A Randomized Trial Evaluating the Prophylactic Activity of DSM265 Against Preerythrocytic Plasmodium falciparum Infection During Controlled Human Malarial Infection by Mosquito Bites and Direct Venous Inoculation

Abstract

Background

DSM265 is a selective inhibitor of Plasmodium dihydroorotate dehydrogenase that fully protected against controlled human malarial infection (CHMI) by direct venous inoculation of Plasmodium falciparum sporozoites when administered 1 day before challenge and provided partial protection when administered 7 days before challenge.

Methods

A double-blinded, randomized, placebo-controlled trial was performed to assess safety, tolerability, pharmacokinetics, and efficacy of 1 oral dose of 400 mg of DSM265 before CHMI. Three cohorts were studied, with DSM265 administered 3 or 7 days before direct venous inoculation of sporozoites or 7 days before 5 bites from infected mosquitoes.

Results

DSM265-related adverse events consisted of mild-to-moderate headache and gastrointestinal symptoms. DSM265 concentrations were consistent with pharmacokinetic models (mean area under the curve extrapolated to infinity, 1707 µg*h/mL). Placebo-treated participants became positive by quantitative reverse transcription–polymerase chain reaction (qRT-PCR) and were treated 7–10 days after CHMI. Among DSM265-treated subjects, 2 of 6 in each cohort were sterilely protected. DSM265-treated recipients had longer times to development of parasitemia than placebo-treated participants (P < .004).

Conclusions

This was the first CHMI study of a novel antimalarial compound to compare direct venous inoculation of sporozoites and mosquito bites. Times to qRT-PCR positivity and treatment were comparable for both routes. DSM265 given 3 or 7 days before CHMI was safe and well tolerated but sterilely protected only one third of participants.

A controlled human malarial infection study was conducted to assess the efficacy of oral DSM265 against preerythrocytic Plasmodium falciparum sporozoite infection. Complete protection was achieved in 33% of volunteers who received DSM265 3 or 7 days before challenge.

Malaria is endemic in 87 countries, threatening 3.3 billion people in the poorest tropical nations [1–3]. Despite control efforts, malaria remains a major global health problem, with 429000 deaths in 2015 [4]. Effective vaccines have not been developed; thus, chemotherapy remains the mainstay of prevention and treatment. Unfortunately, drug resistance to almost every known antimalarial agent has compromised effectiveness of control programs [5, 6]. Artemisinin combination chemotherapies provided new treatment options against drug-resistant parasites [7, 8], but emerging resistance in the Greater Mekong subregion [9, 10] underscores the need for continued drug development.

There are several population sectors in need of effective malaria chemoprophylaxis [11] but only a few prophylactic options: mefloquine, doxycycline, or the fixed combination of atovaquone and proguanil. Mefloquine is dosed weekly, but its use is restricted because of reported neuropsychiatric adverse events (AEs) during prophylaxis and resistance in regions where mefloquine has been used for treatment. Doxycycline and atovaquone-proguanil require daily administration, and poor compliance can lead to reduced protection. Beyond prophylactic options, dihydroartemisinin-piperaquine has shown efficacy for treatment of uncomplicated infections [12–14], for intermittent preventive treatment in children [15], and for prevention of malaria during pregnancy [16].

The malaria genome project [17] accelerated the search for novel antimalarial drug targets. One of the best validated new targets is Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH), an essential enzyme for pyrimidine biosynthesis [18]. While plasmodia rely on salvage pathways for purines, pyrimidines cannot be salvaged as in mammalian cells and must be acquired through de novo synthesis [19–24]. The lack of a redundant pathway in plasmodia and the presence of pyrimidine salvage pathways in the host make pyrimidine biosynthesis a highly vulnerable plasmodial metabolic pathway. An inhibitor of human DHODH leflunomide and its active metabolite teriflunomide are marketed for treatment of rheumatoid arthritis and multiple sclerosis, indicating therapeutic potential of DHODH inhibition [25, 26]. DSM265 is a triazolopyrimidine compound identified by enzyme activity–based high-throughput screens and compound optimization for antimalarial activity in vitro and in vivo [27–30]. DSM265 was designed to selectively inhibit PfDHODH and shows >100-fold selectivity over the human ortholog, owing to structural differences in inhibitor binding [31]. DSM265 has a long elimination half-life in animals [32] and kills preerythrocytic (ie, liver-stage) and erythrocyte-stage parasites by PfDHODH inhibition [32], justifying further clinical development [33]. The compound is only modestly effective against sexual stages of plasmodia [32]. Clinical data indicated a good safety/tolerability profile and confirmed a long half-life (86–118 hours) in humans [34]. In an open-label, phase 2a, proof-of-concept study to assess DSM265 treatment of uncomplicated malaria in Peru, crude adequate clinical and parasitological response analysis showed that 100% of P. falciparum–infected patients treated with 1 dose of DSM265 (400 mg) were cured at day 14, with a median microscopy-determined parasite clearance time of 54 hours (clinical trials registration NCT02123290; unpublished data). In addition, in a controlled human malarial infection (CHMI) study using the induced blood-stage malarial infection model, pharmacokinetic (PK)/pharmacodynamics modeling predicted that a single 340-mg dose of DSM265 would maintain the level of DSM265 above the estimated minimum inhibitory concentration for 7 days and protect against erythrocyte-stage parasite infection [34], consistent with previous PK predictions based on preclinical models [32]. Recently, a single 400-mg dose of DSM265 given 1 day before P. falciparum sporozoite CHMI by direct venous inoculation (DVI) protected all participants from erythrocyte-stage infection. However, dosing 1 week before sporozoite DVI was only partly effective [35]. This recently reported study used DVI of sporozoites, bypassing the parasite’s skin stage. The aim of our study was to evaluate the prophylactic efficacy of different DSM265 dosing intervals against CHMI, using either DVI-based challenge with 3200 P. falciparum strain NF54 sporozoites (PfSPZ-C) or 5 bites from P. falciparum–infected mosquitoes, and to further assess the safety, tolerability, and PK of DSM265.

METHODS

Study Design

The study was a single-center, double-blinded, randomized, placebo-controlled phase 1 trial involving 3 cohorts of human volunteers (Figure 1). The study design is briefly described here; full details are in the Supplementary Materials. In cohort 1, 8 volunteers received 1 dose of 400 mg of DSM265 (n = 6) or placebo (n = 2) 3 days before CHMI via DVI (day −3). In cohort 2a, 8 volunteers received 1 dose of 400 mg of DSM265 (n = 6) or placebo (n = 2) 7 days before CHMI via DVI (day −7). Cohort 2b was identical to cohort 2a except that CHMI was by 5 bites from P. falciparum–infected Anopheles stephensi. Initiation of cohorts 2a/b was contingent on a favorable safety review, including both a lack of serious AEs (SAEs) and favorable efficacy (defined as a geometric mean time to ≥250 estimated parasites/mL of ≥12 days, as determined by quantitative reverse transcription–polymerase chain reaction [qRT-PCR] analysis) in cohort 1. Participants were followed intensively for AEs related to study drug (DSM265 or placebo), CHMI, malaria, and treatment (atovaquone-proguanil) and underwent sampling for drug and Plasmodium 18S ribosomal RNA (rRNA) analyses after dosing and CHMI, respectively (Figure 1).

Figure 1.

DSM265 study design and main interventions. Time line of each of the 3 cohorts in days. Day of sporozoite administration is indicated as day 0. DSM265 or placebo was administered 3 or 7 days before controlled human malarial infection (CHMI), as shown. Pipette symbols indicate days (or hours, on dosing days) with quantitative reverse transcription–polymerase chain reaction (qRT-PCR) testing for parasite 18S ribosomal RNA, and blood drops indicate days with pharmacokinetic testing. Telephone follow-up was conducted 3 and 6 months after CHMI (not depicted). DVI, direct venous inoculation; PK, pharmacokinetic.

The study was performed at the Seattle Malaria Clinical Trials Center at the Fred Hutchinson Cancer Research Center (Seattle, WA) in collaboration with the Center for Infectious Disease Research (CID Research) and the University of Washington, was approved by the Fred Hutchinson Cancer Research Center Institutional Review Board, and is registered with ClinicalTrials.gov (NCT02562872).

Participants, Eligibility, Randomization, and Blinding

Healthy, malaria-naive adults (18–45 years old) were recruited. Eligibility criteria are in the Supplementary Materials. Participants were randomly assigned to receive drug or placebo for all cohorts, using a predetermined randomization schedule known only to the study pharmacist (DSM265 to placebo allocation ratio, 3:1). The safety review team was unblinded after day 60 during an interim database lock, to permit efficacy and safety assessments, but sponsors, investigators, and study volunteers remained blinded until completion of follow-up for cohort 2b and final database lock.

Study Procedures

After screening and providing informed consent (Supplementary Materials), participants fasted for ≥10 hours and then received DSM265 or placebo as a 240-mL oral suspension. Placebo and DSM265 (both supplied by Bend Research, Bend, OR) were indistinguishable in taste and appearance and were reconstituted as reported elsewhere [35]. Participants fasted for 4 hours after dosing, although the impact of food on the blood PK of DSM265 is minimal [32]. Samples for PK analysis were collected at baseline (0 hours) and 1, 2, 4, 6, 8, 12, 24, 48, 72, 144, 240, and 432 hours after dosing. Concentrations of DSM265 and DSM450 (the major inactive metabolite in humans) were measured in plasma specimens and dried blood spots by liquid chromatography–tandem mass spectrometry at the Walter Reed Army Institute of Research.

For cohorts 1 and 2a, CHMI was initiated by DVI of PfSPZ-C (Sanaria, Rockville, MD) as described elsewhere [36, 37]. For cohort 2b, CHMI was initiated by 5 bites from P. falciparum–infected A. stephensi as previously described [38]. Mosquitoes were reared and infected with P. falciparum NF54 in the CID Research Insectary under good manufacturing practice conditions. The presence of sporozoites in blood-feeding mosquitoes was confirmed by microscopy, such that all participants received 5 bites from mosquitoes with sporozoite densities of 2+ in salivary glands, based on the standard grading system [39]. Blood stages of isolates used to make PfSPZ-C and to infect mosquitoes were susceptible to atovaquone-proguanil, chloroquine, and DSM265.

Safety tests (chemistry and hematologic assays), urine drug screening, pregnancy tests, and electrocardiography were performed during screening and at enrollment (Supplementary Materials). AEs and concomitant medications were recorded at all on-site visits, and clinical staff were available by telephone at all hours during the trial.

Participants were monitored immediately after CHMI (day 0) and on days 1 and 3. Infection detection testing by Plasmodium 18S qRT-PCR was conducted when shown in Figure 1 and as in the Supplementary Materials. One positive qRT-PCR result, corresponding to an estimated parasite density of ≥250 parasites/mL, triggered initiation of directly observed treatment with the standard curative dose of atovaquone-proguanil (Malarone, GlaxoSmithKline; 250 mg atovaquone/100 mg proguanil hydrochloride daily for 3 days). qRT-PCR analysis was performed using an unpublished third-generation Plasmodium qRT-PCR assay with performance characteristics equivalent to previously published methods [40, 41]. Thick blood smears (TBS) and qRT-PCR analyses were performed upon initiation of atovaquone-proguanil treatment and on select posttreatment days. Volunteers who remained qRT-PCR negative throughout were treated with atovaquone-proguanil on day 28. Chloroquine phosphate was reserved as second-line therapy [38]. The last in-person visit was on day 42, with follow-up phone calls at 3 and 6 months.

End Points and Statistical Analysis

The study’s primary objective was to assess prophylactic activity of a 400-mg single-dose of DSM265 before CHMI in nonimmune, healthy volunteers. Primary efficacy end points were infection detected by Plasmodium 18S rRNA qRT-PCR within 28 days of challenge, and time from challenge to the first positive qRT-PCR ≥250 estimated parasites/mL (time to treatment eligibility). Secondary efficacy end points were qRT-PCR positivity by day 28 (defined as an estimated ≥20 parasites/mL) and time to qRT-PCR positivity. Participants who did not require treatment with atovaquone-proguanil before day 28 were censored at day 28. Efficacy end points were compared between groups and cohorts, using Fisher exact and log-rank tests. The mean parasite density upon treatment eligibility was estimated using linear models adjusted for intervals between qRT-PCR measurements. Rates of safety events—AEs and their relatedness to study interventions—were estimated by treatment group and cohort. Linear noncompartmental models were fit to individual PK profiles and used to estimate half-life (t_1/2) and area under the curve extrapolated to infinity (AUC_0-∞); the area under the curve through 240 hours (AUC_0-240), the maximum concentration (C_max), and the time of maximum concentration (t_max) were estimated empirically. All P values are 2-sided, with P values of < .05 considered statistically significant. See the Supplementary Materials for details.

RESULTS

The study was conducted from February 2016 to May 2017, with in-person procedures occurring during April–September 2016. Of 53 screened individuals, 24 were eligible, and groups of 8 participants were allocated to cohorts 1, 2a, or 2b and assigned to receive DSM265 or placebo (Figure 2). Fifty percent of participants were female, and the mean age was 28.8 years (range, 20–37 years). Baseline demographic characteristics were similar across all cohorts and treatment arms (Table 1). All volunteers completed all in-person procedures, including DSM265 or placebo dosing and CHMI, and were included in the analysis.

Figure 2.

Study population allocation.

Table 1.

Baseline Characteristics of the Intention-to-Treat Population

Variable	Cohort 1	Cohort 2a	Cohort 2b	Overall
Enrolled participants, no.	8	8	8	24
Female sex	3 (37.5)	6 (75)	3 (37.5)	12 (50)
Age, y
18–20	1 (12.5)	0 (0)	1 (12.5)	2 (8.33)
21–30	5 (62.5)	4 (50)	5 (62.5)	14 (58.33)
31–40	2 (25)	4 (50)	2 (25)	8 (33.33)
Range	20–37	24–34	20–37	20–37
Ethnicity
White	4 (50)	6 (75)	4 (50)	14 (58.33)
Black	0 (0)	1 (12.5)	1 (12.5)	2 (8.33)
Asian	1 (12.5)	0 (0)	2 (25)	3 (12.5)
Other race	1 (12.5)	0 (0)	1 (12.5)	2 (8.33)
Multiracial	2 (25)	1 (12.5)	0 (0)	3 (12.5)
Height, cm	174.6 (164.5–176.8)	171.2 (163.3–173.7)	174.7 (170.2–182.2)	172.9 (164.5–176.5)
Weight, kg	62 (58.2–73.3)	66.6 (64.6–68.5)	75.4 (69.4–84.7)	68.2 (64.1–73.9)
BMIa	22.6 (20.4–23.6)	22.9 (21.8–24.5)	25.7 (24.4–28.1)	23.6 (22.2–25.0)

Data are no. (%) of participants or median (interquartile range), unless otherwise indicated.

^aBody mass index (BMI) was calculated as the weight in kilograms divided by the height in meters squared.

In each cohort, all placebo recipients and 4 of 6 DSM265 recipients required treatment by day 28. Differences in rates of treatment were not statistically significant overall (pooling all cohorts) or within cohorts (P > .28). However, the time from challenge to a parasite density of ≥250 parasites/mL was significantly longer for DSM265 recipients as compared to placebo recipients (P = .004 for each cohort; P < .001 overall; Figure 3A). The median time to a parasite density of ≥250 parasites/mL among DSM265 recipients was longest for cohort 1 (20.9 days; range, 12.4–23.4 days; Figure 3B), in which DSM265 was given 3 days before challenge, compared with cohorts 2a (median, 15.3 days; range, 12.3–15.4 days) and 2b (median, 16.9 days; range, 8.4–20.4 days; Figure 3C), in which DSM265 was administered 7 days before challenge, and was 8.3 days among placebo recipients. Similar trends were apparent at the qRT-PCR limit of detection (an estimated ≥20 parasites/mL): all placebo-recipient controls and 67% of DSM265-treated participants became qRT-PCR positive, and time to positivity was longer for DSM265 recipients relative to placebo recipients (P ≤.02 for each cohort; (Supplementary Figures 1–3). However, the apparent differences in time to ≥250 parasites/mL between cohort 1 (day −3 dosing) and cohort 2 (cohorts 2a/2b combined; day −7 dosing) were not statistically significant (P = .71; Figure 3D). In addition, there were no significant differences when the same challenge route was used (cohorts 1 vs 2a) for the time to ≥250 parasites/mL (Supplementary Figure 4) or for the time to qRT-PCR positivity (≥20 estimated parasites/mL; Supplementary Figure 5). When comparing DSM265 recipients in cohorts 2a (DVI) and 2b (mosquito bite), the same proportion of participants became treatment eligible (≥250 parasites/mL) by day 28, with no apparent differences between cohorts in the time to ≥250 parasites/mL (P = .90; Figure 3C) or the time to qRT-PCR positivity (P = .87; Supplementary Figure 3).

Figure 3.

Primary efficacy assessment of time to ≥250 parasites/mL. Kaplan-Meier estimates of the cumulative proportion of participants who were parasitemic, as determined by quantitative reverse transcription–polymerase chain reaction, with an estimated ≥250 parasites/mL. A, Combined results for all 3 cohorts among placebo-recipient controls (solid black line) and DSM265 recipients (red dashed line). B, Results for cohort 1 (C1) only, with controlled human malarial infection (CHMI) induced by direct venous inoculation (DVI) 3 days after dosing, among placebo-recipient controls (solid black line) and DSM265 recipients (red dashed line). C, Results for cohort 2a (C2a; CHMI induced by DVI) and cohort 2b (C2b; CHMI induced by mosquito bite) among placebo-recipient controls (cohort 2a [C2a], solid black line; cohort 2b [C2b], solid red line) and DSM265 recipients (C2a, dashed black line; C2b, dashed red line). D, Results for C1 (black lines) as compared to C2a and C2b (C2ab; red lines). Log-rank tests were used to compare time to ≥250 parasites/mL between treatment groups and cohorts.

The effect of DSM265 on parasite density was evaluated for participants who required treatment before day 28 (6 placebo recipients and 12 DSM265 recipients). Owing to irregularly spaced study visits, there was some interval variation over time and among participants between qRT-PCR measurements. Upon reaching ≥250 parasites/mL, DSM265 recipients tended to have larger intervals since the last qRT-PCR measurement, as a consequence of less frequent visits later in follow-up, when these participants were treated (Supplementary Figure 6). Without adjustment for the differences in interval between subjects, there was a trend toward a higher mean parasite density among DSM265 recipients as compared to placebo recipients (P = .11; Supplementary Figure 7), driven by differences in cohorts 1 and 2b but not cohort 2a (Supplementary Figure 8). However, after adjustment for differences in intervals, the effect was attenuated (P = .25; Supplementary Figure 7).

All participants were treated with a therapeutic course of atovaquone-proguanil after reaching ≥250 parasites/mL by RT-PCR. In symptomatic participants, all symptoms dissipated without sequelae. One participant in cohort 1 developed erythrocyte-stage parasite infection on day 21, was treated, and unexpectedly developed recurrent Plasmodium 18S rRNA RT-PCR positivity without symptoms on day 33. For this participant, gametocyte-specific pfs25 and pfs0630000 messenger RNAs were detected in blood during days 33–54, as previously reported [42]. The subject tested negative for glucose-6-phosphate dehydrogenase deficiency, was treated with primaquine (45 mg orally once) on day 54, and became negative for Plasmodium 18S rRNA and gametocyte messenger RNAs thereafter.

AEs are summarized in Supplementary Tables 1–7. There were no deaths, SAEs, or clinically relevant changes in vital signs or QTc prolongations (≥30 msec vs baseline or an absolute value of >450 msec). Seventy-two total AEs were reported, with most related to CHMI procedures or malaria symptoms (23 and 22 AEs, respectively). Only 14 AEs were related to test drugs (DSM265 and placebo), 9 of which were reported after DSM265 administration. The most common AE possibly related to DSM265 was mild-to-moderate headache (6 AEs in 5 subjects), while the 3 other AEs consisted of mild-to-moderate gastrointestinal events (ie, vomiting, abdominal discomfort, and gas). AEs considered possibly or definitely related to challenge administration primarily consisted of mild-to-moderate DVI injection site pain or bruising in cohorts 1 and 2a and expected mosquito bite site reactions (ie, redness, itching, swelling, and soreness) in cohort 2b. Notably, 1 cohort 2b subject showed severe bite-site itching on the day of challenge, with a resurgence 26 days after challenge. Some participants with peripheral parasitemia experienced mild-to-moderate malaria-related symptoms (22 AEs in 9 subjects), including headache, backache, muscle pain, cold/shivering, and nausea; there were no severe malaria-related symptoms. AEs after treatment with atovaquone-proguanil (13 AEs in 8 subjects) were all mild to moderate and consisted of nausea, headache, diarrhea, and anorexia. Six additional subjects developed transient neutropenias (defined as ≤1000 neutrophils/µL; nadir, 640–1000 neutrophils/µL) during treatment with atovaquone-proguanil (on days 14–26) that resolved within 1 week. The incidence of transient neutropenia after atovaquone-proguanil treatment was 17% (1 of 6) and 28% (5 of 18) for subjects treated with placebo and those treated with DSM265, respectively. Severe (grade 3) AEs in this study consisted of abnormal laboratory findings, including neutropenia (in 2 of 6 subjects), hypercholesterolemia (2 events in 1 subject), isolated elevated alanine aminotransferase levels (6.3 times the upper limit of normal) and aspartate aminotransferase (3.4 times the upper limit of normal) levels reported in the same participant on day 28, as well as bite site itching (in 1 subject) and a thumb sprain (in 1 subject); none were determined to possibly be related to DSM265 or placebo. There were no additional DSM265- or malaria-related AEs reported at 3- or 6-month follow-up. Full AE line listings can be obtained from the authors upon request. The data indicate that DSM265 was clinically well tolerated. Further analyses will be conducted to understand reports of transient neutropenia during treatment with atovaquone-proguanil.

For PK analyses, plasma samples and blood samples for dried blood spot analysis were collected. Combining data across DSM265 recipients in all cohorts, the mean plasma DSM265 AUC_0-240 was 1242 µg*h/mL, the mean C_max was 11.2 µg/mL, the mean t_max was 14.3 hours, and the mean t_1/2 was 108.6 hours (Figure 4, Table 2, and Supplementary Figures 9–12). There was no apparent association between the overall PK profile for a participant and protection (Figure 5). Summary measures of PK curves, including the DSM265 concentration at the time of challenge (Supplementary Figure 13) and the DSM265 t_1/2 (Supplementary Figure 14), were not predictive of efficacy (P > .48). A previously conducted CHMI study reported a longer t_1/2 of DSM265 in protected versus unprotected subjects [35], which was not replicated in this study.

Figure 4.

Pharmacokinetic assessment of DSM265 plasma concentrations. Individual subject plasma concentrations of DSM265 (A) or metabolite DSM450 (B). Solid lines indicate participants who were not protected and were eventually treated with atovaquone-proguanil, whereas dashed lines indicate sterilely protected participants.

Table 2.

DSM265 Pharmacokinetic Parameters Summarizing Plasma Time-Concentration Curves, Overall and by Cohort

Parameter	Overall	Cohort 1	Cohort 2a	Cohort 2b
AUC0–∞, µg*h/mL	1706 ± 939	1528 ± 630	2463 ± 1148	1126 ± 390
AUC0–240, µg*h/mL	1242 ± 510	1244 ± 427	1661 ± 408	820 ± 335
Cmax, µg/mL	11.2 ± 5.9	12.4 ± 4.6	14.6 ± 6.8	6.5 ± 3.1
t1/2, h	108.6 ± 30.7	89.3 ± 21.6	121.1 ± 33.6	115.5 ± 30.2
tmax, h	14.3 (1–72)	6.5 (1–24)	15.0 (2–72)	21.5 (1–72)

Data are mean ± SD or mean (range). Linear noncompartmental models were fit to individual pharmacokinetic profiles and used to estimate the AUC_0–∞ and t_1/2. The AUC_0-240, C_max, and t_max were estimated empirically.

Abbreviations: AUC_0–240, area under the curve, through 240 hours; AUC_0–∞, area under the curve, extrapolated to infinity; C_max, maximum concentration; t_max, time of maximum concentration; t_1/2, half-life.

Figure 5.

Parasite densities measured by quantitative reverse transcription–polymerase chain reaction for participants in each of 3 cohorts. The bracket in cohort 1 indicates 1 participant with evident gametocytemia after treatment as described in the text. ND, not detected.

DISCUSSION

DSM265 is an antimalarial compound in development for malaria treatment [34] and chemoprophylaxis [35]. Here, a single 400-mg dose of DSM265 was well tolerated with headache being the most common AE. This new PfDHODH inhibitor demonstrated partial protection (33%) against preerythrocytic Plasmodium infection when given 3–7 days before sporozoite challenge by DVI or mosquito bites in malaria-naive, healthy adults. The presence of 2 completely protected individuals in each cohort indicates that DSM265 most likely completely arrests preerythrocytic parasite development in the liver for at least a portion of individuals. However, it is also possible that a small number of erythrocyte-stage parasites were released in qRT-PCR–negative individuals, only to be killed by residual DSM265 erythrocytic activity. Since 2 completely protected individuals were found for cohorts dosed at 3 or 7 days before challenge, we favor the conclusion that DSM265 completely blocked liver-stage parasite development in qRT-PCR–negative persons. This hypothesis is further supported by in vitro and liver-chimeric humanized mouse studies, where DSM265 showed preerythrocytic liver stage activity ([32] and Flannery et al, unpublished data). The overall protection in our study was lower than expected, based on prior modeling studies, and our findings call into question whether the DSM265 dosing regimen can consistently achieve complete protection.

Our findings extend those of another recently completed study [35]. In that study, DSM265 was dosed 1 day before DVI challenge and protected all participants (TBS negative/qPCR negative), but it was only partially protective when dosed 7 days before challenge (3 of 6 participants became TBS positive/qPCR positive, with the remaining 3 intermittently qPCR positive) [35]. We report here that 2 of 6 participants dosed 7 days before challenge were completely protected (qRT-PCR negative). This difference could be indicative of residual erythrocyte-stage parasite activity of DSM265 in some participants in the study by Sulyok et al and/or of differences in molecular test performance. The limit of detection for the qPCR used in the study by Sulyok et al was reported to be lower than that of the qRT-PCR used here, which may explain some of the difference in outcomes defined by molecular methods.

This phase 1 study has some limitations. First, the number of participants is relatively small (6 drug-treated individuals and 2 placebo-recipient controls per cohort). This design is suitable for detecting complete protection and substantial delays in the time to infection, but it is not well powered to quantify partial protection. The study is also limited by the same problems highlighted by the first DSM265 study: reliance on a single laboratory-adapted strain of P. falciparum for CHMI and possibly a challenge dose that is higher than that seen in natural exposures [35].

This is the first chemoprophylaxis study to directly compare treatment of CHMI induced by DVI to that induced by mosquito bite. Cohort sizes were small, and as such there were no statistically significant differences in outcomes for placebo- or DSM265-treated participants, regardless of route. However, the first positive parasite densities in placebo-recipient controls with CHMI due to mosquito bite were higher (approximately 616 and 1074 parasites/mL on day 7) than those after the first positive post-DVI densities (approximately 32, 44, 66, and 90 estimated parasites/mL on days 7 or 8), which suggests that the inoculum delivered by mosquitoes (or its infectivity) may be higher than that we delivered by DVI. This was not unexpected, given that infected mosquitoes may contain >10000 viable sporozoites each.

The qRT-PCR treatment threshold accelerated the time to treatment and reduced malaria-related symptoms as compared to TBS-based studies. The DSM265 study by Sulyok et al [35] initiated treatment in response to TBS positivity (corresponding to ≥5000–10000 parasites/mL), whereas our DSM265 study initiated treatment at a qRT-PCR–defined threshold of 250 estimated parasites/mL. Cohort 2 in the Sulyok et al study was identical to cohort 2a in this study in design and number of subjects. In cohort 2 of the study by Sulyok et al, there were 60 AEs attributed to malaria, including several grade 3 fevers. In contrast, there were only 22 malaria-related AEs and no malaria-related grade 3 AEs in cohort 2a (Supplementary Table 6). DSM265 recipients dosed 7 days before CHMI via DVI became TBS positive at a geometric mean of 15.1 days in the previous study [35] as compared to 13.8 days (range, 12.3–15.4 days) to qRT-PCR detection of ≥250 parasites/mL in our study. Most subjects were treated the following morning in our study (geometric mean time to treatment, 15.2 days), and earlier treatment reduced AEs as compared to TBS-triggered treatment. These findings indicate that molecular end points accelerate infection detection and reduce malaria-related symptoms, compared with TBS.

In the future, higher or multidose DSM265 prophylactic regimens could be tested to determine whether either prevent emergence of erythrocyte-stage parasites. Because single 400-mg DSM265 dosing at 3 or 7 days before challenge provided incomplete protection, drug concentrations likely need to exceed those observed here. Repeated dosing studies could be investigated to further evaluate DSM265-based prophylactic regimens against development of liver-stage parasites. Future studies could add candidate partner drugs for combination therapy and/or prophylaxis.

In conclusion, our study demonstrated that 400 mg of oral DSM265 is safe and well tolerated but confers only partial protection against preerythrocytic infection induced by either DVI or mosquito bite.

Supplementary Data

Supplementary materials are available at The Journal of Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.

Notes

Acknowledgments. We thank the clinical site staff (Claudia Kumai, Tara Ohrazda, Ann Bradshaw, Michaelo Magsipoc, and Angela Modelski), laboratory staff (Glenda Daza and Jose Ortega), and the medical monitor (Stephen Toovey); Lindsay Carpp, for graphic design assistance; Sanaria, for providing cryopreserved sporozoites for DVI challenges; and the insectary staff at CID Research, for rearing infected mosquitoes for the mosquito bite challenge.

Disclaimer. Opinions, interpretations, conclusions, and recommendations are those of the authors and are not necessarily endorsed by the Department of Defense.

Financial support. This work was supported by the Department of Defense Peer-Reviewed Medical Research Program (grant W81XWH-15-2-0010 to the clinical trial), Medicines for Malaria Venture (support to the clinical trial), Takeda Pharmaceuticals (support to the clinical trial), and the National Institutes of Health (grants AI-38858 and P30 AI027757 to the qRT-PCR laboratory).

Potential conflicts of interest. J. G. K. reports receiving grants from the Department of Defense Peer Reviewed Medical Research Program, the Bill and Melinda Gates Foundation (BMGF), and the National Institutes of Health (NIH) during the conduct of the study. S. C. M. received grants from the BMGF and the NIH during this study. T. R., S. C., S. D., N. K., J. J. M., and N. A. are or were employed by MMV. All other authors report no potential conflicts.

Presented in part: Annual Meeting of the American Society of Tropical Medicine and Hygiene, Atlanta, GA, November 16, 2016 (poster 1534); Annual Meeting of the American Society of Tropical Medicine and Hygiene, Baltimore, MD, November 6, 2017 (presentation 11).