TropicalMed, Vol. 7, Pages 396: Safety and Tolerability of an Antimalarial Herbal Remedy in Healthy Volunteers: An Open-Label, Single-Arm, Dose-Escalation Study on Maytenus senegalensis in Tanzania

2.1. Study Design

The study design was an open-label, single-arm, dose-escalation study to evaluate the safety and tolerability of antimalarial herbal remedy M. senegalensis in healthy adult Tanzanian volunteers (ClinicalTrials.gov Identifier: NCT04944966. Accessed on 14 November 2022).

The study volunteers were divided into four groups (1, 2, 3 and 4), with three volunteers in each group. The volunteers from group 1 were enrolled first in the study, given 400 mg of M. senegalensis and followed by a safety evaluation for seven days after the administration. The volunteers of group 2 were enrolled next in the study and given 600 mg of M. senegalensis and followed by a safety assessment for seven days after administration. Then, the volunteers from group 3 were enrolled in the study and given 800 mg of M. senegalensis every 8 h for 4 days, followed by a safety evaluation for 7 days after the first administration. Lastly, volunteers of group 4 were enrolled in the study and given 800 mg of M. senegalensis every 8 h for 4 days (Figure 1). Study volunteers from groups 3 and 4 were given an 8 hourly dose of 800 mg M. senegalensis to generate safety data for the highest dose from the six volunteers.

All medications other than the tested product were recorded as concomitant medications. Neither routine activities nor dieting were restricted during the trial period.

All study volunteers were hospitalized for 7 days. The dose of M. senegalensis was administered under direct observed treatment (DOT). As a precaution, women were not included in the study to reduce the risk of reprotoxicity, because preclinical data on reproduction toxicity were not available.

The trial was carried out from June to September 2021 at the Bagamoyo Clinical Trial Facility (BCTF), located on the Kingani Estate, 74 km north of Dar es Salaam within the coastal region.

2.2. Study Population

The study population included healthy men aged 18 to 45 years with a Body Mass Index (BMI) ranging from 18 to 30 kg/m2 and who were long-term residents of the study area and agreed to release medical information and to be attended by a study clinician prior to and during the study. Other inclusion criteria were the volunteer’s agreement to provide contact information for a third-party household member or close friend to the study team, available by mobile phone 24 h during the study period, agreement not to participate in another clinical trial and donate blood during the study period, health that complied with the study protocol procedures and agreement to undergo HIV, hepatitis B (HBV) and hepatitis C (HCV) tests, demonstrated understanding of the study, signed written informed consent and free of malaria parasitemia by blood smear at enrolment.

Volunteers were excluded if they had any of the following criteria: received an investigational malaria drug in the last 5 years, participation in any other clinical study involving investigational medicinal products within 30 days prior to the onset of the study, history of arrhythmias, prolonged QT-interval or another cardiac disease, clinically significant abnormalities in electrocardiogram (ECG) at screening, a positive family history of cardiac disease in a first- or second-degree relative at the age of 50, a history of psychiatric disease, suffering from any chronic illness (diabetes mellitus, cancer, or HIV/AIDS), any confirmed or suspected immunosuppressive or immune-deficient condition, history of drug or alcohol abuse, use of chronic immunosuppressive drugs or other immune-modifying drugs within three months of study onset except inhaled and topical corticosteroids. Other exclusion criteria were any clinically significant deviation from the normal range in biochemistry or hematology, blood tests or urine analysis; positive HIV, hepatitis B virus or hepatitis C virus tests; suspicion of having clinically active TB history or physical examination with a positive QuantiFERON-TB Gold Test in-tube assay; symptoms, physical signs, and laboratory values suggestive of systemic disorders that could muddle the interpretation of the study results or jeopardize the health of volunteers. The last exclusion criterion was any medical, social or occupational reason that, in the judgment of the study clinician, was a contraindication to participation or impaired the ability to give informed consent, increased the risk to the volunteer due to participation in the study, affected the ability of the volunteer to participate in the study or impaired interpretation of the study data.

2.4. Preparation of M. senegalensis

The root barks of M. senegalensis were harvested on 9 June 2020 from Bugabo buzi village, Misenye District in the Kagera Region. The region has two rain seasons: heavy rain from March to May and short rains from September to December. The plant was identified by Haji O. Selemani, a botanist from the University of Dar es Salaam, with Voucher Specimen Number 5672, which was deposited at the University Herbarium. The root barks were then debarked to obtain the root barks, which were air-dried under shade at room temperature for two weeks and then pulverized by a grinding mill. The pulverized root backs (20 kg) were extracted three times by cold percolation at room temperature using 80% ethanol and filtered by Macherey–Nagel filter paper. The filtrates were distilled under reduced pressure at 40 °C to give 2.24 kg of brown gummy extract.

The presence of an active ingredient (Pristimerin) for the treatment of malaria was qualitatively determined using the Thin Layer Chromatography (TLC) method. The extract (10 mg) and Pristimerin (1 mg) were separately dissolved in methanol (1 mL). The solutions were then spotted on a TLC and eluted with dichloromethane/methanol at 99:1. The Pristimerin in the sample was observed at Rf 0.3, matching the standard Pristimerin (Figure 2). The extract was granulated by mixing it with starch at a ratio of 2:1 (Extract 2: Starch 1) and packed in hard shell capsules. Each capsule contained 300 mg of powder (200 mg of extract and 100 mg of starch). These capsules were packed in blister bags containing 100 capsules (Figure 3).A trial batch of investigational products was analyzed at the Institute of Traditional Medicine-MUHAS, Tanzania Bureau Standards & Government Chemist Laboratory Authority (GCLA) to minimize the risks of misidentification [22]. The test results revealed that the trial batch had met the standards of Tanzania (TZS 2155:2018). Furthermore, no aflatoxins were detected in the trial batch sample submitted to the Tanzania Bureau of Standards (TBS) for analysis. Refer to Table 1.The balance between efficacy and safety was considered during the selection of a dose for the estimation of the human equivalent dose (HED). The toxicity of drugs increases with increasing doses. According to a previous study, a dose of 100 mg/kg body weight was safe and was able to clear parasites by almost 100%; it was also demonstrated that it was safe at doses as high as 1600 mg/kg body weight [4]. Therefore, the no observed adverse effect level (NOAEL) dose during estimation of the maximum recommended starting dose (MRSD) in humans was 1600 mg/kg body weight. Hence, the MRSD in humans extrapolated from the animal NOAEL dose is 7800 mg/10 = 780 mg, or 800 mg. The US FDA guideline for the estimation of the maximum safe starting dose guided the extrapolation of the animal dose to a human dose [23]. 2.5. Safety Assessment

The safety of the tested product was evaluated through taking medical history, physical examination, vital signs, 12-lead ECG, clinical laboratory tests and incidence of AEs. Physical examinations were performed by study clinicians and included the examination of the following: general appearance, eyes, ears, nose, throat, chest/respiratory, heart/cardiovascular, gastrointestinal/liver, musculoskeletal/extremities, dermatological/skin, thyroid/neck, lymph nodes, and neurological/psychiatric systems.

The vital signs observed during the study included systolic blood pressure, diastolic blood pressure, pulse rate, and body temperature at baseline and 0, 1, 2, 3, 4, 5, 6, 7, 14, 28 and 56 days. A 12-lead ECG was performed at baseline and at 3, 7, 14, 28 and 56 days.

Safety laboratory tests (including hematology and chemistry) were performed at baseline and at 3, 7, 14 and 28 days. A total of 1 mL of venous blood sample was collected into a K3 EDTA vacutainer tube (Greiner Bio-one) and a Z-serum clot activator tube (Greiner Bio-one) for hematology and biochemistry analysis, respectively. For hematological analysis, a fully automated 5-part differential hematology analyzer (Sysmex XS-800i, Sysmex Corporation, Japan) was used to analyze samples within 8 h of blood draw. The results printed out were double-checked by two independent, qualified technicians before being handed out to clinicians for clinical management. For biochemical analysis, samples were allowed to stand at room temperature for 30 min before separating the serum by centrifugation at 3000 rpm for 10 min. The serum was analyzed using Cobas Integra 400 Plus (Roche Diagnostic, Switzerland) within 24 h after the blood draw. If the testing was to be delayed, serum for biochemical analyses was stored at −20 °C and subjected to a single freeze–thaw cycle at the time of analysis, and whole EDTA blood for hematological analyses was stored at 4–8 °C and analyzed within 24 h after the blood draw.

For the quality assurance of the analyzers, internal quality control (IQC) and external quality assurance (EQA) quality assessments were performed on the main and back-up analyzers according to standard operating procedures. IQC was carried out daily using quality control materials supplied by the respective analyzer’s manufacturer. EQA was performed monthly using materials supplied by an independent EQA firm (the College of American Pathologists (CAP) for the biochemistry analyzer and the United Kingdom National External Quality Assessment Scheme (UK NEQAS) for the hematology analyzer). Only the analyzers that passed the IQC and EQA assessments were used for clinical sample analysis.

White blood cell count (3.48 to 9.11 × 103/L), red blood cell count (4.39 to 6.5 × 106/L), hemoglobin (12.6 to 17.3 g/dL), platelets (107 to 396.2 × 103/L), neutrophil counts (1.18 to 5.46 × 103/L), lymphocyte counts (1.19 to 3.4 × 103/L), and eosinophil counts (0 to 0.78 × 103/L) were all examined. The analysis of liver function tests included AST (12.3 to 74.7 U/L), ALT (3.5 to 46.8 U/L) and total bilirubin (3.1–31.1μmol/L). The serum creatinine level (49 to 95.3 μmol/L) was measured as a function of the kidney.

These laboratory reference intervals were derived from a local or regional population that was more likely to have similar biological characteristics, as stipulated in the Clinical and Laboratory Standards Institute (CLSI) guidelines [24]. The correct interpretation of laboratory results requires accurate clinical laboratory reference intervals derived from a local or regional population [25].

Solicited adverse events were monitored and recorded up to day 7, while unsolicited adverse events were monitored and recorded up to day 28 after the first administration of the tested product.

All study volunteers were monitored throughout the study for serious adverse events (SAEs). The study clinicians were responsible for evaluating all AEs in terms of intensity (mild, moderate, or severe), duration, severity, outcome, and relationship to the study drug. The Standard Treatment Guidelines & National Essential Medicines List, Tanzania Mainland (STG/NEMLIT-2017); Division of AIDS (DAIDS) Table for Grading the Severity of Adult and Pediatric Adverse Events (Corrected Version 2.1, July 2017); US FDA, Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials: Guidance for Industry (September 2007); and Common Terminology Criteria for Adverse Events (CTCAE) (Version 5.0, November 2017) were used to develop the grading criteria for adverse events.

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