Brian G. Schuster

Medicinal plants in the fight against leishmaniasis

Despite the tremendous progress mode in the understanding o f the molecular biology of Leishmania and the clinical possibilities presented by some experimental chemotherapeutic agents, no new drugs have been developed for the treatment of leishmaniasis since the introduction of the pentovalent antimoniols more than 50 years ago. As reviewed here by Maurice M. Iwu, Joan E. Jackson and Brion G. Schuster, recognition of the current extensive use of herbal therapy in Leishmania-endemic regions has renewed interest in evaluation of plant remedies used in traditional medicine as sources of potential antileishmanials.

Mechanism of cardiotoxicity of halofantrine

To further evaluate the scope and mechanism of potential cardiotoxicity associated with the antimalarial drug halofantrine, case reports submitted to the US Food and Drug Administration Spontaneous Reporting System were examined. Because halofantrine was associated with electrocardiographic prolongation of the QT interval and ventricular arrhythmias, in vitro cardiac electrophysiologic studies (isolated perfused cardiac model and isolated ventricular myocytes) were conducted to test the hypothesis that halofantrine or its metabolite is responsible for cardiotoxicity.

Randomized Dose-Ranging Study of the Safety and Efficacy of WR 238605 (Tafenoquine) in the Prevention of Relapse of Plasmodium vivax Malaria in Thailand

WR 238605 is an 8-aminoquinoline developed for the radical cure of Plasmodium vivax. Forty-four P. vivax-infected patients were randomly assigned to 1 of 4 treatment regimens: 3 groups received a blood schizonticidal dose of chloroquine followed by WR 238605: group A (n=15) received 300 mg daily for 7 days; group B (n=11), 500 mg daily for 3 days, repeated 1 week after the initial dose; group C (n=9), 1 dose of 500 mg. A fourth group (D; n=9) received chloroquine only. Among patients who completed 2-6 months of follow-up (n=23), there was 1 relapse in group B (day 120) and 1 in group C (day 112). Among patients treated with chloroquine only, there were 4 relapses (days 40, 43, 49, and 84). WR 238605 was safe, well tolerated, and effective in preventing P. vivax relapse.

Prophylaxis of Plasmodium falciparum Malaria with Azithromycin Administered to Volunteers

New effective and well-tolerated prophylactic agents against P. falciparum malaria need to be developed. Chloroquine and proguanil are now relatively ineffective [1, 2]. Although the efficacy of mefloquine is approximately 90% [1], this rate may decline as resistance spreads [3], and mefloquine is currently not recommended for pregnant women, infants weighing less than 15 kg, and persons with neuropsychiatric disorders [2]. Sulfadoxine-pyrimethamine (Fansidar, Roche, Nutley, New Jersey) is no longer recommended for prophylaxis [2] because of the possibility of fatal toxic epidermal necrolysis. Doxycycline, 100 mg/d, is approximately 90% effective [4], but this agent is contraindicated in pregnant women and children younger than 8 years old and has gastrointestinal side effects. Plasmodium falciparum initially infects the liver, and, after approximately 7 days, parasites begin to emerge from the liver to parasitize red cells. A prophylactic agent against the liver stage of infection (causal prophylactic agent) needs to be administered for only approximately 7 days or less after infection, whereas an agent effective solely against the blood stage of the infection (suppressive prophylactic agent) needs to be administered for several weeks after infection. Mefloquine and doxycycline are insufficiently effective against liver-stage parasites, and patients require dosing for 4 weeks after leaving the endemic area. Azithromycin is an azalide analog of erythromycin that was recently licensed for the treatment of bacterial and chlamydial diseases. In a rodent model, we found that azithromycin has a causal prophylactic efficacy superior to that of doxycycline [5]. In addition, we did a study in which volunteers were exposed to the bites of P. falciparum-infected mosquitoes and given azithromycin for as long as 5 days after exposure to the bites. Azithromycin, 500 mg administered 2 days before exposure to the bites followed by 250 mg/d administered for 5 days after exposure to the bites, was successful prophylaxis for three of four persons [6]. Because the previous clinical study involved only 4 drug recipients, we did a new study of causal prophylaxis using 10 drug recipients. Because we obtained results showing unexpectedly poor efficacy, we subsequently entered another cohort of 10 drug recipients to determine the combined causal efficacy as well as the suppressive efficacy of azithromycin against P. falciparum malaria in the challenge model. Methods Study Design We did an open-label, controlled phase II study in which two cohorts were entered sequentially. In cohort 1, we evaluated the causal prophylactic efficacy of azithromycin. Ten persons received 500 mg of the drug on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 7 after the challenge. Dosing from day 14 before the challenge to day 0 was intended to allow serum concentrations to reach a steady state; dosing from days 1 to 7 after the challenge was intended to provide causal prophylaxis. In the 12 preceding experiments at our clinical research center, all of approximately 40 control volunteers inoculated with this strain of P. falciparum became infected. In our present study, two controls did not receive azithromycin and were concomitantly infected to verify that the parasitologic challenge was infectious. In cohort 2, we evaluated the combined causal and suppressive prophylactic efficacy of azithromycin. Ten persons received 500 mg of the agent on day 14 before the challenge, followed by 250 mg/d from day 13 before the challenge through day 28 after the challenge. Dosing from days 8 through 28 was intended to provide suppressive prophylaxis. Two controls were concomitantly challenged. Inclusion and Exclusion Criteria Men and women aged 18 through 46 years were eligible if they were healthy on the basis of history, physical examination, and results of laboratory tests (complete blood count; serum levels of sodium, potassium, chloride, urea nitrogen, creatinine, calcium phosphate, total protein, albumin, aspartate aminotransferase, alkaline phosphatase, total bilirubin, lactate dehydrogenase, and uric acid) and had no history of exposure to malaria in the previous 2 years. Persons who did not object to the high likelihood of becoming parasitemic were permitted to be controls. Study Procedures For each cohort of 12 volunteers, all persons were infected [7] by exposure to the bites of five female Anopheles stephensi mosquitoes infected with the chloroquine-sensitive, mefloquine-resistant NF54 strain of P. falciparum. During drug administration, adverse reactions and symptoms of malaria were assessed by means of daily interviews for subjective symptoms (chills, headache, photophobia, back pain, muscle ache, stomach ache, anorexia, nausea, vomiting, diarrhea, vaginal itching), by daily recording of body temperature, and by repetition of the entrance laboratory tests on day 4 before the challenge and day 5 after the challenge. To make the definitive diagnosis of malaria, on days 5 through 28 (for cohort 1) or on days 5 through 35 (for cohort 2), thick blood smears were obtained, stained with Giemsa, and examined for P. falciparum by an investigator blinded to whether or not the study participant had received azithromycin. Positive results were confirmed by two other investigators. In addition, all persons not already diagnosed as having malaria were instructed to contact study personnel until day 70 if they had symptoms that suggested malaria. In each thick smear, 200 fields were examined. Patent malaria was defined as the presence of at least two parasites per smear. If the volunteer had patent parasitemia, he or she was treated with chloroquine (1500-mg base given over 3 days). Treatment Agents Azithromycin in the form of 250-mg tablets was provided by Pfizer Central Research. Groton, Connecticut. Chloroquine in the form of Aralen Phosphate tablets was purchased from Winthrop Pharmaceuticals. New York, New York. Protocol Approval Our protocol was approved by the Office of the Surgeon General of the United States Army and filed under IND with the U.S. Food and Drug Administration. All volunteers gave written informed consent. Statistical Methods Computation of exact CIs of a proportion was done using the binomial distribution. Results Prophylactic Efficacy of Azithromycin Cohort 1 consisted of 10 volunteers who received azithromycin until 7 days after challenge with P. falciparum sporozoites and 2 volunteers who were challenged with sporozoites but received no treatment. Both of the controls who received no treatment and 6 of the 10 volunteers who received treatment with the drug developed parasitemia. One participant was removed from the study on day 25 because of noncompliance with follow-up. He was not patent (parasitemic) on that day, and no other participant became patent between day 23 and the end of the study on day 70. We consider this participant to be a prophylactic success. All but one of the persons who received treatment with the drug and in whom prophylaxis failed had delayed patency (mean day of patency, 19 days; range, 11 to 23 days) compared with the controls (mean day of patency, 10 days; range, 9 to 11 days). In cohort 1, the efficacy of treatment was 40% (95% CI, 12% to 74%). Cohort 2 consisted of 10 persons who received azithromycin until 28 days after exposure to sporozoites and 2 controls who received no treatment. Both controls became patent (on days 11 and 13); none of the participants who received drug treatment became patent. In cohort 2, the efficacy of azithromycin was 100% (lower 95% CI, 70%). Of the 10 participants in both cohorts who became parasitemic, only 3 had temperatures greater than 37.7 C, and 7 had symptoms characterized by chills, headache, myalgia, and anorexia. No volunteer required hospitalization. Adverse Effects of Azithromycin Although each day during the period of drug administration, study participants were interviewed for subjective side effects, we found only four instances of headache, four instances of stomach ache, and one instance of diarrhea not temporally linked to parasitemia. Each of these events was mild and lasted at most 1 day. In addition, one patient developed flulike symptoms 2 days after receiving the last dose of the drug. Neither the one woman in cohort 1 nor the three women in cohort 2 reported symptoms of vaginitis. The only abnormality in laboratory variables was total bilirubin values somewhat higher than the upper limit of normal (24 mol/L) in 3 participants. In 1 volunteer, the value on day 4 before the challenge (37.6 mol/L) had returned to normal by day 5 after the challenge (15.4 mol/L) while the participant was still receiving the drug. In 1 participant, the values were relatively constant during therapy (27.4 mol/L on day 4 before the challenge and 34.2 mol/L on day 5 after the challenge) and then returned to normal after therapy. In the third participant, a value of 30.8 mol/L was first recorded on day 5 and levels returned to normal after therapy. Discussion A causal and suppressive prophylactic regimen of azithromycin, 250 mg/d for 28 days after a single challenge with P. falciparum sporozoites, was successful in 10 of 10 persons studied. In contrast, a causal prophylactic regimen of azithromycin, 250 mg/d for 7 days after challenge, was successful in only 4 of 10 persons. We had hoped that azithromycin would be sufficiently concentrated in the liver to be completely causally prophylactic, because it has been reported that azithromycin liver concentrations are approximately 10 times that of serum concentrations [8]. The results of treatment for 7 days after the challenge show that, as with doxycycline [9], azithromycin has only partial causal prophylactic activity in the human challenge model. The mean percent efficacy determined here (40% [CI, 12% to 74%]) is much less than that recently achieved by Kuschner and colleagues [6] in a tria

Successful topical treatment of murine cutaneous leishmaniasis with a combination of paromomycin (aminosidine) and gentamicin

Cutaneous leishmaniasis is presently treated with 20 days of parenteral therapy with a frequently toxic drug (antimony). Topical formulations of paromomycin (15%) plus methylbenzethonium chloride (MBCL, 12%) or plus urea (10%) in soft white paraffin have been tested for Old and New World disease in humans. We compared the efficacy of a new topical formulation, WR 279,396 (paromomycin [15%] plus gentamicin [0.5%]) to the clinical formulations in the treatment of cutaneous disease in BALB/c mice. Sixty-day-old lesions were treated twice a day for 10 days, and the response to therapy was determined over a further 70 days. For ulcers due to Leishmania major or to Leishmania mexicana, 100% of lesions in the WR 279,396 group healed by day 20 after therapy and did not relapse by day 70; 83% of lesions healed without relapse in the paromomycin-MBCL group. In the paromomycin-urea group, 100% of L. major lesions healed by day 30 but 30% relapsed. For ulcers due to Leishmania panamensis or Leishmania amazonensis, all lesions treated with WR 279,396 healed and did not relapse; < 50% of lesions treated with paromomycin-MBCL healed by day 30, and all lesions relapsed by day 70. In addition to being active, WR 279,396 was not toxic in this model and appears to have a cosmetic effect (promoting hair growth, healing, and limiting the size of the scar).

Population pharmacokinetics and pharmacodynamics of pyridostigmine bromide for prophylaxis against nerve agents in humans

This study was conducted to determine the pharmacokinetics and pharmacodynamics of pyridostigmine given as 30 mg of pyridostigmine bromide every 8 hours in healthy subjects. Plasma pyridostigmine concentration and red blood cell acetylcholinesterase activity were measured in blood samples collected during a 3‐week period. Population analysis was performed using standard pharmacokinetic and pharmacodynamic models with the nonlinear mixed‐effect modeling software (NONMEM). The pharmacokinetic model that best fit the pyridostigmine plasma levels was a two‐compartment open model with first‐order absorption, a lag time, and first‐order elimination from the central compartment. The pharmacodynamic model that best fit red blood cell acetylcholinesterase activity was an inhibitory Emax model with an effect compartment linked to the central compartment. The results showed that the pharmacokinetics of pyridostigmine bromide are both gender and weight dependent. The pharmacodynamic effect does not lag significantly from the plasma concentration and returns to near normal within 8 hours. With the present dosage regimen of 30 mg every 8 hours, 30% of individuals may not have red blood cell acetylcholinesterase inhibition >10% at the time of the trough.

Failure of Doxycycline as a Causal Prophylactic Agent against Plasmodium falciparum Malaria in Healthy Nonimmune Volunteers

With the growth of international travel, increasing numbers of nonimmune travelers may be at risk for exposure to Plasmodium falciparum malaria [1-3]. Since the emergence and spread of multidrug-resistant P. falciparum parasites [4, 5], the choice of drug to prevent malarial infections has become problematic [6, 7]. In most areas endemic for malaria, chloroquine is no longer the drug of choice. Chemoprophylaxis with Fansidar (Roche Laboratories; Nutley, New Jersey) has been discouraged in recent years after reports of severe life-threatening side effects [8] and the emergence of parasite resistance to this combination drug [9, 10]. The Centers for Disease Control and Prevention (CDC) list of alternative preventive agents for travelers to areas with chloroquine-resistant P. falciparum malaria includes mefloquine, doxycycline, or the combination of proguanil plus chloroquine [11]. Mefloquine is currently the drug of choice. However, reports of increasing parasite resistance to this drug [12-14] and possible mefloquine-associated neuropsychiatric side effects [15-17] may limit its usefulness as a prophylactic agent. Proguanil, although ineffective alone, is widely used overseas in combination with chloroquine. It is not, however, commercially available in the United States. Doxycycline is recommended for prevention of malaria in persons traveling for short periods to areas with chloroquine-resistant P. falciparum malaria who cannot tolerate mefloquine or for whom the drug is contra-indicated [11]. In addition, travelers to areas where mefloquine resistance has been identified, such as the Thai-Burmese border area, may be particularly good candidates for doxycycline prophylactic therapy. Current recommendations advise starting the drug 1 to 2 days before travel and taking it daily throughout the period of potential exposure to infected mosquitoes and for 4 weeks after returning from an endemic area. The terminal use of doxycycline for 4 weeks after possible exposure is intended to eliminate any erythrocytic parasites. This use is based on doxycyclines presumed activity as a suppressive prophylactic agent, that is, an agent effective against blood stages of the parasite. Continued daily drug ingestion for 4 weeks in the absence of symptoms, however, requires well-disciplined compliance and is expensive. It is important, therefore, to ensure that the use of doxycycline for 4 weeks after potential exposure has ceased is necessary. A drug that is active against the pre-erythrocytic liver stage, thereby preventing erythrocytic malaria, is called a causal prophylactic drug. If it is effective against P. falciparum parasites, such a drug prevents symptomatic malaria and needs to be taken only during the hepatic phase of infection, normally about 1 week from the time of mosquito bite. Studies in the early 1970s [18, 19] suggested that tetracycline compounds were effective as causal prophylactic agents. Minocycline, a semi-synthetic, long-acting tetracycline (T1/2, 18 h 4 h) (mean SD), when administered as 100 mg daily for 7 days, starting 1 day before exposure to P. falciparum parasites, prevented parasitemia in four of four persons [18]. Two other persons receiving 100 mg on the day of mosquito challenge and on day 3 after challenge were also protected. Doxycycline, another long-acting tetracycline analog (T1/2, 16 h 6 h) [20], has been shown in field trials to have prophylactic activity [21, 22]. Whether its mechanism of action was causal or suppressive, however, could not be determined. We designed our study to determine whether oral doxycycline, 100 mg administered daily, is effective as a causal prophylactic agent against P. falciparum malaria. If so, it would justify shortening the duration of terminal prophylactic therapy from 4 weeks to 1 week. Methods Volunteers were recruited under a protocol approved by the Human Use Review Committee, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, and the Surgeon Generals Human Subjects Research Review Board of the Department of the Army. Eighteen healthy men (age, 21.7 2.9 [SD] years) were selected after giving written informed consent. The screening evaluation included a baseline medical history, physical examination, electrocardiogram, and chest roentgenogram. Exclusion criteria included a history of malaria; splenectomy; allergy to doxycycline, tetracycline, or chloroquine; abnormal results of a complete blood count or tests for aspartate aminotransferase, alanine aminotransferase, bilirubin, lactic dehydrogenase, alkaline phosphatase, blood urea nitrogen, or creatinine; hematuria or proteinuria; glucose-6-phosphate-dehydrogenase deficiency; antibody to hepatitis B surface antigen; or antibody to human immunodeficiency virus. Informed Consent Prospective volunteers received a briefing and a written information sheet describing the procedures and potential risks associated with the study. Those willing to participate signed a consent form. None of the volunteers received compensation. They were assured of their right to withdraw from the study at any time without penalties. The study was designed to minimize the number of volunteers that might develop malaria. The number enrolled was based on the presumption that doxycycline protection rate would exceed 90% when administered as a causal prophylactic agent. Such a level of efficacy would have justified, in our opinion, changing the current CDC recommendation for terminal prophylactic therapy. The number of volunteers assigned to receive placebo was the minimum needed to ensure infectivity of the mosquitoes. To minimize morbidity, all participants were closely monitored throughout the study. Monitoring was intensified during periods of greatest likelihood of emergence of parasitemia by hospitalizing the participants on the medical ward. Given the close monitoring and the early treatment when parasitemia was low, the risk to the participants was felt to be minimal. Study Design The study was a randomized, double-blind, placebo-controlled trial. Doxycycline, 100 mg daily, was administered orally, starting 3 days before and ending 6 days after exposure to P. falciparum-infected mosquitoes. The study was implemented in two phases, 2 months apart. In phase 1, eight persons (group 1) were randomly assigned to receive doxycycline (six persons) or placebo (two persons). In phase 2, ten persons (group 2) were randomly assigned to receive either doxycycline (seven persons) or placebo (three persons). The drug used was doxycycline hyclate (Vibramycin, Pfizer Inc.; New York, New York), which was given in 100-mg capsules; identical placebo capsules were prepared by the University of Iowa Pharmaceutical Services Division. After an overnight fast the participants were hospitalized on the medical ward for 24 hours. Within 30 minutes after breakfast, either a doxycycline or a placebo capsule was administered to each participant. Nine samples for plasma doxycycline level measurements were then collected from each person during the first 24 hours, with eight additional samples collected during the following 12 days. On the afternoon of day 4 of drug administration, each participant was exposed to Anopheles stephensi mosquitoes infected with chloroquine-sensitive P. falciparum malaria. Giemsa-stained malaria smears were prepared daily from day 5 to day 15 after exposure and at least once weekly for 2 months. Thick and thin malaria smears were examined by two of the investigators. When present, parasitemia was quantified on a thick smear by the method of Earle and Perez [23]. All volunteers were hospitalized during week 2 after exposure, the time of highest risk for developing malaria. Those who remained well were discharged on day 15 and followed as outpatients. Persons who became febrile during the outpatient period were rehospitalized and evaluated for malaria with twice-daily blood smears. If parasites were found, standard chloroquine therapy, 1500-mg base for 48 hours, was administered. The participants were discharged after their blood films were clear of parasites and their symptoms resolved for 3 days. Induction of Malaria Infection Anopheles stephensi mosquitoes were used to transmit NF54 strain P. falciparum parasites. Female mosquitoes, 4 to 7 days after emergence, were membrane-fed on a mixture of cultured gametocytes [24], defibrinated blood, and human serum negative for hepatitis B surface antigen and human immunodeficiency virus antibody. Thirty-five days and 21 days after membrane feeding, the mosquitoes inoculated the first and second group of participants, respectively. To increase the survival rate of the mosquitoes in the first colony to 35 days, the environmental temperature was decreased from room temperature to 20 C in the last 2 weeks before human exposure. Mosquitoes used to induce infection in the second group were given a second blood meal when they were found to have low oocyst counts on day 6 after membrane feeding. On day 4 of drug administration, 6 to 8 hours after the morning medication, a cage containing five A. stephensi mosquitoes was placed on the forearm of each volunteer for 5 minutes. Those mosquitoes ingesting a blood meal were dissected and the sporozoite density of the paired salivary glands was quantified on a log-based categorical scale of 0 to 4. Each volunteer was then exposed to additional mosquitoes, as necessary, until five mosquitoes with sporozoite densities of 2 or greater had successfully taken a blood meal. Previous experience in our laboratory using these procedures resulted in an infection rate of 100% [25-27]. Doxycycline Plasma Concentration Seventeen plasma samples were collected for pharmacokinetic analysis: a sample before dosing and at 1, 2.5, 3, 4, 6, 9, 12, 24, 72, 75, 168, 215, 218, 240, 264, and 288 hours after the first dose. Three of the samples were collected at times corresponding to steady-state trough levels. The last three sam

Antimalarial activity of WR 243251, a Dihydroacridinedione.

WR 243251 is a dihydroacridinedione that was evaluated for antimalarial blood schizonticidal activity in vitro and in vivo. The in vitro doses calculated to kill 50% of organisms were 11 nM for a chloroquine-susceptible, mefloquine-resistant standard strain and 25 nM for a chloroquine- and pyrimethamine-resistant standard strain. The total dose needed to cure 100% of mice infected with a drug-susceptible strain of Plasmodium berghei was 12 to 20 mg/kg of body weight for both oral and subcutaneous administration. The regimen needed to cure 100% of Aotus monkeys infected with Plasmodium falciparum was 8 mg/kg/day for 3 days (chloroquine-susceptible strain) and 16 mg/kg/day for 3 days (chloroquine-resistant strain). The 100% curative doses for Aotus monkeys did not increase for parasites previously exposed to subcurative doses. The absolute value of the curative doses of WR 243251 was comparable to or lower than the values for clinical antimalarial agents. The high absolute activity, comparability of activities against susceptible and resistant parasites, and inability to induce resistance by exposure to subcurative doses suggest that WR 243251 has strong potential as a blood schizonticidal agent.

Pharmacokinetics of an extended‐dose halofantrine regimen in patients with malaria and in healthy volunteers

The pharmacokinetics and tolerance of a 4.5 gm 7‐day halofantrine loading dose regimen were evaluated in 10 Thai patients with malaria and in 10 noninfected volunteers. Halofantrine peak plasma concentrations and bioavailability on the first day of treatment were significantly lower in patients with malaria than in healthy volunteers. Halofantrine elimination half‐life was significantly shorter in patients with malaria than healthy control subjects (9.5 versus 15.8 days). These data show a distinct effect of acute malaria on the absorption and elimination of the drug. In addition, marked intersubject and intrasubject variability in peak and trough halofantrine levels was observed, indicating variable drug absorption. This dosing regimen was effective and well tolerated, with mild transient diarrhea during the first few days of treatment in both groups. To produce consistently effective drug levels, the currently recommended dosing regimens may be suboptimal. Slow halofantrine elimination raises concern for induction of parasite resistance when the drug is used in endemic areas of the world.

Preclinical studies with halofantrine

Summary Habfantrine is one of a series of phenanthrene methanols investigated by WRAIR for us antimalarial activity. It has been shown to be highly active in vitro and to cure malaria in animal models. Its putative metabolite desbutyl halofantrine was shown to be equipotent against chloroquine-resistant and -sensitive P. falciparum strains. At present there is little evidence of cross-resistance between it and other antimalarials in human malaria species. Halofantrine is absorbed to a varying degree by animal species and is widely distributed in tissues. Elimination half-lives vary considerably among species with most of the drug apparently eliminated in the faeces. Significant changes in physiological systems were not observed. Toxicological studies, both acute and sub-acute (28 days), showed effects on a number of target organs at higher doses. No-effect doses were determined to be several times greater than expected human doses considering the systemic exposure to halofantrine. Reproductive studies showed embryotoxic but not teratogenic potential at high doses, and halofantrine was apparently excreted in breast milk. Mutagenicity studies were negative. Halofantrine in preclinical studies therefore appears to be an active antimalarial, with a satisfactory toxicological profile.