Objectives
The primary objective of this study was to assess the safety and efficacy of a weekly CQAZ regimen for prophylaxis against CQ resistant Plasmodium falciparum. Secondary objectives were to assess the tolerability and pharmacokinetics of the regimen.
Study Participants
This study was an open-label, randomized controlled trial utilizing a human malaria challenge model. The investigation was conducted at the WRAIR Clinical Trials Center, Silver Spring. MD. Healthy, non-pregnant, non-breastfeeding adults aged 18–50 (inclusive) were eligible for participation. Potentially eligible participants were screened utilizing medical history, physical examination and standard hematologic, renal and liver laboratory evaluations. Laboratory evaluations for human immunodeficiency virus, hepatitis B and C were also conducted. Cardiac risk factors and screening electrocardiogram were assessed. Main exclusion criteria included any chronic medical condition as determined by history, physical examination, or laboratory evaluation that would affect the study results or put the subject at an unacceptably increased risk. Female subjects had to have a negative urine pregnancy test at initial screening and prior to first treatment drug administration and malaria challenge. Prior to enrollment, study subjects could not have visited a malaria endemic country in the previous 3 months, received any malaria prophylaxis in the previous 2 months, been diagnosed with malaria within the past 3 years, or have ever received an experimental malaria vaccine. Concomitant medications that could potentially affect the pharmacokinetics of either treatment drug or the prescribed medication such as cimetidine or other antacids, atorvastatin, or fluconazole or have potential anti-malarial activity were prohibited during the study. The study was registered on ClinicalTrials.gov- NCT03278808 Registered 12 September 2017 - Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT03278808?cond=cq%2Faz&draw=2&rank=1
Endpoints
The primary endpoint was symptomatic parasitemia within 28 days of being challenged. Parasitemia was determined by microscopic evaluation of thick blood smears. Symptomatic was defined as any one of the following solicited adverse events (AEs) that occurred concurrently with parasitemia: fever (temperature > 100.4°F), chills, headache, arthralgia, myalgia, nausea, vomiting, or abdominal pain. Secondary safety endpoints included solicited, unsolicited AEs and electrocardiogram (ECG) findings. Secondary pharmacokinetic endpoints included area under the curve (AUC), maximum concentration (Cmax), time to maximum concentration (Tmax), and half-life (t½) for AZ, CQ and chloroquine’s major metabolite, desethylchloroquine (CQm).
Controlled Human Malaria Infection (CHMI) Challenge Product
The 7G8 clone was established by Thomas R. Burkot, Department of Entomology, WRAIR in April 1982. It was one of several clones, obtained by limiting dilution, from a Brazilian isolate, IMTM 22. The latter was isolated from a 12-year-old boy near Manaus, Brazil, on 2 March 1980 and cryopreserved.
7G8 was successfully tested for infectivity to mosquitoes in May 1982. It was later tested for drug sensitivity by personnel in the Division of Experimental Therapeutics, WRAIR and was found to be resistant to CQ and susceptible to atovaquone-proguanil. 7G8 has subsequently been used by WRAIR and the Navy Medical Research Institute (Bethesda, MD) for challenges requiring chloroquine resistant P. falciparum parasites. (18)
CHMI
The CHMI was conducted at the WRAIR, an institution where over 100 malaria challenges have been conducted since 1985. Each subject’s forearm was exposed to five 7G8-infected mosquitoes for a period of 5 minutes. Subsequent dissection of the mosquito midgut and salivary gland was used to determine if an infectious bite occurred. If none or fewer than 5 infectious mosquitoes bit a subject, then the subject’s forearm was exposed to an additional number of mosquitoes to reach 5 infectious bites total.
Design
After enrollment, subjects were randomized into either the CQAZ group or CQ only control group. The CQAZ group received 2 gm of AZ (Greenstone®) and 300 mg of CQ base (Natco Pharm Ltd) and the CQ only group received 300 mg of CQ base. Subjects received their treatment medication weekly for 3 weeks prior to the malaria challenge. All treatment medications were administered by study staff and directly observed. Although a standard meal was not provided, all participants were instructed to have eaten prior to taking the CQAZ or CQ, and were provided food if they had not eaten. The challenge occurred midway between weeks 3 and 4 of the study. Eight days after the malaria challenge, subjects checked into and were followed closely at a local hotel with 24-hour on-site medical support. Daily Giemsa-stained thick blood film smears for microscopy were obtained on post-challenge days 8–21, in addition to any time a subject reported possible malaria-related symptoms. Microscopists reading the smears were blinded to the treatment group. Parasitemia was determined using Giemsa-stained blood slides using World Health Organization (WHO)-recommended methods. (19) Subjects that had a positive blood smear for malaria and reported any symptom consistent with possible malaria were considered treatment failures. These subjects stopped receiving weekly CQAZ or CQ and received rescue treatment with 1000 mg atovaquone/400 mg proguanil for 3 days under DOT by study staff. They were then followed until they had 3 consecutive daily negative blood smears. Subjects without symptoms (regardless of smear results) continued to receive weekly CQAZ or CQ for 3 weeks following the challenge. After post challenge day 21, subjects were discharged from the hotel and received follow-up blood smears on day 28 and day 56 and whenever they reported possible malaria-related symptoms. (Fig. 1) After the 6th and final dose, any subject who had a positive smear for malaria received rescue treatment with atovaquone/proguanil regardless of whether they were symptomatic, as it was deemed unethical to wait for symptoms given they were no longer scheduled to receive any post-exposure prophylaxis.
Solicited and unsolicited AEs were collected throughout the study as well as hematologic, metabolic, liver and kidney function laboratory evaluations. AEs were graded according to the FDA Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials. All AEs were coded using the Medical Dictionary for Regulatory Activities version 21.0.
After the 5th weekly dose of CQAZ (3 pre-exposure and 2 post-exposure doses), for any subject who had not yet been deemed a treatment failure, blood samples were collected at 0, 1, 2, 4, 6, 10, 24, 48, 72, and 96 hours post dosing. The same subjects also had an ECG performed approximately 6 hours after this dose. QT measurements were corrected using Fridericia’s (QTcF) formulas.
Figure 1 CQAZ Study Design Schema
Bioanalytical Methods
Calibration Standard Curve and Sample Preparation
1.00 mg/ml standard stock solutions of CQ, CQm and AZ (US Pharmacopeia, Rockville, MD) were prepared in dimethylsulfoxide (DMSO) and were used to make up a 10 µg/ml mixture of CQ, CQm and AZ in acetonitrile. The calibration standard curve and quality controls (QC) were prepared by spiking blank human plasma (Li-heparin; BioIVT, Westbury, NY) with this 10 µg/ml stock. The calibration standard curve consisted of matrix and an internal standard, mefloquine, with analytes ranging from 0–1000 ng/ml concentrations, with QC samples covering the low, medium, and high concentration ranges of the standard curve. 100 µL of sample was placed in a microcentrifuge tube and 200 µL of acetonitrile with internal standard was added. Each sample was vigorously vortexed for 15 seconds and centrifuged at 13,000 rpm for 10 minutes at 4 °C. 220 µL of undisturbed supernatant was transferred to a 96-well plate for liquid chromatography - mass spectrometry (LC-MS) analysis.
The human plasma samples were extracted in the same manner with 200 µL of internal standard added to 100 µL of sample. Concentrations of CQ, CQm and AZ in samples were interpolated from each corresponding standard curve. Samples with concentrations greater than the highest point of the calibration curve were diluted with blank human plasma and extracted in the same manner.
Liquid Chromatography - Mass spectrometry (LC-MS/MS) Methods
A Waters (Milford, MA) ACQUITY UPLC system was coupled with an AB Sciex (Framingham, MA) QTrap 4000 linear ion trap spectrometer equipped with a Turbo-V source. A Waters CORTECS C18 (2.1 × 50 mm, 2.7 µm) column was maintained at room temperature while the autosampler was maintained at 4oC to minimize evaporation. Samples were eluted using a linear gradient going from 5–95% acetonitrile/0.1% formic acid in water over the course of 1.50 minutes followed by 1.75 minutes of isocratic gradient of 95% acetonitrile/0.1% formic acid in water at the flow rate of 0.400 ml/min.
The analysis was performed in multiple reaction monitoring in positive electrospray ionization mode by monitoring the ion transitions from m/z 320.200→247.100 (CQ), m/z 292.120→114.100 (CQm), m/z 749.601→591.400 (AZ), and m/z 379.100→361.100 (mefloquine).
Compound parameters and source/gas parameters were optimized to obtain the highest intensity of the analytes. The instrument was controlled and data was collected using Analyst® software.
Pharmacokinetic Analysis
The measured plasma concentrations of AZ, CQ and the main chloroquine metabolite (CQm), from each study subject, based on 10 time points per subject, were evaluated. Using the Phoenix WinNonlin 8.1 software (Certara USA, Inc., 100 Overlook Center, Suite 101, Princeton, NJ 08540 USA), non-compartmental analysis (NCA) with the linear up-log down trapezoidal method was performed to calculate the mean pharmacokinetic parameters. Calculated parameters included the t1/2, Tmax, Cmax, AUC from 0 to 96 hours (AUC0 − 96), extrapolated AUC from 0 to infinity hours (AUC0−∞) and the elimination rate constant (Kel).
Sample Size Estimate
The desired prophylactic efficacy for antimalarial drugs in general is defined as approximately 95% compared to placebo. However, given that this was an exploratory proof of concept study efficacy rates > 90% were to be considered a success. The CQ control group was utilized as a measure of malaria challenge success with greater than 1/6 (17%) of subjects not getting study malaria defined as a malaria challenge failure. This number was based on the previous history of malaria challenges at the WRAIR. The study was designed to enroll 12–15 subjects in the CQAZ group with prophylactic success defined as equal to or greater than 91.6% (11/12), 92.3% (12/13), 92.8% (13/14) or 93.3% (14/15).
Statistical Analysis
All reported study data was recorded on the electronic Case Report Forms supplied by Statistics and Data Corporation (SDC) using an Electronic Data Capture clinical database called iMedNet. After data was entered into the clinical study database, electronic edit checks and data review were performed.
Since the measure of success for the primary efficacy analysis was pre-set at greater than 1/12 treatment failures and the CQ group was a control for measuring challenge success, no inferential statistics were performed comparing the efficacy of CQAZ versus CQ. Group characteristics were compared between the two groups using the intention to treat population (ITT). The ITT population was defined as any subject that was enrolled, randomized and received at least one dose of either CQAZ or CQ. Variables were compared using the using Fisher’s Exact Test or two sample T-test. Subjects in the CQAZ group were further analyzed based on whether they were considered protected from malaria. Variables in each group (protected (P) vs. non-protected (NP)) were compared using Fisher’s Exact Test or two sample T-test. Pharmacokinetic variables in these two groups were compared using two sample T-test. All inferential tests were performed at the α = 0.05 significance level.