatory autoimmune processes and immune compromised states such as in HIV and non-HIV states1. Other positive effects of LDN has been observed in cancer patients, such as the reduction of nausea and vomiting associated with chemotherapy, which enables increased quality of life for cancer sufferer and therefore promotes effectiveness of the chemotherapy.

There are some tolerable side effects that have been discovered mostly to do with sleep disturbances, such as early wakefulness and vivid dreams; however, nothing that warrants discontinuation given its positive effects. When the 4.5mg cannot be tolerated, the 3.0mg is suggested and both have shown to be equally effective.

The value of Naltrexone as an immune correcting and balancing modulator was recognized by Dr. Ian Zagon at the Pennsylvania State University2,3and the late Dr. Bernard Bihari, a Neuropsychiatrist from New York, USA (who passed away on May 16th, 2010) who began treating his (HIV/AIDS) patients in the late 1980s4,5 with low dose of the drug. Since their pioneering work, many doctors throughout the United States have been prescribing LDN for a number of indications including Multiple Sclerosis (MS), Parkinson’s disease, and Crohn’s disease, HIV/AIDS, cancer and other autoimmune and inflammatory diseases. In regards to LDN immunotherapies, a number of research and clinical trials have been completed and undergone with phase I and phase II Clinical Trials successfully run at a number of universities in the United States and Europe, including Pennsylvania State University Medical School at Hershey; University of Chicago; State University of New York; SUNY Upstate Medical University; London Health Sciences Centre - University Hospital; Alpert Medical School of Brown University; Department of Neurology, San Raffaele Scientific Institute; Division of Rheumatology, St. Louis College of Pharmacy; Department of Internal Medicine, University of Utah; Jondi-Shapoor University of Medical Sciences; Department of Psychiatry & Behavioral Sciences, Duke University Medical Center; and Multiple Sclerosis Center at UCSF6. Leading immunologists Dr. Nicholas Plotnikoff, Dr. Ronald Herberman, Dr. Bernard Bihari, Dr. Angus Dalgleish, Dr. Ian S. Zagon, Dr. Jill Smith, Dr. McLaughlin, Dr. Jacqueline McCandless, and Moshe Rogosnitzky, among others, pioneered these efforts. The combination of its proven safety and efficacy in the United States necessitated approval of patents in the United States.

In reference to Africa, studies have been conducted in Mali by Dr. Jacqueline McCandless that resulted in two papers published in the Journal of AIDS and HIV Research (JAHR) late in 2011, which describe the two phases of the Mali Project initiated five years earlier. The first paper: Page 2/27

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“Single cohort study of the effect of Low Dose Naltrexone on the evolution of immunological, virological and clinical state of HIV+ adults in Mali” describes the results of giving LDN to a group of 57 HIV positive adults, all of whose CD4 count fell between 350 and 600 cell/mm3 and who showed no symptoms of AIDS. The second paper: “Impact of Low Dose Naltrexone (LDN) on antiretroviral therapy (ART) treated HIV+ adults in Mali: A single blind randomized clinical trial” describes the results of comparing the health of two groups of 57 adults each, all of whom were HIV positive, had a CD4 count below 350 cells/mm3 and were receiving standard ARV medications. One group was also given a 3.0 mg dose of LDN and the other a placebo. The purpose of this phase of the study was to see if LDN improved the standard ARV treatment in a significant way. In the first study her team found that LDN significantly helped HIV positive individuals to maintain their CD4% over a nine-month period. Although the same was not true for the absolute CD4 count, the increase in that measure was less than for a similar population of untreated patients. Since the CD4% is increasingly being seen as a more stable and indicative measure of immune system health, they found the results encouraging. In the second study involving a population in ARV treatment, LDN improved the CD4 count significantly relative to the control group after six months and marginally after nine months. Taken together, the conclusion from the two studies strongly suggests that LDN has a promising role to play in treating HIV/AIDS. Between the 3.0 and 4.5mg daily dose is what is known to produce positive attributes listed including CD4 elevation and reduction of opportunistic infections.

The mechanism and action of naltrexone, in autoimmune diseases and cancer, is still being researched, but there are theories as to the mechanism of action that both explain why LDN works on both autoimmune diseases, cancers, as well as in inflammatory and infectious diseases.

According to Mark J. Donahue’s paper on LDN that uses interviews from Dr. David Gluck, Dr. Jacquelyn McCandless, Dr. Jarred Younger, and Dr. Ian Zagon: “LDN is an opioid antagonist that not only blocks the reception of opiates, but also the body’s own endogenous opioids – endorphins. However, because LDN is administered in such a 'low dose' it is believed that LDN only briefly (for 3-4 hours) obstructs the effects of endorphins. Sensing an endorphin deficit, the hypothalamus signals for increased production of endorphins in what is called 'the rebound effect.” The rebound effect results in three things happening that promote overall immune balance and correcting: ๏ Opioid receptor production increases in order to try and capture more endorphins. ๏ Opioid receptor sensitivity increases, also in order to try and capture more endorphins. ๏ Production of endorphins is increased in order to compensate for the perceived shortage.

Once LDN is metabolized by the liver and eliminated from the body (after 3-4 hours), the elevated levels of endorphins produced, as a result of the rebound effect, can now interact and bind with the more sensitive and more plentiful opioid receptors. These opioid receptors, are found throughout the body, including virtually every cell of the body’s immune system. The elevated levels of endorphins will usually last around 18-20 hours. During this time the elevated endorphins act by up-regulating vital elements of the body’s immune cells. By doing so clinical trials have shown the following: ๏ Down regulating inflammatory cytokines ๏ Reducing inflammation and oxidative stress Page 3/27

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๏ Facilitating tissue repair and wound healing ๏ Restoring T-helper/CD4 levels ๏ Restoring the balance between Th1 & Th2 lymphocytes ๏ Increasing cytotoxic T cells and natural killer (NK) cells ๏ Regulating cell growth & inhibiting tumor growth ๏ Reducing excitotoxicity and microglial activation ๏ Reducing apoptosis of the myelin-producing oligo-dendrocytes ๏ Stimulating mucosal healing (lining of bowel)”7 Research, consistently over many countries, diseases and disorders, supports that LDN can serve as an adjunct treatment for improving general health and prognosis in HIV+ patients as well as assist in the decrease of opportunistic infections, which is an important result for HIV+ patients.

According to the recently published UNAIDS 2015 aggregated report on HIV/AIDS, of the 35 million people living with HIV in the world, 19 million do not know their HIV-positive status.

Although the number of people who are newly infected with HIV is continuing to decline in most parts of the world, there were 2.1 million [1.9 million–2.4 million] new HIV infections in 2013—a decline of 38% from 2001. Some 86% of people living with HIV who know their status in sub-Saharan Africa are receiving antiretroviral therapy, and nearly 76% of them have achieved viral suppression but in many cases are still immune compromised. There are an estimated 24.7 million [23.5–26.1 million] people living with HIV in sub-Saharan Africa, nearly 71% of the global total. Ten countries—Ethiopia, Kenya, Malawi, Mozambique, Nigeria, South Africa, Uganda, the United Republic of Tanzania, Zambia and Zimbabwe—account for 81% of all people living with HIV in the region and half of those are in only two countries—Nigeria and South Africa. There are also more women living with HIV in sub-Saharan Africa than HIVpositive men: women account for 58% of the total number of people living with HIV. Fifteen countries accounted for more than 75% of the 2.1 million new HIV infections that occurred in 2013.Nigeria being one of the 15 at second place with 10% of the population of newly affected people, lead only by South Africa with 16%. HIV treatment coverage is only 20% in Nigeria.

Of all AIDS-related deaths in the sub-Saharan Africa region, 19% occur in Nigeria. Additionally Nigeria is not noted for significant decrease in AIDS deaths compared to the decreases as seen in South Africa (76%), Eritrea (67%), Botswana (58%), Burkina Faso (58%), Ethiopia (63%), Kenya (60%), Zimbabwe (57%), Malawi (51%) and the United Republic of Tanzania (44%).Moreover, more than 75% of all estimated HIV incident tuberculosis people live in just 10 countries, nine of them in sub-Saharan Africa. These include Ethiopia, Kenya, Mozambique, Nigeria, South Africa, United Republic of Tanzania, Uganda, Zambia and Zimbabwe. Research has shown that an HIV+ has a 66% chance of developing tuberculosis but is able to reduce their risk of death by 50% when receiving ART. However, ART medications are costly and at times complex to administer, particularly for children (Cross Continents Collaboration for Kids, 2008).

Moreover, the great diversity of HIV strains and the emergence of resistant hybrids limit this therapeutic arsenal (Bennett et al., 2008). As a consequence, new therapeutic approaches are being explored, including immune-regulatory molecules that may have the potential to delay or prevent the onset of AIDS symptoms in HIV positive individuals (Gekker et al., 2001; Steele et al., 2003, Zagon 2011). LDN has shown the capacity for immune-enhancement in HIV infected Page 4/27

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subjects with no significant side effects, thus preventing or delaying the progression of the disease (Bihari et al., 1988; Mathews et al., 1983; Puente et al., 1991).

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2.0 Methodology 2.1 Study Objective To confirm that LodonalTM has a beneficial effect on the immune system of patients in Nigeria with a compromised immune system (refer to Inclusion/Exclusion Criteria). This will be determined by the primary objectives of demonstrating: ๏ The number of subjects with CD4 counts stabilization (less than 10% changes from baseline mean values) or 25% increase in CD4 counts on LodonalTM compared to observation group from baseline to Days 30, 60 and 90. ๏ The CD4 percent stabilization or increase from baseline to Days 30, 60 and 90. ๏ To evaluate the safety of LodonalTM in adult subjects with a compromised immune system, using standard measures of safety [physical examinations, vital signs, clinical laboratory parameters (chemistry & hematology), and AE reporting].

2.2 Design In order to evaluate the efficacy of Low Dose Naltrexone [marketing name LodonalTM when used in conjunction with ART medication for Human Immunodeficiency Virus positive (HIV+) adults in Nigeria, one-hundred and fifty [150] subjects with active HIV disease were enrolled in the study. With the approval of the National Agency for Food and Drug Administration and Control (NAFDAC) to conduct the trial and having registered same in the Nigeria Clinical Trial Registry (NCTR), the study was conducted as a Phase 3, open labeled, randomized two-group clinical trial during the period of May 2015 through December 2015. The control group received the standard ART medication plus a placebo, while the treatment group received 4.5 mg of LodonalTM [daily at bedtime] in addition to the standard ART medications. Informed consent was obtained from the participants after the study protocol had been well explained and understood by them. All these were done before any study procedures including screening and pre-dose baseline evaluations were conducted on the participants. All the participants were covered with health insurance during the study period as required by NAFDAC and Osun State Ministry of Health Ethics Committee.

The sample size of 150 (75 for each group) was established using available best estimates of expected dropout rates and the variability of CD 4 count measurements. Participants were randomized between the two groups. The patients were enrolled in a staggered fashion, starting in May of 2015. The last few patients who were enrolled in September of 2015 completed their ninety-day clinical evaluation in December of 2015. Testing was conducted for all subjects starting with the first day in the program (baseline) and then on the 30th day of the first month, second and third months. The impact of Lodonal +ART was evaluated in BMI, Triglycerides, Cholesterol, Hemoglobin, Electrolytes, Creatinine, BUN and CD4 count.

Onset of AIDS symptoms or pregnancy was conditions requiring removal of the subject from the clinical study (with treatment initiated as appropriate). Since this population was very small, no survival analysis (OS) was performed on these patients. Future large studies to document the Page 6/27

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overall effect on survivors could be conducted, but for this study only safety and efficacy were the focus.

2.3 Study Site Recruitment, drug dispensing, sample collection and evaluations were conducted at one site: State Specialist Hospital Asubiaro, Osogbo, Osun State, Nigeria. The site is one of the primary treatment centers for HIV/AIDS and infectious diseases in Nigeria. The hospital specializes in infectious diseases and the skin diseases that are common in HIV/AIDS. 2.4 Patient Selection Eligible persons were invited and were given detailed information about the drug trial. The process of recruiting patients based on exclusion criteria took some time as patients were located in municipalities outside of the city and in villages. Subjects were recruited from a population of HIV+ male and female adults between the ages of 18 and 60.

Exclusion criteria included: ๏ Women of childbearing potential unless surgically sterile or using adequate contraception (either IUD, oral or depot contraceptive, or barrier plus spermicide), and willing and able to continue the same contraception during the study and for at least 3 months after the completion of the study; ๏ Women who are pregnant or breast-feeding; Elevated (i.e. >3x ULN) liver enzymes at screening visit; Chronic kidney disease (CKD) as defined by GFR <60 mL/min/1.73m2 ; ๏ Any disease, concomitant injury, or condition that interferes with the performance or interpretation of protocol-specified assessments; ๏ Unlikely to be available for follow-up as specified in the protocol; ๏ Participated in a previous clinical study and received another investigational product within 30 days of screening; ๏ Allergy to naltrexone HCl or any of its excipients; ๏ Patients taking high-dose opioid-based narcotics, unless they discontinued for at least 2 weeks prior to study initiation; ๏ Active infection or neoplastic process; Evidence of active hepatitis C infection determined by HCV Ab/RNA test. ๏ Active drug or alcohol abuse; ๏ Concurrent illness that in the opinion of the principal Investigator(s) may interfere with trial completion; ๏ Any issue which, in the opinion of the Investigator(s), will make the patient unsuitable for study participation ; Once patients were selected, consents forms were presented to all willing to participate in the trial. After signing the Informed Consent Form (ICF), participants were grouped into 2 - the first 75 were selected as the "treated group" (Group A - labeled as ‘LDN Group’ in the analysis) and were placed on LodonalTM (Naltrexone 4.5mg) alongside their regular Highly Active Antiretroviral Therapy (HAART) drugs. The remaining 75 were selected as the "delayedtreatment group" (Group B - controls - labeled as ‘Placebo’ in the analysis) as they continued Page 7/27

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their HAART drugs only and were only to receive LodonalTM after the 90-day trial, so that they too may receive the benefits of LodonalTM. Also, upon selection, patient’s medical records, blood and urine samples necessary for the study were collected and compiled for all 150 patients. The blood and urine samples were collected from all 150 participants under standard conditions.

Samples were transferred to the laboratory for examination and donors were dismissed with instructions regarding when they were next required to come back. Tracking of the participants was done via telephone, which means all reminders, dismissals or inquiry as to the nature of an absence was conducted via telephone. We attempted to align their Clinical trial follow-up with their regular check-up. During the 30, 60 and 90-day mark, following the normal clinic flow chart, the patients would submit their appointment cards and from there they were led to the nurses’ station where the vital signs such as blood pressure, weight, height ,BMI and temperature would be recorded. The patients were then led to see the clinician in turn to discuss their qualitative status [complaints/improvements or difficulties], and all points of the discussion were recorded in the patient’s file. Thereafter they were given a laboratory request form that they presented to the laboratory technicians. Prior to drawing blood, the patient was always made to feel comfortable always and received an explanation of ‘why’ and how ‘of the study at the phlebotomy unit. The phlebotomist carefully wrote the bio-data of the patients on the 3 sample bottles (hematology, chemistry and urinalysis bottles). Injection site for each patient was cleaned with methylated spirit after breaching the blood flow. The heamatology bottles were gently rolled to mix with anticoagulant to avoid clotting and the serum separation bottle left serum above the clotted sample. The heamatology sample was divided into two parts. The first part was used for CD4 where they were processed, fixed and read in the BD- Facs count machine and the second part for CBC with differentials. The serum was also collected and read in the Pro-Selectra machine. The urinalysis kits were used to read the urine samples. All sample results were recorded in the laboratory register. From the laboratory, the patient proceeded to the adherence unit and finally to the pharmacy unit where drugs were dispensed and the next appointment date was given. Two weeks prior to the next appointment date, patients were already being tracked through calling and visitation.

2.5 Laboratory Analysis The following analyses were conducted: ๏ Chemistry ๏ Pro-Selectra/ion Selective Electrode ๏ HAEMATOLOGY ๏ BD FACS COUNT (CD4) ๏ SYSMEX/ABACUS These laboratory analyses were conducted by trained, certified and experienced laboratory technicians.

Quality Assurance (QA) was conducted on a daily basis, equipment was calibrated as required by the set QA standard and the analytic process through defined Quality Control procedures. It was important to use these laboratory techniques in order to measure not only CD4 count but also other important organ function and levels so as to ascertain drug safety in regards to liver function, bone marrow suppression, electrolytes, kidney function and other systemic dysfunction.

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2.6 Test Results Entry Results of the test were received from the laboratory and was sorted, collated, and imputed into the electronic data spreadsheet. Two weeks after the initial stage, participants were being tracked (follow-up) to return for day-30 evaluations. During the course of the follow-up, a brief physical examination was performed to determine their eligibility to continue their participation in the trial. On day 30, the participants were received again at the testing site and laboratory examination processes were carried out. The results were collated and recorded. The same collection methods and laboratory examination processes were repeated for day 60 and day 90 respectively. QA was assured at four levels, from the bottom upwards: 1.Laboratory Technician, 2-Head of the Laboratory, 3-Data Entry Technician and 4- Principal Investigators.

2.7 Statistical Analysis [Based on Clinical Protocol Document submitted to NAFDAC) The main time point of interest is Day 90, but treatment comparisons over all visits, when available, will be conducted.

The primary endpoints for the study are: ๏ The number of subjects with CD4 [T cell] count stabilization (less than 10% change from base line mean values) or 25% increase in CD4 counts on LodonalTM compared to observation group from baseline to Days 30, 60 and 90. ๏ CD4% stabilization or increase from baseline to Days 30, 60 and 90.

The secondary endpoints for this study are: ๏ Hemoglobin stabilization or increase ๏ Body weight stabilization or increase ๏ Interferon alpha levels stabilization The safety endpoints for this study: ๏ Physical examinations, vital signs and clinical laboratory testing (chemistry, hematology, and urinalysis)will be performed at baseline and every 4 weeks until the final clinical trial visit (or early termination visit if applicable). ๏ Adverse events and concomitant medications.

2.7.1 Sample Size Considerations The sample size is based on previous experience with comparable bridging studies and practical considerations to determine safety and efficacy of LodonalTM in the Nigerian population.

2.7.2 Actual Analyses

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One hundred fifty (150) enrolled subjects entered into the open-label study. It was anticipated that premature withdrawal rate by Day 90 will be no more than 30 subjects yielding 120 evaluable subjects. Given the nature of the study, there was no imputation of missing data and there were no formal statistical criteria; rather, the change in laboratory values from baseline was evaluated to determine if the change in values at Days 30, 60 and 90 are statistically significant.

2.7.3 Statistical Methods Summary statistics for discrete variables included number of subjects, counts, and percentages for each category. Descriptive statistics for continuous variables included means, standard deviations, medians, minima, and maxima calculated for CD4 measurements at the baseline visit and at Days 30, 60 and 90, for both the those subjects initially randomized to LodonalTM compared to those randomized to observation.

As hemoglobin levels [part of the safety indices] were within normal limits, further analysis was not conducted. Analysis of IFN-Îą levels was not possible as data collection was hampered by a lack of technical capabilities and mechanism to assure sample integrity if shipped overseas for analysis. Comparisons between treatment groups and changes from baseline values were calculated and tested for statistical significance with two samples and paired t- tests, respectively.

The proportion of those achieving a response with a CD4 count of 200 at each time point and those achieving a CD4 count of >500 was compared between LodonalTM and observation subjects using the Fisherâ&#x20AC;&#x2122;s exact test. Analysis was performed with the intent-to-treat criteria.

Clinical significance was accepted if the difference meets 95% Confidence (p<0.05). It was anticipated that outcomes exhibiting a skewed distribution would be analyzed with Wilcoxon Signed-Rank and Mannâ&#x20AC;&#x201C;Whitney tests, but this was not necessary.

2.7.4 Analysis Sets The Intent-to-Treat (ITT) analysis set consists of all subjects who were enrolled into the study.

This was considered the primary analysis set for efficacy analysis. Subjects were analyzed under the same treatment plan. The Safety Population analysis set was the set of ITT subjects who received at least one dose of study drug. Subjects were analyzed according to the treatment received.

2.7.5 Safety:

Analyses of all safety data was performed on the Safety Population and was presented by the treatment received. The occurrence of TEAEs and serious TEAEs was presented by treatment group overall and within each MedDRA system organ class and preferred term.

Adverse event severity (within each system organ class and preferred term, subjects with more than one incidence was categorized into the maximum severity experienced) and relationship to study drug (subjects categorized into the most-related relationship) was provided.

Use of prior and concomitant medications was summarized. Descriptive statistics of laboratory variables at each visit and on the change from baseline to each visit was presented on mean change from baseline using analysis of covariance at each visit adjusting for baseline. Summary of abnormalities using shift tables was presented for safety laboratory tests. Similar analyses were carried out for vital signs. Notable physical findings were also identified.

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2.7.6 Efficacy:

As mentioned above the descriptive statistics of laboratory variables for CD4 count, CD4%, at each visit and on the change from baseline to each visit was presented on mean change from baseline using analysis of covariance at each visit adjusting for baseline. Summary statistics for continuous variables included means, standard deviations, medians, minima, and maxima. Full specifications regarding all designated analyses were provided in the Statistical Analysis Plan for safety and efficacy.

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3.0 Results: 3.1 Descriptive Statistics Of the 150 patients originally enrolled, seven ‘Treatment’ patients and zero ‘Placebo’ patients were excluded from the study [See Table 1]. Of the seven treatment patients excluded, two dropped because of relocation, one did get pregnant, two went on vacation, one was removed due to severe depression due to bereavement (loss of mother) and one dropped without a trace. Subsequent reduction in patient participation was due to the pending holiday season when most people travel to their home of origin.

Table 1: Patient Dropout during study

Enrollment (time=0) n1 (Treatment) = 75, n2 (Placebo) = 75 Tested Day One

Dropped from study/ missed appointment n1=68, n2=75

Tested Month 1 (30-Days)

Dropped from study/ missed appointment n1=67, n2=62

Tested Month 2 (60-Days)

Dropped from study/ missed appointment n1=64, n2=53

Tested Month 3 (90-Days)

Dropped from study/ missed appointment n1=53 n2=52

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Table 2: Descriptive Analysis Overview Mean

Day-1

Day-30

Day-60

Day-90

LDN

366

514

537

534

Placebo

463

526

506

517

LDN

185

281

300

300

Placebo

269

334.21

266

277

LDN

305

505

502

534

Placebo

435

427.5

477

459

LDN

21

27

14

13

Placebo

24

10

11

29

880

1,422

1,394

1,366

1,222

1,829

1,322

1,378

STD

Median

Minimum

Maximum LDN Placebo

Although we went below the anticipated total number of subjects [120] for the study by day, results were robust early enough in the study and maintained to suggest that they were not by chance. Looking at the means between groups for each day, it is clear to see that Day-1 differences between the groups might suggest a significant difference whilst the other test days do not. To check these differences we conducted unpaired t-test analysis between the groups for each of the test days to determined statistical significance. As assumed, only the first day showed a significance between the groups which was purely by chance as patients were randomly assigned to each of the groups [Table 3]. This result is of no consequence to the primary objective, which is to show change in CD4 count overtime time within a group. All values were rounded off to the nearest whole number.

Table 3: Unpaired t-test comparison of means between groups for each test day Day-0

Treatment

Placebo

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t-test results Day-0

The t-value is 366 -2.50737. The p-value is .

013298. The result is 463 significant at p < .05.

Day-30

t-test results Day-30

t-value is 514 -0.22364. The p-value is .

823394. The result is not 526 significant at p < .05.

Day-60

t-test results Day-60

The t-value is 537 0.59449. The pvalue is .

553354. The result is not 506 significant at p < .05.

Day-90

t-test results Day-90

The t-value is 534 0.15431. The p-value is .

877672. The result is not 517 significant at p < .05.

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3.2 CD4 Count Analysis The selection of patients between the Treatment and Placebo group was random, however the measurement at Day-1 shows that the Treatment group started the study with a lower absolute mean CD4 count [366 vs.

463], which makes their increase to ‘catch-up’ with the Placebo group at Day-30 even more dramatic [Chart 1]. The change between Day 1 and Days 30, 60 & 90 for the Treatment group was above the 25% increase we aimed to demonstrate in this study, whereas the Placebo group maintained an average increase of about 11% which we had pre-determined would be considered a ‘no-change’ effect, [Table 4].

Chart 1 - Absolute Mean CD4 (cell/mm3) Count Over 90 Days Treatment

Placebo Group

600

Absolute Mean CD4 Count

540

480

420

360

300 Day-1

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Day-30

Day-60

Day-90

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Table 4: Percentage Change Between each test day T

P

T

Day-1 N Mean

68 366

P

T

Day-30 75

P

T

Day-60

67

62

463 513(+40%)**

526(+14%)**

64 537(+5%)**

P Day-90

53

53

506(-4%)** 534(-.01%)**

52 517(+1%)**

N= Number of patients Mean = Absolute Mean CD4 (cell/mm3 ) Count; T = Treatment Group; P = Placebo Group; ** Percentage Change in between test days

Although there is an impressive increase in absolute mean CD4 count from Day-1 compared to the other evaluation points at Day 30, 60 and 90, the increases between the days were negligible, Table 5.

Table 5: Percentage Change Between Day1 and Days 30, 60 and 90.

Day 1 Vs. Day 30

Day 1 vs. Day 60

Mean % Change from Day 1 to 30, 60 & 90

Day 1 vs. Day 90

Treatment

40%

47%

46%

44%

Placebo

14%

9%

11%

11%

We conducted paired t-tests with only the patients with complete data sets between each of the days within each group. As a paired t-test can only be conducted with associated values only 50 patients from the Treatment group and 40 from the Placebo group were part of the analysis, however their means were within range of the total group [Table 6] so the analysis’ results were still demonstrated valid results.

Table 6: Percentage Change Between test days paired t-test Day-1 vs. Day 30 % change and t-test

Treatment

40% The value of t is 5.014338. The value of p is < 0.00001. The result is significant at p ≤ 0.05. [Mean diff = 203]

Placebo

14% The value of t is

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3.162597. The value of p is 0.003025. The result is significant at p ≤ 0.05.

Day-1 vs. Day 60 % change and t-test

47% The value of t is 4.357167. The value of p is 6.7E-05. The result is significant at p ≤ 0.05. [mean diff = 190] 9% The value of t is

1.619905. The value of p is 0.113312. The result is not significant at p ≤ 0.05.

Day-1 vs. Day 90 % change and t-test

44% The value of t is 4.042417. The value of p is 0.000186.

The result is significant at p ≤ 0.05. [mean diff= 177.8] 11% The value of t is 1.547536. The value of p is 0.129811.

The result is not significant at p ≤ 0.05.

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Table 7: Means of sub-group of patients for paired t-test Day-1

Day-30

Day-60

Day-90

Treament [N=50]

352

555

542

529

Placebo [40]

486

569

521

520

Because of the severe attrition towards the end of the study it is difficult to make inferences in regards to patterns exhibited by patients with more than 200cell/mm3 or 500cell/mm3 between the groups over time.

Even with a Fisherâ&#x20AC;&#x2122;s exact test the N would have been too low, not paired and somewhat scattered. The objective of showing the more than 25% increase CD4 count and percentage in the Treatment group between Day-1 and Day-90 was demonstrated, whilst the Placebo group only demonstrated an average of 11% increase.

3.3 Side Effects/Quality of Life There were no reported side effects. There were no reported opportunistic infections and no toxicity levels uncovered. Liver function remained normal and there wasnâ&#x20AC;&#x2122;t any negative impact on other systems based on blood results. The patients were able to go about their daily routine unhindered. No significant sleep disturbance or vivid dreams were present enough to justify trial discontinuation. No significant adverse CNS, renal, cardiac, hepatic, musculoskeletal, hematopoietic side effects were present.

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3.4 Meetings Pre-Trial Meeting in Osogbo (September 11, 2015): Present: The full team comprised of Dr. Richards Afonja, Dr.Olanrewaju Ibigbami, John B. Aiyegbo, Dr. Abayomi Oni, Pharm Babatunde Alaka, Olagunoye Adekola Gafar, Omotosho Bukola R., Kolawole L. I., Joy Anokam, Dr Aina Oluwafemi, Damilola Tugbobo, Pharm Akinrinmade D. B., Isamot I. A., Ogunbode O. J., Adewole M. O., Dr Ajibade Adewunmi., Ogunjenyo A. A., Mr Alabi (represented), Mrs Omoloye K. S., Mrs Odebiyi F. O., Mrs Oyedotun Y. M. The meeting began at exactly 10.15am with opening remarks said by the Principal Investigator (PI) Dr. Abayomi Oni. He welcomed all, acknowledging the presence of the clinical and nonclinical members of the clinical trial team. He reiterated the fact that the approval of the NAFDAC and the protocol for the Trial will be strictly adhered to. He stated the fact that all will be done to ensure that the trial subjects are given the best consideration with regards to meeting international ethical standards. He subsequently introduced the Sponsor Representative and CoInvestigator Dr. Afonja.

The Sponsor Representative, Dr. Afonja also welcomed all. He stated that the investigative product (LodonalTM) has been shipped in and the packages verified hence, all is set to commence the clinical trial as scheduled.The protocol for the study was then briefly presented and reviewed by all present. He thereafter requested for clarifications on the protocol.

Dr. Aina the Project Manager itemized the issues that needed to be clarified to facilitate the smooth running of the trial in regards to power supply to the laboratory, and the need to ensure that there is adequate backup for the Laboratory staff to smoothly run the laboratory tests.

Dr. Oni stated that all the staff who will be involved in the trail will be favorably motivated by the sponsors and further vouched to ensure that all who carry out extra duties as a result of the trial will be duly compensated.

Dr. Afonja further emphasized the need to adhere strictly to the study protocol and ensure the principles of Good Clinical Practice are maintained. Further on this point, the trial design was reviewed in great detail to be sure that everyone was clear on all procedures.

The issue of the safety and accountability of the investigative product was also discussed and it was agreed that the drugs be kept in Dr. Oniâ&#x20AC;&#x2122;s Office and the drug dispensed by the Pharmacist with an inventory kept for proper monitoring.The PI thanked all for their presence and contributions. The meeting ended at 1:05 pm.

Pre-Trial Meeting in Osogbo (September 21, 2015): Present: Dr. Abayomi Oni& Dr. and Dr. Femi Aina from State Specialist Hospital Asubiaro; Dr. Richards Afonja, Mr. John Aiyegbo, Ms. Joy Anokam and Mr. Damilola Tugbobo from AHAR; Dr. Ibigbami from the Osun State Ethics committe. Four other support staff also joined

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Dr. Oni opened the meeting as usual exactly at 10:33am, stating that in seven days the treatment group will be receiving their first round of LodonalTM. There is great anticipation and as such several issues and challenges were discussed in order to decide a resolution. These included the following: Power issue - Reinforcing the generator infrastructure became a vital part of the study. The risk of samples not being properly refrigerated and analyzed at the designated time frame would negate the whole study’s design and compromise the data.

Communication Issue- In order to curb attrition it was decided that all patients will be checked through telephone on a fairly regular basis [at least twice between testing days]. Investigative team members that had any cultural connections were paired with those patients in order to reduce language and other disparities. It was also decided that each team member who was tracking patients be given a phone card, logged locally and given access to transportation in the event that a visit was necessary.

Transportation- A designated car for the study was at hand a few days prior to the 30, 60 & 90 day mark in order to facilitate any logistical requirements in so far as patients, reagents etc.

Day 0 (zero) was marked as Monday September 28, 2015. On this day the treatment group of 75 patients would receive 4.5mg LodonalTM [Low Dose Naltrexone] and the control group will receive a placebo.

Actions still do be attended to are: To print the remaining informed consent forms ๏ Include names of AHAR staff members ๏ Damilola will convert data from Monitoring and Evaluation [M&E] into a ๏ spreadsheet The operational staff for the study will be designated as follows: M&E Unit - three people ๏ Pharmacy - two people ๏ Counseling - one person ๏ Doctors - two people ๏ Laboratory technicians - 5 people ๏ Dr Ibigbami and Dr Oni have oversight at all times, which means 15 people altogether to be paid monthly for the extra hours that they would be accruing as a result of the study. *Names of all staff members to be provided by Dr. Aina Meeting ended at 1:46pm.

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Trial Meeting in Osogbo (October 26, 2015): Present: Dr. Abayomi Oni& Dr. and Dr. Femi Aina from State Specialist Hospital Asubiaro; Dr. Richards Afonja, Mr. John Aiyegbo, Ms. Joy Anokam and Mr. Damilola Tugbobo from AHAR; and support staff members Dr. Oni opened the meeting as usual exactly at 9:10am, stating that the trial has commenced and that we were off to an excellent start despite some issues with a number of patients, all of whom coincidentally were in the treatment group. Patients in this group were not treated in any way differently than the placebo group, nor were they aware of which group they were assigned.

Day-0 (zero) blood was drawn from 150 patients however, leading up to Day-30 we already know that seven treatment patients will not be present due to the following reasons: - One patient was overcome by their HIV/AIDS illness and died, two days after blood was drawn - One patient was declared pregnant - One patient opted out due to bereavement [loss of parents] - Two patients went on vacation.

- Two patients relocated Given this attrition, it made clear that follow-up is paramount and patients must be contacted close to 30-day marks but also in-between. This will add an extra burden on the budget of the trial but it is very necessary in order to reduce significant reduction of patients. It was also realized that leading up to the holiday season it will be paramount to keep in contact via telephone and home visit with patients. Patients must be reminded of their trial protocol and with traveling we know that many things seen as â&#x20AC;&#x2DC;optionalâ&#x20AC;&#x2122; may not be adhered. It was agreed that the aim is not to drop lower than 50 patients per experiment group by Day-90.

All procedures where further reviewed and no issues where found. At the end of the week the Day-30 milestone will be reached and a very good turn out of patients is anticipated.

Dr. Oni dismissed the meeting at 11:35 and reminded everyone that only after Day-90 there will be a committee meeting, which will be in the new year given the holiday season. Between now and the end of the trial any operational issues will be dealt with immediately.

Post-Trial Meeting in Osogbo (January 06 2016 ): Present: TDr. Abayomi Oni& Dr. and Dr. Femi Aina from State Specialist Hospital Asubiaro, Mr. John Aiyegbo, Ms. Joy Anokam and Mr. Damilola Tugbobo from AHAR; and support staff members Dr. Oni opened the meeting at exactly 10:00am. The spirit was positive and everyone congratulated each other on the new year and conclusion of the bridging trial. Dr. Oni acknowledged that getting patients for the 90-Day testing was definitely the toughest round, but was very pleased with the turn out almost 110 patients from the original 150. Logistics was certainly the most challenging aspect of the trial and there was agreement to reflect upon the trial so that the institution can better serve another clinical trial in this area or others. Dr. Oni expressed his pride and respect for the team. He mentioned that they worked Page 19/27

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under harsh circumstances and yet in unison, which was critical to achieving complete data collection according to the defined protocol. Several staff members became ill and still had to continue. Dr. Afonja sent a note to express his gratitude for a job well done and took note of the fact that all were committed to quality, which was the corner stone of success. M&E mentioned that the last set of data is yet to be logged and would be completed some time within the next week. Dr. Oni thanked everyone and dismissed the meeting at exactly 11:05am.

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4.0 Discussion In line with other studies that showed a positive effect of LodonalTM in HIV+ patients, this 90day trial once again solidifies the therapeutic role LodonalTM can have on HIV+ patients.The shorter duration of this study demonstrated that even within limited time frame, LodonalTM’s effects can be felt qualitatively and observed clinically. The lack of abnormal liver function means that the drug was well tolerated in the local population and the positive effects of LodonalTM in the first 30 days sustained after 30 days. Our results also seem to indicate that patients with lower CD4 count may fair better with LodonalTM than patients with values above 500. An increase in CD4 count would have a stabilizing effect on the immune system, thus reducing the chances of opportunistic infections and prolong life in patients with HIV. Dr. Cyril A. Allen, former Director of United Medical Center Washington DC and leading specialist in immune therapy, stated that “ …the results of the study showed extremely good consistency with previous studies and it is important to note that this drug as an opiate antagonist has been used in studies since the 1970s. Moreover, the immunomodulatory effects of this drug have been well described in previous studies…”.

As more people are retained on HIV treatment over the long term, dealing with co-morbidities is becoming increasingly important. In particular, TB was responsible for 31% of the estimated 1.2 million HIV-related deaths globally in 2014. The African Region accounted for about three quarters (73%) of the people dying from HIV- and TB-associated causes globally in 2014.

Alongside communicable diseases, people living with HIV are at increased risk of developing a range of noncommunicable diseases as a consequence of their HIV infection or of side effects of their treatment. These noncommunicable diseases include cardiovascular disease, diabetes, liver and pulmonary disease, hypertension and a range of non-AIDS-associated malignancies, notably cancer. Because of greater access to effective ART, people with HIV are living longer and experiencing the noncommunicable diseases associated with aging, which is posing new challenges to health-care systems. For non-communicable diseases related to depressed immunity such as cancer, LodonalTM can serve as a preventative therapy, alleviating treatment requirements that cannot be met due to a lack of medical infrastructure in oncological treatment.

Lastly, based on several clinical studies, one can extrapolate that LodonalTM could be an effective mechanism for stabilizing patients experiencing ART resistance. Although the occurrence of HIV + status has been dramatically reduced by 38% within the past decade, it is apparent that alongside ART treatment which has significantly decreased morbidity also within the past decade, the emergence of HIV drug resistance has to be minimized to preserve the long-term populationlevel effectiveness of ART. Drug resistance has been modest, but there are signs that it has been rising in some countries and regions in recent years [ref WHO report].

Given these robust results, LodonalTM should be considered as an adjunct immune therapy given its proven potential to maintain or increase CD4 count levels, which suggest an increase or stabilization of patient’s immunity that can potentially prevent opportunistic infections, Page 21/27

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tolerability and efficacy. LodonalTM’s affordability [at about $1/day], effective ‘immune balancing and correcting’ attribute, safety, ease in administration and storage makes it a formidable adjunct therapy that needs to be considered for all HIV+ patients. Dr. Allen further mentions that the compelling evidence to date is consistent with this study and while further indepth studies may provide further insights, it would be a shame to deprive patients of this medication any further.

4.1 Trial Challenges and Mitigation Strategies: Conducting the clinical trial had several mitigating factors that contributed to the start of trial, drop out or removal of patients and processing of collected samples such as the following: ๏ Suspicion and trust issues amongst some patients. Patients were use to receiving their HAART medication and that process already has issues of trust associated with it.

Convincing a less educated segment of the patient population of participating in a clinical drug trial was not something they were familiar with and were afraid of how it would negatively affect their current medications. ๏ The lesser educated patients were a challenge, however they were much easier to convince as you could have then read some literature. The non-literate patients caused the most difficulty in communication. More time was required over a period of time [repeated conversations] with them to explain drug trials, side effects, potential benefits and consent signing. As with the patients described above, translators were used and repeated conversations were put in place and within a group of like minded individuals. Constantly being reiterated was the potential benefit of LodonalTM as far as the increased CD4 count and protection from opportunistic infections are concerned. ๏ We also know that all patients whether recipients of placebo or LodonalTM where at times Non-compliant with taking drugs, which could be related to - memory impairment secondary to HIV and also family pressure. It was one thing to convince the patients, but the patients sometimes had a difficult time explaining further to their families, which would lead to uncertainty with our trial patients. The team took its time to explain the potential benefit of LodonalTM as far as the increased CD4 count and protection from opportunistic infections are concerned. ๏ Non-compliance with follow-up at the clinic for blood work, side effect/complication monitoring and evaluation mostly due to the cost of transportation to and from the clinic.The HIV Clinic at Asubiaro is a regional clinic that draws patients from far and near. Most patients outside the town incur so much cost coming back and forth for the trial that we facilitated their presence at the hospital. ๏ Samples were threatened by the lack and loss of electricity, however the hospital generator was refurbished, backup generator was put in place and there was always sufficient fuel on-site to run the generator for at least 24 hours ๏ Although most patients had telephone access, there were a few for whom at home visits were necessary because they either lacked access to a cell phone or preferred not to use one. For these patients transportation or gas was provided for M&E team, physician,

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pharmacist and phlebotomist for home visits for patients who could not afford to come to the clinic or for whom telephone access was challenging. ๏ At times there were challenges with internet connection in order to facilitate real time reporting of results. When these ‘outages’ lasted longer than a day, staff travelled to the next major city to communicate the situation and connect via an internet cafe. ๏ Some American Hospital staff permanently situated in Osogbo for the 90-day duration of the trial contracted Malaria and Salmonella Typhoid food poisoning and had to be hospitalized and treated. The hardship on the trial staff was anticipated, therefore there was sufficient backup of staff to continue with study requirements when there was hospitalization. No one suffered any lasting health consequences and all treatments were successful. ๏ Difficulty choosing staff from the hospital to work in the trial (budget!) which meant the other staff felt sidelined.

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Acknowledgements The authors acknowledge members of the support team in Nigeria led by Ms. Joy Anokam and the individuals who supported the program through their fiscal sponsorship, those who acted as medical advisers, program coordinator and support to the principal investigators, the medical team and other staff [Dr Olanrewaju Aina, Babatunde Alaka, Temitayo Petra Akintaro, Ademola Gafari Olagunoye, Bukola Omotosho, Toyin Irigho, Idayat Ajoke Isamot, Lydia labo kolawole, Monsurat Olubunmi Adewole, Oluseyi Joel Ogunbode, Olukemi Olukunle].Furthermore, the authors are grateful to the Nigerian Ministry of Health, NAFDAC and the Osogbo Ministry of Health Ethics Committee whose guidance insured that the trial was conducted according to the quality, safety and protocol parameters set out by the Nigerian government.

Finally, the authors want to express their deep admiration and gratitude to the late Dr. Bernard Bihari, the late Dr. Ronald Herberman andJacqueline McCandless whose pioneering work with LodonalTM for the treatment of HIV/AIDS guided the design of the Nigeria Trials. In addition to the above we wish to acknowledge Dr. Terry Grossman, Dr. Jay Ellenby, Dr. Jack Zimmerman, Ms. Jaquelyn Young.

Conflict of interest Immune Therapeutics have intellectual property rights and have a patent for the use of Lodonal (“Low Dose Naltrexone”) in Human Immunodeficiency Virus (HIV), cancer, MS, Crohn’s Disease, adjunct to chemotherapy, Parkinson Disease, IBS and have filed pending applications for malaria, TB, Sickle Cell, myasthenia gravis. This disclosure was provided to all study participants. The statistical analysis of the entire data sets pertaining to efficacy (specifically primary and major secondary efficacy endpoints) and safety (specifically, serious adverse events as defined in federal guidelines) have been independently confirmed by a biostatistician who has no conflict of interest.

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