This study was reviewed and approved by the Johns Hopkins Medical Institutional Review Board, conducted under an Investigational New Drug Application sponsored by Immtech Pharmaceuticals, Inc., and at its inception registered with ClinicalTrials.gov (NCT00408369). The study was a single-center, randomized, double-blind, placebo-controlled clinical trial conducted entirely on an outpatient basis. Volunteers were screened, dosed, and followed as a single cohort in the outpatient clinic at the Drug Development Unit, Johns Hopkins Hospital. The malaria challenge was conducted at the insectary of the Johns Hopkins Bloomberg School of Public Health. Day 0 signified the day of challenge. Patency was defined as detection of parasitemia by quantitative buffy coat (QBC) and/or malaria blood smear. All units of pafuramidine and DB75 are expressed as base, not salt.
Candidates were recruited by printed advertisements. Eligibility criteria included an age of 18–55 years, a body mass index of 19–31, blood type A or O that supported in vitro growth of P. falciparum, women with no reproductive potential (e.g., postmenopausal or surgically sterile), ability to score at least 80% on a written examination to test comprehension of malaria and the study, ability to comply with the follow-up schedule, and ability to provide a responsible person to assist with follow-up. Volunteers were excluded for clinically significant abnormalities on medical history, physical or laboratory examination, taking prescription or over-the-counter drugs, alcohol or drug abuse, history of malaria or significant malaria exposure, glucose-6-phosphate dehydrogenase deficiency, hemoglobin C or S, use of anti-infective drugs or quinine-containing beverages in the week preceding dosing, and allergy to mosquito bites or intolerance to antimalarial drugs. Written informed consent was obtained for the screening procedures and for human immunodeficiency virus testing. For candidates who passed the screening, informed consent for study participation was obtained at enrollment.
Dosing regimen and rationale
In a sporozoite challenge study, attribution of prophylactic activity to a causal mechanism requires that blood levels of DB75 be “unequivocally subtherapeutic” against erythrocytic parasites at their first possible appearance. For the NF54 strain used in the study, this is 6.5 days after challenge.23
Unequivocally subtherapeutic plasma levels of DB75 were defined as less than 0.25 ng/mL on the basis of two observations. First, DB75 consistently showed no detectable antimalarial activity in vitro
at a concentration of 1 ng/mL (), a level that is four times higher than our defined subtherapeutic level. (Of note, in vitro
assays are conducted in 10% serum, which results in a higher proportion of free drug than is seen in vivo
, and the possible overestimation of antimalarial activity attainable in vivo
; DB75 is 77% protein-bound.) Second, in patients with acute P. falciparum
malaria treated with multiple doses of pafuramidine, trough plasma concentrations of DB75 at or below 3 ng/mL were associated with an 83% failure rate, and all patients with mean trough plasma concentrations less than 2 ng/mL (a level eight times higher than our defined unequivocally subtherapeutic concentration) failed treatment (Immtech Pharmaceuticals, Inc., unpublished data). Accordingly, the designation of 0.25 ng/mL as subtherapeutic is conservative.
FIGURE 2 In vitro activity of DB75 against asexual erythrocytic NF54 Plasmodium falciparum, as assessed by [3H] hypoxanthine incorporation.28,29 The 50% effective concentration is 7.4 ng/mL. Results of three independent assays are shown.
Conversely, for a dosing regimen of pafuramidine to be effective as a causal prophylactic, DB75 levels in the liver must be high enough to act against exoerythrocytic parasites during this period of development. Studies in rats and monkeys have shown that high levels of DB75 accumulate in the liver, and persist for weeks after dosing.15
In cynomolgus monkeys given a single oral dose of 5 mg/kg [14
C] pafuramidine, 10% of the administered dose still remained in the liver in the form of DB75 at 43 days after dosing. When normalized by surface area, this dose is equivalent to 145 mg of pafuramidine for a 70 kg (1.85 m2
) human (assuming monkeys weigh 5 kg and have a surface area of 0.32 m2
Given that human liver has a mean weight of 1.5 kg and 50% water composition,25
and if in humans 10% of a dose is associated with the liver, then after a single oral dose of 100 mg of pafuramidine, the levels of DB75 in liver will be 13,300 ng/mL. This is 1,800 times greater than the 7.4 ng/mL 50% effective concentration (EC50
) for erythrocytic P. falciparum in vitro
(). Accordingly, a single oral dose of 100 mg of pafuramidine should provide substantial levels of DB75 in liver cells.
Three treatment arms were evaluated: placebo, day −1 dosing (a single tablet containing 100 mg of pafuramidine administered the day before challenge), and day −8 dosing (a single tablet containing 100 mg of pafuramidine administered 8 days before challenge). A 100-mg dose was selected because this dose appeared to be safe and likely to attain high DB75 levels in the liver. The placebo arm was essential to demonstrate infectivity of the challenge and to reduce observer bias in interpreting efficacy and toxicity data. The day −1 arm was included to provide the greatest possible drug exposure after infection (thus, greatest chance of efficacy) that has a reasonable likelihood of falling to subtherapeutic plasma levels by day 6.5. The day −8 arm almost certainly ensures subtherapeutic plasma levels by day 6.5 and was included to exploit the anticipated persistence of high liver concentrations. The two pafuramidine-treated groups were thus designed to provide maximal levels of DB75 in the liver during exoerythrocytic development and to ensure that plasma levels of DB75 were unequivocally subtherapeutic on day 6.5 after challenge. If both treatment arms were successful, this regimen would not only support a causal mechanism, but also suggest a once-weekly prophylaxis regimen.
A total of 10 volunteers was required, assuming one-sided significance α = 0.05, to detect at least 20% difference in infection rate between placebo and either of the pafuramidine-treated groups, with a statistical power of 80%. With these criteria, a placebo:drug ratio of 2:3 does not alter the required number. Therefore, four placebo recipients and six in each of two treatment groups (total of 16 volunteers) were selected. This number of volunteers provided a study small enough to be managed safely and large enough to provide an estimate of efficacy that has an exact binomial 95% confidence interval of 54–100% if six of six in a treatment arm are protected and four of four placebo recipients are not. To ensure the availability of 16 volunteers at the time of challenge, 19 were recruited and randomly assigned to three treatment arms: placebo (five persons), day −1 (seven persons), or day −8 (seven persons).
Randomization and drug dosing
Pafuramidine (coated tablets of pafuramidine maleate containing 100 mg of pafuramidine base) and matching placebo were provided by Immtech Pharmaceuticals, Inc. Preparation of unit doses and randomization were performed by the Johns Hopkins Hospital Investigational Pharmacy. Unit doses were packaged, labeled with the study day and subject number, and provided to study personnel. At the time the randomization scheme was developed, one person from each arm was randomly selected for exclusion from challenge. These persons were followed for safety and pharmacokinetics. The code and the identity of those selected for exclusion were available only to the investigational pharmacy.
Volunteers were asked to fast after midnight before and four hours after dosing. All treatments were administered with high-fat breakfast (900 calories, 50% fat) and under direct observation. The placebo group received a placebo tablet on days −8 and −1. The day −8 treatment group received a pafuramidine tablet on day −8 and a placebo tablet on day −1. The day −1 group received a placebo tablet on day −8 and a pafuramidine tablet on day −1. All study personnel and the volunteers were blinded to the treatment assignment throughout the study.
Mosquito infection and malaria challenge
were propagated and maintained in the environmentally controlled insectary of the Johns Hopkins Bloomberg School of Public Health by standard method.26
They were infected with 16–18-day-old gametocyte cultures of the NF54 strain of P. falciparum27
and used for challenge 17 days after infection. Sensitivity to chloroquine (EC50
= 3.0 ng/mL) and DB75 (EC50
= 7.3 ng/mL) were confirmed28,29
After pafuramidine/placebo dosing, 16 volunteers (four placebo, six in each treatment arm) were selected for challenge from the 19 who were dosed. Each person was bitten by five infected mosquitoes using a previously described method.26
After feeding, the mosquitoes were dissected to ascertain the presence of blood in the abdomen and of sporozoites in the salivary glands. Additional mosquitoes were allowed to feed until the volunteer was bitten by five infected mosquitoes. Each volunteer was then given follow-up instructions, a pager and wallet card listing names and contact number of investigators who were available at all times, and was fitted with a medical alert bracelet.
Assessment of efficacy and safety
Clinic visits were scheduled for all subjects on days −8 to 0. Those challenged returned daily on days 1–21, every other day on days 23–35, and weekly until 12 weeks after challenge, unless parasitemia was detected (below). Those not challenged were seen on days 7 and 14.
Efficacy evaluations were conducted on the 16 persons who were challenged. Efficacy was defined as the absence of parasitemia by all five diagnostic methods (Giemsa-stained thick and thin blood smears, QBC, PCR, and blood culture). For real-time evaluation and treatment decisions, blood for thick and thin blood smears and QBC was drawn and examined daily from day 5 to day 21, every other day from day 23 to day 35, and weekly until 12 weeks after challenge. Baseline QBC was evaluated on day −1. All microscopy methods were conducted in accordance with the Clinical Laboratory Improvement Amendments. Blood for PCR was collected on day −1, day 1, daily from day 5 to day 21, and at 6 weeks after challenge; these were not assayed in real-time. Blood was cultured on days 7 and 8.
Assessment of safety for all 19 persons included a subjective clinical interview conducted in a non-directed fashion on each visit, and objective laboratory evaluation on days −8, −1, 7, and 14. Additional laboratory evaluations were done at patency and when deemed necessary. Those who developed symptomatic or laboratory abnormalities were evaluated every 1–3 days until the abnormalities resolved. Safety laboratory evaluation included hematocrit, hemoglobin, leukocyte count and differential count, platelet count, reticulocyte count, prothrombin time, international normalized ratio, activated partial thromboplastin time, Na, K, Cl, HCO3
, creatinine, glucose, albumin, total bilirubin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, magnesium, calcium, amylase, and lipase. Severity of adverse events was graded on the basis of the World Health Organization Toxicity Criteria (2000) (http://www.accessdata.fda.gov/scripts/cder/onctools/whotox.cfm
Before the study blind was broken, all clinical symptoms, signs, and laboratory findings were reviewed and evaluated for severity and judged for their relationship to pafuramidine, malaria, and/or other factors.
Polymerase chain reaction
Blood was collected in sodium citrate and stored at −80°C. DNA was extracted from 200 µL of whole blood using the GenElute™ Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St. Louis, MO) per the manufacturer’s instructions and eluted into a final volume of 200 µL. DNA was amplified using subtelomeric variable open reading frame (STEVOR) primers and nested PCR conditions as previously described.30
A 20-µL aliquot of DNA was amplified in a 100-µL reaction in the first round using primers P5, P18, P19, and P20, and 2 µL of the reaction product was then amplified in a 50-µL reaction in the second round using primers P17 and P24. Five microliters of the final reaction was used for electrophoresis in a 3% agarose gel containing 0.5 µg/mL of ethidium bromide.
Patency and treatment of patent infection
Either positive blood smear (as determined by two independent examiners) or QBC (as determined by two independent examiners) was considered prophylactic failure and treated as patent infection. At patency, blood was drawn for smears, QBC, PCR, parasite culture and drug sensitivity testing, and pafuramidine and DB75 levels. Volunteers then received chloroquine phosphate (1 g salt followed by 500 mg salt at 6, 24, and 48 hr after diagnosis), and had the option to take acetaminophen, up to 1.3 g every 8 hour until symptoms resolved. Volunteers were instructed to return for clinical assessment and parasitologic examination (blood smears, QBC, and PCR), daily until three consecutive negative QBCs were obtained, and at six weeks after diagnosis. Blood was obtained for safety monitoring at diagnosis, when indicated during malaria illness, at the time of third negative QBC, and six weeks after diagnosis.
Drug assay and pharmacokinetic analyses
Blood for pafuramidine and DB75 determinations was collected from all persons who were dosed, immediately before dosing (time 0) and at 4 hours after dosing on both dosing days (day −8 and day −1), daily from day −7 to day 9, inclusive, and at patency. Plasma was collected and stored at −70°C prior to shipping for liquid chromatography/mass spectrometry analyses at Tandem Laboratory (Salt Lake City, UT).15
Lower limits of quantitation for both pafuramidine and DB75 are 0.25 ng/mL. Concentration-time plots for pafuramidine and DB75 were visually examined at all sampling periods. Pharmacokinetic parameters and frequent dosing simulation were obtained by a non-compartmental analytical method using WinNonlin software (Professional Version 4.1; Pharsight Corporation, Mountain View, CA). The maximal plasma concentration (Cmax
) was identified by linear trapezoidal rule, and single dose pharmacokinetic parameters were calculated for the area under the concentration-time curve (AUC0–144 hours
from time zero to 144 hours after dosing), terminal elimination rate constant and elimination half-life (t1/2
) (by linear regression of log-plasma concentration-time curves), total apparent oral clearance (Cl/F), and apparent volume of distribution (V/F).
Urine was assayed for other antimalarial drugs using a previously described method.31
This test, which does not detect pafuramidine or DB75, was performed once before challenge and on day 7. An additional assay was conducted on day 21 for the challenged volunteer who did not develop patent parasitemia.
Descriptive and statistical analyses were performed using Stata (Intercooled Stata 9.2;StataCorp, College Station, TX) and SPSS version 9.0.1 (SPSS Inc., Chicago, IL). The median and interquartile ranges (IQR) were used to describe Cmax, AUC0–144 hours, elimination t1/2, V/F, and Cl/F of pafuramidine and DB75. The proportion of volunteers diagnosed with malaria in the placebo arm was compared using Fisher’s exact test with that of the day −1 or day −8 treatment arms. Time to parasitemia in each treatment arm and by each diagnostic method was plotted and analyzed using Kaplan-Meier survival curves with the log rank test and Wilcoxon signed rank analytical methods.