Review. Development of novel drugs for human African trypanosomiasis. Future Microbiology

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1 For reprint orders, please contact: Development of novel drugs for human African trypanosomiasis Reto Brun, Robert Don 1, Robert T Jacobs 2, Michael Zhuo Wang 3 & Michael P Barrett 4 1 Drugs for eglected Diseases initiative, Geneva, Switzerland 2 Department of Chemistry, SCYEXIS, Inc., P Box 12878, Research Triangle Park, C, , USA 3 Eshelman School of Pharmacy, University of orth Carolina, Chapel Hill, C, USA 4 Wellcome Trust Centre for Molecular Parasitology, University of Glasgow, Glasgow, Scotland Author for correspondence: Department Medical Parasitology & Infection Biology, Swiss Tropical & Public Health Institute, and, University of Basel, CH-4002 Basel, Switzerland Tel.: Fax: reto.brun@unibas.ch Human African trypanosomiasis (HAT) or sleeping sickness is a neglected tropical disease caused by the parasite Trypanosoma brucei. ovel models for funding pharmaceutical development against HAT are beginning to yield results. The Drugs for eglected Diseases initiative (DDi) rediscovered a nitroimidazole, fexinidazole, which is currently in Phase I clinical trials. ovel benzoxaboroles, discovered by Anacor, Scynexis and DDi, have good pharmacokinetic properties in plasma and in the brain and are curative in a murine model of stage two HAT with brain infection. The Consortium for Parasitic Drug Development (CPDD) has identified a series of dicationic compounds that can cure a monkey model of stage two HAT. With other screening programs yielding hits, the pipeline for new HAT drugs might finally begin to fill. Future Microbiology HAT disease Human African trypanosomiasis (HAT) is caused by subspecies of the protozoan parasite Trypanosoma brucei spp. [1], which belong to the taxonomic order Kinetoplastida, named for the presence of a densely intercatenated network of circular DA that comprises their mitochondrial genome. The biochemical physiology of these parasites is of great interest, particularly from the perspective of chemotherapy where there has long been a view that the peculiar aspects of biochemistry in these organisms may be exploited as specific drug targets [2]. The parasites are transmitted by tsetse flies of the genus Glossina. HAT has two defined stages. The first stage involves trypanosomes in the hemolymphatic system. At this point symptoms are rather nonspecific, including general malaise, fever and headache [1]. The second stage begins once parasites cross into the brain and provoke neurological function breakdown, including psychological changes and also the disruptions to sleep/wake patterns, which has led to the common name of sleeping sickness being used for the disease [3]. Diagnosis of the disease involves a primary serological screen using the card agglutination test for trypanosomiasis (CATT), which targets antibodies produced against one of the trypanosome s surface antigen types. However, variable sensitivity of this test means that positive microscopical identification of parasites in lymph or in blood is usually required. Since the disease is treated using different drugs, depending on whether the infection is at stage one or two, it is also necessary to perform a lumbar puncture, where the presence of parasites or else white cells (usually using a cut off of five per microliter) leads to diagnosis of stage two disease. There are two forms of HAT. The form prevalent in Central and West Africa is caused by the sub-species T. b. gambiense. Generally speaking the parasitemia associated with gambiense HAT remains low and a chronic infection is established. It takes on average 2 years before the parasites establish within the CS and a further 2 years of progressive deterioration during stage two disease prior to death [4]. T. b. rhodesiense is found in Eastern and Southern Africa and normally causes a more acute disease, lasting just several weeks to months before death. ther trypanosome species are vulnerable to a trypanosome lytic factor comprising apolipoprotein L1 (apol1) [5] in the context of high density lipoprotein where a haptoglobin-like protein (hpr) is also critical in ensuring uptake. Rhodesiense parasites avoid lysis through expression of a serum resistance associated (sra) gene that neutralizes the lytic activity of apol1 [5]. Gambiense parasites do not contain the sra gene. However, it has recently been proposed that these parasites show reduced expression levels of the hpr receptor that allows incorporation of the lytic molecule explaining how these parasites resist lysis [6]. Keywords aromatic diamidines benzoxaboroles disease elimination drug targets fexinidazole human African trypanosomiasis novel drugs part of /FMB Reto Brun, Robert Don, Robert T Jacobs, Michael Zhuo Wang & Michael P Barrett Future Microbiol. (2011) 6(6), ISS

2 Brun, Don, Jacobs, Wang & Barrett Countries reporting no cases Countries reporting under 100 cases Countries reporting between 100 and 1000 cases Countries reporting more than 1000 cases A process of antigenic variation [7] allows the trypanosome to elude the adaptive immune response, and this process has so far precluded vaccines against HAT meaning chemotherapy remains the only option in disease intervention in infected individuals. In this article we aim to describe work over the last few years that has seen the emergence of several compounds active against murine models of stage two disease and thus reaching a position where entry into clinical trials is ongoing or planned. HAT epidemiology The disease is restricted to sub-saharan Africa and overlaps with the distribution of the tsetse flies that act as vector. ver 95% of the cases are attributable to T. b. gambiense and less than 5% to T. b. rhodesiense (Figure 1). During the last 120 years three major epidemics occurred [8]. The most devastating one killed an estimated 800,000 Africans around the turn from the 19th to the 20th century. Between 1920 and 1940 another major epidemic took place and prompted the colonial forces to intensify vector control and surveillance of affected communities. These measures led to a reduction of HAT T. b. gambiense T. b. rhodesiense Figure 1. Distribution of human African trypanosomiasis. The black line divides the areas where Trypanosoma brucei gambiense and T. b. rhodesiense occur. Reproduced with permission from [68]. cases and a situation close to elimination. After the independence of African countries the prevalence of HAT increased, based on neglected control activities due to civil wars, political instability and a general neglect of the health systems. As a consequence, the numbers of HAT patients steadily increased to an estimated 300,000 or more by the end of the 20th century. Since 1998 the prevalence of HAT has been declining in all affected African countries. The reasons for this decline are a better political stability, international collaboration of national control programs, WH and nongovernmental organizations and donations of all antitrypanosomal drugs [9]. The number of reported cases dropped from over 36,000 in 1997 to below 10,000 in 2009 [101]. However, these are only the registered cases, the majority of patients remain undetected due to only poor coverage by surveillance systems of the affected countries. WH estimated the prevalence in 2009 to be 30,000 40,000 using a ratio of 1:3 for reported to undetected cases. A HAT control program of Médecins sans Frontières identified villages in Central African Republic and in Chad with prevalence over 14% [9]. This example illustrates that the epidemiology of HAT is heterogeneous and not easy to understand. The disease can flare up in areas where no control is taking place, which can lead to the activation of old foci or the appearance of new ones. Among tourists HAT is very rare. f those cases more infections were with T. b. rhodesiense than T. b. gambiense and were reported mainly from the ational Parks in Tanzania. Current drugs Current drugs for HAT show varying degrees of toxicity, all require parenteral administration and often show efficacy problems. For first stage disease, pentamidine is used for T. b. gambiense, while suramin is preferred for T. b. rhodesiense infection. Until recently, melarsoprol was the first-line choice for second stage gambiense disease and the only option for second stage rhodesiense disease. Eflornithine has replaced melarsoprol for T. b. gambiense in many endemic countries, when available, and recently its use in combination with nifurtimox has been recommended [10,11]. Pentamidine Pentamidine has been used for over 60 years to treat first stage T. b. gambiense disease [1,3]. It is usually given by intramuscular injection. A 7 day, once daily 4 mg kg -1 dosing is often prescribed. In vitro trypanocidal activity is excellent with 678 Future Microbiol. (2011) 6(6)

3 Development of novel drugs for human African trypanosomiasis IC 50 values in the order of 1 10 nm in a typical 3 day drug sensitivity assay. Pentamidine is concentrated to high (millimolar) levels by trypanosomes using the P2 amino-purine permease and other transporters that include a high affinity pentamidine transporter (HAPT1) and a low affinity pentamidine transporter (LAPT1) [12]. How it actually kills trypanosomes is not certain, although diamidines are known to bind to DA and mitochondrial dysfunction has been associated with diamidine treatment. Extensive tissue retention and binding to serum proteins contribute to a large volume of distribution and long terminal half-life. The drug is charged resulting in a lack of oral availability and minimal brain permeation. The drug is also extensively metabolized. Toxicity is an issue with pain at the site of injection. ephrotoxicity, leucopenia and liver enzyme abnormalities are all common as well as the risk of hypoglycemia. Suramin Suramin was first used against HAT in It is usually given by slow intravenous injection [3]; for example, a course of five injections, every 3 7 days, over a 4-week period is typical. More than 99% of the drug binds to serum proteins and the terminal half-life is very long (41 78 days). Blood brain barrier (BBB) permeation is minimal meaning it is useful only in stage one. In spite of many hypotheses on modes of action, it remains uncertain how suramin kills trypanosomes [3]. Reports on suramin resistance in the field are rare, although it is easily selected in the laboratory. Pyrexia, reversible nephrotoxicity, nausea, urticaria, neuropathy and anemia are all common side effects, especially at increased doses, although HAT regimens are short enough to make safety tolerable. Melarsoprol Melarsoprol, a melaminophenyl-based organic arsenical, was introduced in 1949 [3]. It is one of the most noxious compounds legally administered to man and 5 10% of patients taking melarsoprol suffer a frequently fatal reactive encephalopathy. Administered by intravenous injection as a 3.6% solution in propylene glycol, the drug is increasingly given using a 10-day course, which superseded earlier regimens [1]. It is not known how arsenicals kill trypanosomes, although rapid uptake via the P2 aminopurine transporter and at least one other carrier (probably the HAPT1 transporter [13]), contributes to selective activity. Loss of transport may lead to resistance. Treatment failures have reached alarming levels in many foci although definitive studies relating treatment failure to parasite drug resistance are still lacking. Melarsoprol converts rapidly to an active metabolite, melarsen oxide, in vivo, which shows a mean elimination halflife of 3.5 h. Accumulation of active metabolite across the BBB occurs but to levels of only approximately 1 2% of maximum plasma levels. This could have implications in treatment failure where a relatively low drop in sensitivity to drug could render parasites in cerebrospinal fluid (CSF) nonsusceptible to those levels accumulating in this compartment. Side effects are severe. Convulsions and other neurological sequelae can precede coma and death in the reactive encephalopathy mentioned previously. ther adverse events include pyrexia, headache, pruritus and thrombocytopenia. Heart failure has also been reported. Coadministration of corticosteroids (e.g., prednisolone) yields some protection against the reactive encephalopathy. Eflornithine Eflornithine (d,l-a-difluoromethylornithine), an analog of ornithine, inhibits the polyamine biosynthetic enzyme ornithine decarboxylase (DC) [3]. The drug is active against T. b. gambiense but less so against T. b rhodesiense. The usual regimen involves 100 mg kg -1 body weight at 6 h intervals (i.e., 400 mg kg -1 per day) by intravenous infusion for 14 days. The specificity of the drug against trypanosomes relates to the enzyme target being far less rapidly turned over in trypanosomes than in mammalian cells, hence the inactivation leads to long-term loss of polyamine biosynthesis in trypanosomes while enzyme activity is continuously replenished in mammals. IC 50 growth inhibitory values for eflornithine of µm in vitro are poor when compared with the other licensed drugs and in vivo activity probably depends also on the immune system. The drug enters trypanosomes via a transporter-mediated process and recent evidence points to the loss of a particular amino acid transporter as underlying eflornithine resistance [14 16]. The mean half-life in plasma following intravenous injection of eflornithine is only in the order of 3 h. CSF to plasma ratios in man have been reported between 0.1 and 0.9 [3], but in mice very little eflornithine apparently enters the brain [17]. Adverse effects include fever, headache, hypertension, macular rash, peripheral neuropathy, tremor and gastrointestinal p roblems, including diarrhea

4 Brun, Don, Jacobs, Wang & Barrett ifurtimox eflornithine combination therapy Antimicrobial combination chemotherapies are increasingly popular as they improve efficacy, decrease dosing and reduce the risk of drug resistance emerging. A series of clinical trials, involving various combinations of the registered trypanocides and also nifurtimox (registered for Chagas disease), concluded that a combination of eflornithine with nifurtimox was the most successful treatment [10]. A regimen using eflornithine by intravenous infusion at 200 mg/kg every 12 h for 7 days (rather than every 6 h for 14 days as in monotherapy), with nifurtimox being given orally three times a day for 10 days, offers significant advantages in cost and convenience whilst remaining of equal efficacy to the longer eflornithine monotherapy [10]. Curiously, eflornithine and nifurtimox are not synergistic in in vitro assays [14], possibly because nifurtimox s mode of action appears to involve reduction by an unusual nitroreductase in T. brucei [18] followed by further metabolism to a highly reactive nitrile derivative [19], rather than through oxidative stress as previously assumed. Loss of the nitroreductase causes resistance to nitroheterocycles [18,20]. Clearance of nifurtimox is fast with a plasma elimination half-life of approximately 3 h, but the drug can accumulate across the BBB to levels approximately half of those found in plasma. African trypanosomes are not very susceptible to nifurtimox with IC 50 values of approximately 5 µm in vitro and resistance is easily selected in the laboratory [20]. Toxic effects to the central and peripheral nervous systems have been reported with nifurtimox [3]. In the nifurtimox-eflornithine combination therapy trial the only statistically significant (p < 0.05) adverse events reported to occur at higher frequency in the nifurtimoxeflornithine combination therapy cohort versus the eflornithine monotherapy cohort were an increased incidence of tremors and increased gastrointestinal disturbance, including anorexia and nausea [10]. ew drug candidates for HAT Fexinidazole Fexinidazole, a 2-substituted 5-nitroimidazole, was originally synthesized by Hoechst in the 1970s as part of an anti-infective drug discovery program and was shown to have trypanocidal activity [21]. This was further substantiated in 1983 by Jennings and Urquhart but clinical development was not pursued at that time [21]. In 2006, it was rediscovered by the Drugs for eglected Diseases initiative (DDi) during an extensive data mining project that reviewed more than 700 new and existing nitroheterocyclic compounds as potential drug candidates for treatment of stage two HAT [22]. It is currently undergoing assessment by DDi in Phase I clinical trials with the ultimate goal to develop the compound as an oral treatment for stage one and two HAT. Fexinidazole has an IC 50 of 0.32 µg/ml against the reference laboratory strain of T. b. gambiense (STIB 930). Compared with the reference drug melarsoprol, with an IC 50 of µg/ml, the in vitro potency of fexinidazole is considerably less. In contrast to melarsoprol, fexinidazole showed very little in vitro cytotoxicity with an IC 50 against a rat myoblast cell line (L-6) of more than 90 µg/ml. Fexinidazole has also been screened against a number of recent T. b. gambiense clinical isolates and has shown IC 50 values in the range of 0.30 to 0.93 µg/ml [22]. The stage one STIB900 (T. b. rhodesiense) model was used to test the in vivo efficacy of fexinidazole. Fexinidazole elicited cure with an oral dose of 100 mg/kg once daily or 50 mg/kg twice daily (bid). In a T. b. brucei GVR35 murine model of stage two trypanosomiasis, oral dosing of fexinidazole showed a dose-dependent increase in efficacy with complete cure at 100 mg/kg bid for 5 days [22]. Fexinidazole is metabolized to yield a sulfoxide metabolite followed by subsequent metabolism to a sulfone derivative (Figure 2). Both metabolites have similar potency to the parent compound with IC 50 values against T. b. rhodesiense of µg/ml for the sulfoxide and µg/ml for the sulfone versus µg/ml for the parent compound [22]. In vitro studies using hepatocytes from mouse, rat, dog, monkey and human and in vivo pharmacokinetic studies in mouse, rat and dog showed that this metabolic profile is the same in all of these species [22]. Recent Phase I clinical trials, [Tarral A, Unpublished Data] confirmed a similar in vivo metabolic profile in man. The parent compound is rapidly metabolized to the sulfoxide by a number of cytochrome P450 enzymes, including cytochrome P450s 1A2, 2B6, 2C19, 3A4 and 3A5. The metabolic pathways for the sufoxide and sulfone derivatives have not been established but none of the cytochrome P450 enzymes tested metabolized either compound to any significant degree. The in vivo clearance of the metabolites was considerably lower than that for fexinidazole. As such, it could be considered 680 Future Microbiol. (2011) 6(6)

5 Development of novel drugs for human African trypanosomiasis 2 CYPs 2 2 S S S Fexinidazole Fexinidazole sulfoxide (M1) Fexinidazole sulfone (M2) Figure 2. Fexinidazole, fexinidazole sulfoxide and fexinidazole sulfone. CYP: Cytochrome P450. that fexinidazole acts as a prodrug with the primary pharmacodynamic activity attributed to the sulfoxide and sulfone metabolites. Fexinidazole is orally available with absolute bioavailabilities in mice, rats and dogs of 14, 30 and 10%, respectively. To assess the potential for CS exposure, membrane permeability was initially screened in the in vitro MDR1-MDCK model of cell permeability and fexinidazole showed a high predicted brain permeation (Papp = cm/s with no significant efflux). Subsequent pharmacokinetic experiments in mice showed the presence of fexinidazole and both metabolites in the brain in the same relative concentrations as those in plasma after oral dosing. This is consistent with the data showing efficacy in the murine model of stage two HAT. The drug and its metabolites appear to distribute evenly through the body, as indicated by quantitative whole body autoradiography in the rat. A radiolabeled excretion balance study indicated that excretion is largely via the bile following extensive hepatic metabolism (see Supplementary Information Dataset S11 in [22]). Fexinidazole and its metabolites belong to the 5-nitroimidazole class of compounds that are known to be associated with genotoxicity in bacterial cells. Their bacterial mutagenic potential was confirmed in the Salmonella strains used in an Ames study, but this activity was highly reduced or abolished in nitroreductase deficient mutants. This has been interpreted to suggest that the mutagenic activity is the result of activation by bacterial nitroreductases and is not a property that is inherent to the compounds themselves. All mammalian mutagenicity/clastogenicity assays conduced with fexinidazole were negative. These included an in vitro micronucleus test with human peripheral lymphocytes, and the presence and absence of rat liver microsomal enzymes, an in vivo bone micronucleus test in mice and an ex vivo rat-liver unscheduled DA synthesis study (see Supplementary Information Datasets S20 and S21 in [22]). In addition to the genotoxicity studies described and as part of the preparations for Phase I clinical trials, an investigational new drug enabling package of safety studies was completed. These included: Safety pharmacology (in vitro and in vivo cardiac safety, in vivo respiratory safety profiling and in vivo neurobehavioral safety profiling); Repeat dose toxicokinetics. With respect to safety pharmacology, fexinidazole and the sulfoxide metabolite showed no effect on herg peak tail current at the doses tested, and the sulfone metabolite elicited a 33% decrease in current at the highest dose of 30 µm. Follow-up assessment in beagle dogs showed no effect on blood pressure, heart rate and ECG intervals after single oral doses up to 1000 mg/kg. At the same doses in rats, no effects were observed on behavioral characteristics and on respiratory parameters (see Supplementary Information Datasets S14 and S15 in [22]). The proposed regimen for human treatment with fexinidazole is oral administration for 14 days or less. To support this, 28 day repeat dose toxicokinetic studies were carried out with rats and dogs at daily doses of 50, 200 and 800 mg/kg. With rats, minimal-to-moderate changes in liver histopathology were observed at the high dose of 800 mg/kg and the intermediate dose of 200 mg/kg was specified as the no observed adverse effect level (AEL). In dogs, a slight-to-moderate loss in body weight and food consumption was observed at the highest dose and a 200 mg/kg AEL was also specified for dogs (see Supplementary Information Dataset S17 in [22]). In 2009, an application to conduct Phase I clinical trials was submitted to the European Medicines Agency and these trials are currently underway in France. If fexinidazole completes clinical development, it will have a significant impact on treatment of patients with this neglected disease. First, it will be the first oral treatment for stage two HAT and will lead to a 681

6 Brun, Don, Jacobs, Wang & Barrett revolutionary improvement in patient access over the current first-line treatment, which includes 7 days of bi-daily infusions of eflornithine [10]. Second, if it proves to have an attractive safety profile, it could also be used to treat stage one disease and in doing so, the need for an invasive lumbar puncture to differentiate the stages of disease would be eliminated. Dicationic molecules Pentamidine has been used to treat stage one HAT since the 1940s, but the drug requires parenteral administration via intra muscular injection and is inactive against stage two disease [12]. Efforts to improve the dicationic molecule, led by the Consortium for Parasitic Drug Development (CPDD), have focused on structural modifications that afford oral and/or CS activity against HAT. A prodrug approach proved to be effective in improving oral absorption by masking the positive charge of the amidine group with -alkyl moieties [23]. ral pafuramidine (the methoxy prodrug of furamidine; Figure 3) achieved cures in rodent and nonhuman primate HAT animal models and in clinical trials. Recently, promising efficacy of aza analogs, including CPD-0802 (or DB-829 as the hydrochloride salt) and its prodrug DB-868 (Figure 3), in the models of CS infection in mice and monkeys offered hope for dicationic molecules to treat stage two HAT [24]. ew aza analogs possess nanomolar IC 50 values against T. b. rhodesiense and T. b. gambiense isolates (Table 1). CPD-0802 showed a dose response and achieved complete cure at 10 mg/kg 4 days intraperitoneal (i.p.) in the STIB900 acute mouse model [Brun R & Wenzler T, Unpublished Data], similar to its hydrochloride salt preparation DB-829 [24]. Surprisingly, a single dose of CPD-0802 at 10 mg/kg i.p. also cured all mice in this model. Using the GVR35 CS mouse model, DB-829 cured all four mice at 20 mg/kg 10 days i.p. (Table 2). Due to the H H H 3 C CH 3 H 2 Pentamidine H 2 H 2 H 2 Pafuramidine (DB-289) H H H 2 H 2 Furamidine (DB-75) H 3 C H 2 DB-868 H 2 CH 3 H H 2 CPD-0802 (diacetate) DB-829 (hydrochloride) H H 2 Figure 3. Prodrugs (A) and active cationic diamidines (B). Reproduced with permission from [25]. 682 Future Microbiol. (2011) 6(6)

7 Development of novel drugs for human African trypanosomiasis Table 1. In vitro antitrypanosomal activities against different subspecies. Compound IC 50 (nm) T. b. rhodesiense T. b. gambiense T. b. brucei STIB900wt STIB900pent STIB900mel K03048 ITMAP STIB930 BS221 AT1K Pentamidine Furamidine DB (HCl salt) CPD-0802 (diacetate salt) P2 transporter knockout of Trypanosoma brucei BS221 strain. Adapted with permission from [24]. promising efficacy in mice, CPD-0802 was further examined in the vervet monkey model of stage two HAT. In this model, monkeys are infected with T. b. rhodesiense and demonstrate trypanosome presence and elevated white cell counts in CSF prior to drug treatment that starts 28 days postinfection [25]. CPD-0802, given intramuscularly at 5 mg/kg 5 days or 5 mg/kg every other day five doses (over 9 days), cleared trypanosomes from blood and CSF immediately after completing drug treatment [Thuita JK, Unpublished Data]. More importantly, these treated monkeys have remained free of detectable parasites in both blood and CSF for 300 days post last day of treatment and are considered cured. DB-868 is the methoxy prodrug of CPD and itself is inactive against trypanosomes in vitro. After oral administration (p.o.), DB-868 is absorbed and biotransformed to the active metabolite CPD-0802, exhibiting oral efficacy in animal models of HAT. DB-868 completely cured mice in the STIB900 acute model (50 mg/kg 4 days p.o.) and the GVR35 CS model (Table 2) [24]. This compound also progressed to the vervet monkey model of stage two HAT, where it cleared blood and CSF immediately after completing drug treatment at 20 mg/kg 10 days p.o. and has kept them free of detectable parasites for at least 75 days post last day of treatment [Thuita JK, Unpublished Data]). A definitive antitrypanosomal target for cationic diamidines remains elusive, but kinetoplast intercatenated circular DA has been proposed as a possible target [12]. Diamidines bind avidly to the minor groove of AT-rich DA via hydrogen bonding, van der Waals contacts and electrostatic interactions and are concentrated to high levels (in the low millimolar range) within trypanosomes exposed to low micromolar concentrations in the medium or plasma [26], properties that are essential for diamidines to kill the parasite. Due to the perpetual positive charge on the molecule at physiologic ph, diamidine uptake into trypanosomes is generally considered as a carrier-mediated process. This process has been described for pentamidine in detail [12], where three distinct transporters (i.e., P2 aminopurine transporter, high affinity pentamidine transporter and low affinity pentamidine transporter) mediate pentamidine uptake by T. brucei. However, variability among diamidines in resistance factors (range from 2.4-fold to 19-fold) in T. brucei AT1K cells (P2 transporter knockout of T. b. brucei BS221 strain) indicates that secondary transporter(s) and/or distinct transport kinetics exist, which may have important implications in determining drug resistance liability [26, 27]. Resistance factors for DB-829 and CPD 0802 ranged from 2 to 10 in resistant lines of T. b. rhodesiense (Table 1), appreciably less than those of pentamidine (~100), indicating that resistance liability of the aza analog might be no worse than pentamidine. Table 2. In vivo antitrypanosomal efficacy in the GVR35 CS mouse model of second stage human African trypanosomiasis. Compound GVR35 CS mouse Dose (mg/kg per o. of mice cured MSD no. of days) DB-829 (HCl salt) 20/10 i.p. 4/4 >180 20/5 i.p. 1/4 >132 10/10 i.p. 0/5 96 CPD-0802 (diacetate salt) 10/10 i.p. Toxic 20/5 i.p. 2/7 DB /10 p.o. 5/5 >180 50/10 p.o. 4/5 >153 25/10 p.o. 1/5 >132 10/10 p.o. 0/5 70 i.p.: Intraperitoneal; MSD: Mean survival day; p.o.: ral. Adapted with permission from [24]

8 Brun, Don, Jacobs, Wang & Barrett Plasma concentration (nm) 10, To improve oral absorption, a prodrug strategy was employed to mask the positive charge of the amidine group with -alkyl moieties, resulting in increased lipophilicity and enhanced intestinal permeability [12]. For example, at the same dose of 100 µmol/kg in mice, maximal plasma concentration and area under the concentration time curve (AUC) of DB-829 after a single oral dose of the methoxy prodrug DB-868 were approximately 12- and 24-fold greater than those after a single oral dose of DB-829 itself (Figure 4). This indicated that DB-868 was readily absorbed and subsequently biotransformed to the active metabolite DB-829 in vivo. DB-868 has a short half-life of approximately 2 h and clearance of its metabolite DB-829 appears to be elimination-limited as its half-life is approximately five-times greater. Biotransformation of DB-868 to DB-829 is expected to require multiple steps of oxidative -demethylation and reductive -dehydroxylation reactions catalyzed by cytochrome P450 enzymes and cytochrome b 5 /ADH b 5 reductase system, similar to the biotransformation of pafuramidine to furamidine [28 30]. Mechanisms that regulate the transport of cationic diamidines into mammalian cells and across the BBB remain relatively unexplored. Recently, pentamidine, furamidine [31] and their Time (h) DB-829 DB-829 formed from DB-868 DB-868 Figure 4. Plasma concentration versus time profiles for DB-868 (square) and DB-829 (circles) in mice. Healthy mice were administered a single oral 100 µmol/kg (~37 mg base/kg) dose of DB-868 and plasma concentrations of DB-868 (square) and its metabolite DB-829 (open circle) were quantified by high performance liquid chromatography/mass spectrometry/mass spectrometry analysis. In a separate experiment, healthy mice were administered a single oral 100 µmol/kg (~31 mg base/kg) dose of DB-829 directly and plasma concentrations of DB-829 (solid circle) were quantified by high performance liquid chromatography/mass spectrometry/mass spectrometry analysis. Symbols and error bars denote the mean and standard error of three mice. Dashed lines represent the best fit of a one-compartment pharmacokinetic model to the data. aza analogs DB-829 [Wang MZ, Unpublished Data] have been demonstrated to be substrates of the human facilitative organic cation transporter 1 (CT1), which is primarily expressed in the sinusoidal membrane of hepatocytes. Studies using P-gp (Mdr1)-deficient mice and adenosine competition suggested that pentamidine is a substrate of efflux transporters P-gp and MRP [32]. evertheless, uptake mechanism of the aza analog DB-829 (or CPD-0802) across the BBB is unknown and of great interest in understanding how to deliver polar chemotherapeutic molecules to the brain. Special attention needs to be paid to determine the risk of renal toxicity with new cationic diamidines, as an unexpected delayed-onset renal insufficiency observed in an expanded Phase I trial resulted in the discontinuation of the pafuramidine program [12]. It should be noted that the novel aza analog CPD-0802 accumulates in the rat kidney at concentrations more than ten-times less than furamidine 48 h after a single intravenous dose at 10 µmol/kg [33]. The observed lower kidney exposure of CPD-0802 may provide the necessary safety margin and favorable risk benefit profile for developing these CS-active diamidines to treat stage two HAT. Discovery of SCYX-7158, a benzoxaborole-6-carboxamide for treatment of stage two HAT Benzoxaboroles were initially found by Anacor Pharmaceuticals to have in vitro activity against T. brucei through collaboration with James McKerrow at the Sandler Center (University of California, San Francisco, CA, USA). Discussions with the DDi led to the DDi being granted a license for the use of these compounds for treatment of kinetoplastid diseases such as HAT, leishmaniasis and Chagas disease. As such, the DDi brought forth this class of compounds to an integrated, collaborative HAT drug discovery program being executed by SCYEXIS and Pace University in March 2008 for further optimization. Initial efforts at SCYEXIS encompassed screening of approximately 50 representative benzoxaboroles provided by Anacor in whole cell T. b. brucei viability, cytotoxicity, in vitro metabolic stability, in vitro membrane permeability and physicochemical property assays. These studies revealed that three classes of benzoxaboroles, namely the 6-arylthio (2), 5-aryloxy (3) and 6-carboxamido (4), series were particularly attractive starting points for optimization (Figure 5) [34]. 684 Future Microbiol. (2011) 6(6)

9 Development of novel drugs for human African trypanosomiasis Representative compounds from each of these series were progressed to a murine model of stage one HAT infection at Pace University, which allowed for prioritization of lead optimization efforts to the 6-carboxamido series, as a greater breadth of activity was observed in this class relative to either the 6-arylthio or 5-aryloxy classes. Concurrent with evaluation of in vivo efficacy, we examined the in vivo pharmacokinetic properties of representative compounds from these series, which demonstrated the superior exposure observed for the 2-trifluoromethyl benzamide (4a A3520) relative to the 6-arylsulfinyl analog (2a A2920). Plasma concentrations of 4a were maintained above the in vitro MIC (0.12 µg/ml) for trypanocidal activity for at least 8 h following a single 10 mg/kg oral dose, as compared with less than 4 h above MIC (0.36 µg/ml) for A2920 [34]. Further improvements in pharmacokinetic properties were observed through preparation of the 4-fluoro-2-trifluoromethyl benzamide 4b (SCYX-6759), which exhibited sufficient exposure in both plasma and brain to warrant progression to a murine model of stage two HAT, where it was found to cure the CS infection following i.p. administration at 50 mg/kg, bid for 14 days [35]. Subsequent studies in this CS efficacy model revealed that SCYX-6759 was orally active, with 80% cure following 50 mg/kg, bid 7 day administration (Figure 6). The need for twice-daily dosing to achieve efficacy in the CS model could be understood based on the brain pharmacokinetics of this compound, which fell below the in vitro trypanocidal MIC within h following a single 50 mg/kg dose [35]. Introduction of small alkyl substituents at C(3) of the benzoxaborole ring was pursued next as an approach to improve brain exposure. A single methyl group, such as in compound 4c, had little effect on brain pharmacokinetics, but cytotoxicity was significantly greater than in the C(3)-unsubstituted series. Compounds containing larger C(3) alkyl groups, such as isobutyl (4d) or cyclopentyl (4e) were essentially inactive as trypanocides. A significant breakthrough in achievement of extended brain exposure was found through introduction of a second alkyl group at C(3) to afford compounds such as the gem-dimethyl analog 4f (SCYX-7158) [36]. As observed in the C(3)-monosubstituted analogs, substituents larger than methyl were not tolerated at this position, but unlike the C(3)- methyl compounds, the gem-dimethyl compounds were not cytotoxic. Most importantly, the R 4 R 6 R 5 R 2 H H B Figure 5. Benzoxaboroles. pharmacokinetic properties of 4f were predictive of superior efficacy in the CS mouse model, which was confirmed. In this model, 4f effected % animals parasite free , R 5, R 6 = H 2, R 5 = H, R 6 = -SAr 3, R 5 = -Ar, R 6 = H 4, R 5 = H, R 6 = -HCAr 4a, R 2 = CF 3, R 4 = H 4b, R 2 = CF 3, R 4 = F B H Berenil D4 Berenil D mg/kg Days postinfection mg/kg mg/kg mg/kg % animals parasite free F F Cl CF 3 CF3 S H 2a H R3a B B H H H 4c, R 3a = CH 3, R 3b = H 4d, R 3a = cyclopentyl, R 3b = H 4e, R 3a = isobutyl, R 3b = H 4f, R 3a = R 3b = CH 3 7 Days postinfection R 3b H Berenil D4 Berenil D mg/kg mg/kg mg/kg mg/kg Figure 6. SCYX-6759 and SCYX-7158 cure stage two trypanosomiasis in mice. Kaplan-Meier parasitemia plots for female Swiss-Webster mice (n = 10 per group) after infection with Trypanosoma brucei brucei TREU 667 (inoculum 10 4 parasites/mouse). (A) ral treatment with SCYX-6759 (twice daily) started on day 21 after infection at the indicated doses for 7 days. (B) ral treatment with SCYX-7158 (four-times daily) started on day 21 after infection at the indicated doses for 7 days. Berenil (diminazene) was administered as a single 10 mg/kg dose intraperitoneally on either day 4 (positive control) or day 21 (negative control) in both studies. Parasitemia was assessed weekly starting on day 21 by microscopic examination of a blood sample. Animals in which parasites were detected in the blood were sacrificed. Reproduced with permission from [37]

10 Brun, Don, Jacobs, Wang & Barrett 100% cure following a 25 mg/kg, 7 day oral administration, with 80% cure following a 12.5 mg/kg, 7 day oral paradigm (Figure 6), which could be understood based on maintenance of drug concentration in the brain above the MIC (0.95 µg/ml) for 24 h [36,37]. Given the impressive activity of 4f in the CS mouse model, this compound has been fully profiled in a broad array of in vitro and in vivo biological and pharmacokinetic assays. In whole cell assays, 4f exhibits potent trypanocidal activity versus numerous strains of T. b. brucei, T. b. rhodesiense and T. b. gambiense, including drug resistant strains as summarized in Table 3 [36]. The potential interaction of SCYX-7158 with mammalian biochemical targets (e.g., enzymes, receptors and ion channels) was also explored through radiochemical binding and functional assays. At a concentration of 10 µm, no significant inhibition of ligand binding was observed for over 100 target proteins, including cytochrome P450 (CYP) isoforms 1A2, 2C19, 2D6 and 3A4, serine and cysteine proteases, cell-cycle kinases or the herg potassium channel [36]. Functional assays for herg channel blockade and CYP inhibition confirmed the lack of significant interaction with these proteins at concentrations up to 20 µm. In a bacterial reverse mutation (Ames) assay, SCYX-7158 was found to be nonmutagenic, both in the presence or absence of metabolic activation, at all concentrations tested opposite five test strains. Pharmacokinetic characterization of SCYX across mouse, rat, cynomolgus monkey and dog suggest the compound to be slowly metabolized both in vitro and in vivo, with the primary metabolite identified as the product of oxidative deboronation, 7. This metabolite is present in plasma (mouse, rat, cynomolgus monkey), brain (mouse, rat) and CSF (rat, cynomolgus monkey) at concentrations approximately 1 5% of SCYX 7158 (Figure 7) [36]. At therapeutically relevant doses, the pharmacokinetics of SCYX-7158 are essentially dose proportional, although at higher doses required for toxicity studies, slightly less than proportional increases in exposure have been observed, most likely due to solubility-limited absorption. n repeated dosing of SCYX-7158 over 7 days, modest (~threefold) accumulation of both parent drug and metabolite 7 have been observed [Don R, Pers. Comm.]. Preliminary toxicological evaluation of SCYX-7158 is ongoing. In conclusion, an integrated drug discovery program focused on a series of benzoxaborole 6-carboxamides initially identified by Anacor Pharmaceuticals and executed through a collaboration between DDi, Scynexis, Pace University and Swiss Tropical and Public Health Institute have resulted in the identification and characterization of SCYX-7158 (4f) as an orally-active drug candidate for treatment of stage two HAT [36]. ngoing experiments to further profile the efficacy, pharmacokinetic and toxicological properties are poised to support submission of SCYX-7158 to regulatory authorities for clinical evaluation of this compound in Current targets, hits & leads Some drugs act through the specific inhibition of particular targets. For example, in trypanosomes DC is the target of eflornithine. Targets for the other registered antitrypanosomals are not known. onetheless, much contemporary drug discovery is focused on identifying individual targets that can be hit with small molecules that can then be modified into even more potent inhibitors with drug-like characteristics. Trypanosomes, as anciently diverging eukaryotic organisms, are replete with biochemical peculiarities that have long held interest among those developing drugs for HAT [2]. Table 3. Activity of SCYX-7158 (4f) in whole cell trypanocidal assays. Strain IC 50 (µm) Comments Trypanosoma brucei brucei 1.10 ± 0.22 Routine screening strain S 427 (n= 5) T. b. rhodesiense STIB Isolated from a patient in Tanzania in 1982, adapted to cell culture at Swiss Tropical Institute T. b. gambiense 40R 0.99 Isolated from a patient in DRC in 2005, relapse 6 months after melarsoprol treatment T. b. gambiense 108R 0.45 Isolated from a patient in DRC in 2005, relapse 8 months after melarsoprol treatment T. b. gambiense DAL Isolated from a patient in Cote d Ivoire in 1990 T. b. gambiense ITMAP Isolated from a patient in DRC in 1960 T. b. gambiense Drani 0.35 Isolated from a patient in Uganda in 1995 DRC: Democratic Republic of Congo. Data taken from [37]. 686 Future Microbiol. (2011) 6(6)

11 Development of novel drugs for human African trypanosomiasis It has become routine to identify genes encoding enzymes and to validate their essentiality through genetic means using gene knockout or RAi. It has also become relatively straightforward to test the ability of chemicals in large libraries to inhibit potential drug targets purified after overexpression in heterologous systems [38]. It is also important to consider the druggability of targets, that is to assess the likelihood that they will bind chemicals carrying those features making them useful as drugs (i.e., with suitable pharmacokinetic and toxicological properties [39]). Bloodstream form trypanosomes are absolutely dependent upon substrate level phosphorylation through glycolysis to generate energy. The fact that the enzymes of glycolysis are compartmentalized within a membrane-bound organelle, the glycosome, endows them with structural and regulatory features different from those of mammalian counterparts. With structural information available for most of the enzymes, several efforts have been made to design selective inhibitors. For example, targeting the ADH binding site of glyceraldehyde 3-phosphate dehydrogenase allowed development of some selective inhibitors of the trypanosomal enzyme that had trypanocidal activity. ther enzymes, including phosphofructokinase [40] and aldolase [41], have also been targeted. A screen of more than 220,000 compounds against T. brucei hexokinase identified several potent selective inhibitors that targeted the enzyme s ATP binding site and also demonstrated trypanocidal activity in vitro [42]. The pentose phosphate has also been shown to be important to bloodstream form trypanosomes, and 6-phosphogluconate dehydrogenase, in particular, has been targeted with various inhibitors, including those with profound trypanocidal activity [43]. To date, however, the various hits that have emerged from such screens have not progressed to genuine leads. Trypanothione is a redox active metabolite specific to the trypanosomatid protozoa. It comprises two molecules of glutathione linked by the polyamine spermidine and plays multiple essential roles in trypanosomes. The reduced form of the metabolite is maintained by the essential enzyme trypanothione reductase (TR) that has been subject to multiple focused and also diverse chemical library screens [44]. umerous hits have been identified but not progressed through lead optimization programs. The biosynthetic enzyme, trypanothione synthetase has also been screened for hits and showed some promise [45]. Concentration (µg/ml) PL mg/kg PL mg/kg MIC 7158 Time post dose (h) The one enzyme that is known to be a target for one of the currently used drugs is DC, the target of eflornithine. Various other inhibitors of DC were described including 3-(aminooxy)propanamine (APA) [46]. In the case of APA it was questioned whether enhanced uptake of the drug could increase activity hence the melamine motif known to carry drugs into trypanosomes via the P2 transporter [47, 48] was added and some improvement was seen. DC has also recently been subject to high throughput screening to identify novel inhibitors, and a library of more than 316,000 compounds yielded four new families of inhibitors, although trypanocidal activities of these compounds were not reported [49]. In addition to DC, the essential polyamine pathway enzyme S-adenosylmethionine decarboxylase has also been subject to inhibitor analysis and several potent inhibitors of this enzyme, such as several S-adenosylmethionine analogs [50], could be found. ther polyamine pathway enzymes, including g-glutamyl cysteine synthetase and spermidine synthase [51,52] have also been shown to be essential. Protein farnesyl transferases (PFT) have been of interest in anticancer and antimalarial drug campaigns, and emphasis has been put into learning about the trypanosome PFT enzyme and specific inhibitors with potent trypanocidal activity have been identified [53]. BR mg/kg BR mg/kg Figure 7. Time versus concentration curves for SCYX-7158 (4f) and SCYX 3109 (7) in mice. Uninfected male CD-1 mice received a single oral 50 mg/ kg dose of SCYX Blood (solid line) and brain (dashed line) samples were collected and analyzed by liquid chromatography/mass spectrometry/mass spectrometry. Data points represent a single mouse at each time point [37]

12 Brun, Don, Jacobs, Wang & Barrett -myristoyl transferase plays an important role in adding myristate to various membrane proteins in trypanosomes and the enzyme was shown to be essential in T. brucei [54]. An initial screen against a diverse library of around 60,000 compounds identified hits that were converted to increasingly efficacious leads using crystallography and molecular modelling to direct synthesis until highly potent inhibitors of the enzyme were generated that showed great efficacy against T. brucei both in vitro and in vivo [55]. The relative simplicity in determining whether genes are essential has provided evidence on many enzymes as potential drug targets in addition to the examples discussed earlier. Drug-like inhibitors of an unusual RA ligase [56] involved in another trypanosomatidspecific process, RA editing of mitochondrial transcripts, were reported. Focused chemical set screens for glycogen synthase kinase 3 also unearthed novel inhibitors [57]. Multiple enzymes involved in the lipid biosynthetic pathways [58], pyrimidine biosynthesis [59], pterin metabolism [60], intracellular trafficking [61], protein kinases [62], phosphodiesterases [63] and cellular proteases [64], have all been validated and some progress in identifying chemical inhibitors has been made. The target-based approach clearly has an important role in drug discovery. However, there is an increasing realization that many good drugs in use today actually exert their activity not through the clean inhibition of individual enzymes but through the simultaneous inhibition of multiple targets within a host. Selective uptake can stimulate such processes and it might be of no accident that drugs like pentamidine and melarsoprol, which are potent inhibitors of many cellular processes, are actively and selectively accumulated into trypanosomes via the P2 transporter [48]. The concept of polypharmacology [65] emphasises a need to also screen compounds directly against the parasites whereby lethal effects associated with inhibition of multiple targets becomes clear. A screen of 2160 known drugs and bioactives [66] returned suramin and pentamidine among the most potent compounds, along with a diverse set of other trypanocides, and another screen including additional commercial libraries of bioactive compounds similarly returned known trypanocides along with some additional hits [67]. atural Executive summary Epidemiology of human African trypanosomiasis & current treatment The number of patients has been declining during the last 10 years and is currently at less than 10,000/year. Disease surveillance should be maintained otherwise flare-ups are a possible threat. Currently used drugs are old, toxic, of limited efficacy or difficult to administer. ew drugs are urgently needed. The only new medication is the combination of nifurtimox and eflornithine with the drawback of 14 intraveneous applications over 7 days. Fexinidazole (drug candidate) Fexinidazole was rediscovered by Drugs for eglected Diseases initiative during a screening campaign of nitroimidazoles. It cured a CS mouse model after oral administration of 100mg/kg twice daily for 5 days and showed no mammalian mutagenicity. It passed Phase I clinical trials and is now entering Phase II. Dicationic molecules as preclinical candidates CS active aromatic diamidines have been identified by a concerted effort to improve the bioavailability and brain penetration of dicationic molecules, led by the Consortium for Parasitic Drug Development. CPD-0802 cured both CS mouse and monkey human African trypanosomiasis (HAT) models after parenteral administration. DB-868 cured a CS mouse HAT model after oral administration of 100 mg/kg once daily for 10 days. An improved renal safety profile is needed for further development of CPD-0802 and DB-868. SCYX-7158, a benzoxaborole, as a preclinical candidate Benzoxaboroles from Anacor identified as active in a screening campaign at the Sandler Center (University of California, San Francisco, CA, USA), have been optimized by Scynexis in a Drugs for eglected Diseases initiative-sponsored consortium. Integration of medicinal chemistry, parasitology and pharmacokinetics expertise is key to rapid progression of a lead optimization program. SCYX-7158 exhibits excellent exposure in plasma and brain and is curative in a mouse model of CS HAT following oral administration. Preliminary toxicological evalution of SCYX-7158 is ongoing in anticipation of progression to clinical trials in Current targets & lead compounds It is now trivial to validate, clone, express and purify potential drug targets for chemical library screening. In vitro assays to determine activity of chemicals against trypanosomes are straightforward. ew chemical hits are emerging through screening programs at an unprecedented rate. 688 Future Microbiol. (2011) 6(6)