Tafenoquine is not neurotoxic following supertherapeutic dosing in rats.

BACKGROUND: Tafenoquine is a new drug for malaria prevention. The goal of the present work was to conduct a specific neurobehavioral study in rats with histopathological assessment of the brain. METHODS: The clinical, hematological, behavioral, motor activity, and neurohistopathologic changes induced by different dose levels of tafenoquine were evaluated following single super-therapeutic dose administration. Toxicokinetic data were generated to allow extrapolation to clinical exposures. RESULTS: At the highest dose (500 mg/kg), two animals (of 12) died. Surviving animals showed clinical signs of toxicity and had reduced body weight 7-8 days after dosing. Decreases in motor activity were observed on more than one occasion at doses > 9-fold higher than the clinical exposure. No statistically significant changes were observed for other behavioral endpoints. No neurohistopathological changes were noted. Changes in hematological and clinical pathology endpoints were observed at the lowest dose level (125 mg/kg). For context, the human dosing regimen is a 10 mg/kg load followed by 3.3 mg/kg weekly (in a 60 kg person). CONCLUSIONS: As in humans, adverse events other than neurotoxicity were dose-limiting for tafenoquine in rats. This raises the prospect that a new weekly prophylactic, without neurologic liability, may become available in the near future.

Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model.

Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model.

Summary of anti-malarial prophylactic efficacy of tafenoquine from three placebo-controlled studies of residents of malaria-endemic countries.

BACKGROUND: Tafenoquine is a long half-life primaquine analog being developed for malaria prophylaxis. The US Army recently performed a unified analysis of efficacy in preparation for a regulatory submission, utilizing legacy data from three placebo-controlled studies conducted in the late 1990s and early 2000s. The subjects were residents of Africa who were naturally exposed to Plasmodium falciparum for 12-26 weeks. METHODS: The prophylactic efficacy of tafenoquine and mefloquine (included in some studies as a comparator) was calculated using incidence density among subjects who had completed the three-day loading doses of study drug, had at least one maintenance dose and had at least one blood smear assessed during the prophylactic period. The three placebo-controlled studies were analysed separately and then in two pooled analyses: one for tafenoquine versus placebo (three studies) and one for tafenoquine and mefloquine versus placebo (two studies). RESULTS: The pooled protective efficacy (PE) of a tafenoquine regimen with three daily loading doses plus weekly maintenance at 200-mg for 10 weeks or longer (referred to as 200-mg weekly hereafter) relative to placebo in three placebo-controlled studies was 93.1 % [95 % confidence interval (CI) 89.1-95.6 %; total N = 492]. The pooled PEs of regimens of tafenoquine 200-mg weekly and mefloquine 250-mg weekly relative to placebo in two placebo-controlled studies (total N = 519) were 93.5 % (95 % CI 88.6-96.2 %) and 94.5 % (95 % CI 88.7-97.3 %), respectively. Three daily loading plus weekly maintenance doses of 50- and 100-mg, but not 25-mg, exhibited similar PEs. The PEs of tafenoquine regimens of a three-day loading dose at 400-mg with and without follow-up weekly maintenance doses at 400-mg were 93.7 % (95 % CI 85.4-97.3 %) and 81.0 % (95 % CI 66.8-89.1 %), respectively. CONCLUSIONS: Tafenoquine provided the same level of prophylactic efficacy as mefloquine in residents of Africa. These data support the prophylactic efficacy of tafenoquine and mefloquine that has already been demonstrated in the intended malaria naive population.

A retrospective analysis of the protective efficacy of tafenoquine and mefloquine as prophylactic anti-malarials in non-immune individuals during deployment to a malaria-endemic area.

BACKGROUND: In 2000/2001, the Australian Defense Forces (ADF), in collaboration with SmithKline Beecham and the United States Army, conducted a field trial to evaluate the safety, tolerability and efficacy of tafenoquine and mefloquine/primaquine for the prophylaxis of malaria amongst non-immune Australian soldiers deployed to East Timor (now called Timor Leste) for peacekeeping operations. The lack of a concurrent placebo control arm prevented an internal estimate of the malaria attack rate and so the protective efficacy of the study regimens was not determined at the time. METHODS: In a retrospective analysis of the trial results, the all species malaria attack rate was estimated for the prophylactic phase of the study which was defined as the period between administration of the first prophylactic dose and the first dose of post-deployment medication. First, the Plasmodium vivax attack rate was estimated during the prophylactic phase of the deployment by adjusting the observed P. vivax relapse rate during post-deployment to account for the known anti-relapse efficacies (or effectiveness) of the study medications (determined from prior studies). The all species malaria attack rate (P. vivax and Plasmodium falciparum) was then determined by adjusting the P. vivax attack rate based on the ratio of P. falciparum to P. vivax observed during prior ADF deployments to Timor Leste. This estimated all species malaria attack rate was then used as the 'constant estimated attack rate' in the calculation of the protective efficacy of tafenoquine and mefloquine during the prophylactic phase of the deployment. RESULTS: The estimated attack rate during the prophylactic phase of the study was determined to be 7.88%. The protective efficacies of tafenoquine and mefloquine, with corresponding 95% confidence intervals (95% CI), were determined to be 100% (93%-100%) and 100% (79%-100%) respectively. CONCLUSIONS: The protective efficacy of tafenoquine (200 mg per day for three days, followed by weekly 200 mg maintenance doses) is similar to that of the weekly standard of care (mefloquine, 250 mg).

Ketotifen is an antimalarial prodrug of norketotifen with blood schizonticidal and liver-stage efficacy.

Ketotifen is known to exhibit antimalarial activity in mouse and monkey malaria models. However, the low plasma levels and short half life of the drug do not adequately explain its in vivo efficacy. We synthesized most of the known metabolites of ketotifen and evaluated their antimalarial activity and pharmacokinetics in mice. Norketotifen, the de-methylated metabolite of ketotifen, was a more potent antimalarial in vitro as compared to ketotifen, and exhibited equivalent activity in vivo against asexual blood and developing liver-stage parasites. After ketotifen dosing, norketotifen levels were much higher than ketotifen relative to the IC50s of the compounds against Plasmodium falciparum in vitro. The data support the notion that the antimalarial activity of ketotifen in mice is mediated through norketotifen.

Radical curative efficacy of tafenoquine combination regimens in Plasmodium cynomolgi-infected Rhesus monkeys (Macaca mulatta).

BACKGROUND: Tafenoquine is an 8-aminoquinoline being developed for radical cure (blood and liver stage elimination) of Plasmodium vivax. During monotherapy treatment, the compound exhibits slow parasite and fever clearance times, and toxicity in glucose-6-phosphate dehydrogenase (G6PD) deficiency is a concern. Combination with other antimalarials may mitigate these concerns. METHODS: In 2005, the radical curative efficacy of tafenoquine combinations was investigated in Plasmodium cynomolgi-infected naïve Indian-origin Rhesus monkeys. In the first cohort, groups of two monkeys were treated with a three-day regimen of tafenoquine at different doses alone and in combination with a three-day chloroquine regimen to determine the minimum curative dose (MCD). In the second cohort, the radical curative efficacy of a single-day regimen of tafenoquine-mefloquine was compared to that of two three-day regimens comprising tafenoquine at its MCD with chloroquine or artemether-lumefantrine in groups of six monkeys. In a final cohort, the efficacy of the MCD of tafenoquine against hypnozoites alone and in combination with chloroquine was investigated in groups of six monkeys after quinine pre-treatment to eliminate asexual parasites. Plasma tafenoquine, chloroquine and desethylchloroquine concentrations were determined by LC-MS in order to compare doses of the drugs to those used clinically in humans. RESULTS: The total MCD of tafenoquine required in combination regimens for radical cure was ten-fold lower (1.8 mg/kg versus 18 mg/kg) than for monotherapy. This regimen (1.8 mg/kg) was equally efficacious as monotherapy or in combination with chloroquine after quinine pre-treatment to eliminate asexual stages. The same dose of (1.8 mg/kg) was radically curative in combination with artemether-lumefantrine. Tafenoquine was also radically curative when combined with mefloquine. The MCD of tafenoquine monotherapy for radical cure (18 mg/kg) appears to be biologically equivalent to a 600-1200 mg dose in humans. At its MCD in combination with blood schizonticidal drugs (1.8 mg/kg), the maximum observed plasma concentrations were substantially lower than (20-84 versus 550-1,100 ng/ml) after administration of 1, 200 mg in clinical studies. CONCLUSIONS: Ten-fold lower clinical doses of tafenoquine than used in prior studies may be effective against P. vivax hypnozoites if the drug is deployed in combination with effective blood-schizonticidal drugs.

Central nervous system exposure of next generation quinoline methanols is reduced relative to mefloquine after intravenous dosing in mice.

BACKGROUND: The clinical use of mefloquine (MQ) has declined due to dose-related neurological events. Next generation quinoline methanols (NGQMs) that do not accumulate in the central nervous system (CNS) to the same extent may have utility. In this study, CNS levels of NGQMs relative to MQ were measured and an early lead chemotype was identified for further optimization. EXPERIMENTAL DESIGN: The plasma and brain levels of MQ and twenty five, 4-position modified NGQMs were determined using LCMS/MS at 5 min, 1, 6 and 24 h after IV administration (5 mg/kg) to male FVB mice. Fraction unbound in brain tissue homogenate was assessed in vitro using equilibrium dialysis and this was then used to calculate brain-unbound concentration from the measured brain total concentration. A five-fold reduction CNS levels relative to mefloquine was considered acceptable. Additional pharmacological properties such as permeability and potency were determined. RESULTS: The maximum brain (whole/free) concentrations of MQ were 1807/4.9 ng/g. Maximum whole brain concentrations of NGQMs were 23 - 21546 ng/g. Maximum free brain concentrations were 0.5 to 267 ng/g. Seven (28%) and two (8%) compounds exhibited acceptable whole and free brain concentrations, respectively. Optimization of maximum free brain levels, IC90s (as a measure or potency) and residual plasma concentrations at 24 h (as a surrogate for half-life) in the same molecule may be feasible since they were not correlated. Diamine quinoline methanols were the most promising lead compounds. CONCLUSION: Reduction of CNS levels of NGQMs relative to mefloquine may be feasible. Optimization of this property together with potency and long half-life may be feasible amongst diamine quinoline methanols.

Synthesis and biological evaluation of the first pentafluorosulfanyl analogs of mefloquine.

Two novel SF5 analogs of the antimalarial agent mefloquine were synthesized in 5 steps and 10-23% overall yields and found to have improved activity and selectivity against malaria parasites. This work also represents the first report of SF5-substituted quinolines.

Antimalarial activity of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors.

The antimalarial activity and pharmacology of a series of phenylthiazolyl-bearing hydroxamate-based histone deacetylase inhibitors (HDACIs) was evaluated. In in vitro growth inhibition assays approximately 50 analogs were evaluated against four drug resistant strains of Plasmodium falciparum. The range of 50% inhibitory concentrations (IC(50)s) was 0.0005 to >1 microM. Five analogs exhibited IC(50)s of <3 nM, and three of these exhibited selectivity indices of >600. The most potent compound, WR301801 (YC-2-88) was shown to cause hyperacetylation of P. falciparum histones, which is a marker for HDAC inhibition in eukaryotic cells. The compound also inhibited malarial and mammalian HDAC activity in functional assays at low nanomolar concentrations. WR301801 did not exhibit cures in P. berghei-infected mice at oral doses as high as 640 mg/kg/day for 3 days or in P. falciparum-infected Aotus lemurinus lemurinus monkeys at oral doses of 32 mg/kg/day for 3 days, despite high relative bioavailability. The failure of monotherapy in mice may be due to a short half-life, since the compound was rapidly hydrolyzed to an inactive acid metabolite by loss of its hydroxamate group in vitro (half-life of 11 min in mouse microsomes) and in vivo (half-life in mice of 3.5 h after a single oral dose of 50 mg/kg). However, WR301801 exhibited cures in P. berghei-infected mice when combined at doses of 52 mg/kg/day orally with subcurative doses of chloroquine. Next-generation HDACIs with greater metabolic stability than WR301801 may be useful as antimalarials if combined appropriately with conventional antimalarial drugs.

A series of potent and selective, triazolylphenyl-based histone deacetylases inhibitors with activity against pancreatic cancer cells and Plasmodium falciparum.

The discovery of the rules governing the inhibition of the various HDAC isoforms is likely to be key to identifying improved therapeutics that act as epigenetic modulators of gene transcription. Herein we present results on the modification of the CAP region of a set of triazolylphenyl-based HDACIs, and show that the nature of substitution on the phenyl ring plays a role in their selectivity for HDAC1 versus HDAC6, with low to moderate selectivity (2-51-fold) being achieved. In light of the valuable selectivity and potency that were identified for the triazolylphenyl ligand 6b in the inhibition of HDAC6 (IC50 = 1.9 nM), this compound represents a valuable research tool and a candidate for further chemical modifications. Lastly, these new HDACIs were studied for both their anticancer and antimalarial activity, which serve to validate the superior activity of the HDACI 10c.

Clinical development of new prophylactic antimalarial drugs after the 5th Amendment to the Declaration of Helsinki.

Malaria is of continuing concern in nonimmune traveling populations. Traditionally, antimalarial drugs have been developed as agents for dual indications (treatment and prophylaxis). However, since 2000, when the 5th Amendment to the Declaration of Helsinki (DH2000) was adopted, development of new malaria prophylaxis drugs in this manner has ceased. As a consequence, there may not be any new drugs licensed for this indication in the foreseeable future. Major pharmaceutical companies have interpreted DH2000 to mean that the traditional development paradigm may be considered unethical because of doubt over the likelihood of benefit to endemic populations participating in clinical studies, the use of placebo, and the sustainability of post-trial access to study medications. In this article, we explore the basis of these concerns and suggest that the traditional development paradigm remains ethical under certain circumstances. We also consider alternative approaches that may be more attractive to sponsors as they either do not use placebo, or utilize populations in endemic countries who may unambiguously benefit. These approaches represent the way forward in the future, but are at present unproven in clinical practice, and face numerous regulatory, logistical and technical challenges. Consequently, in the short term, we argue that the traditional clinical development paradigm remains the most feasible approach and is ethical and consistent with the spirit of DH2000 under the appropriate circumstances.

Transcriptional profiling of mefloquine-induced disruption of calcium homeostasis in neurons in vitro.

Mefloquine is associated with adverse neurological effects that are mediated via unknown mechanisms. Recent in vitro studies have shown that mefloquine disrupts neuronal calcium homeostasis via liberation of the endoplasmic reticulum (ER) store and induction of calcium influx across the plasma membrane. In the present study, global changes in gene expression induced in neurons in response to mefloquine-induced disruption of calcium homeostasis and appropriate control agents were investigated in vitro using Affymetrix arrays. The mefloquine transcriptome was found to be enriched for important regulatory sequences of the unfolded protein response and the drug was also found to induce key ER stress proteins, albeit in a manner dissimilar to, and at higher equivalent concentrations than, known ER-tropic agents like thapsigargin. Mefloquine also down-regulated several important functional categories of genes, including transcripts encoding G proteins and ion channels. These effects may be related to intrusion of extracellular calcium since they were also observed after glutamate, but not thapsigargin, hydrogen peroxide, or low-dose mefloquine treatment. Mefloquine could be successfully differentiated from other treatments on the basis of principle component analysis of its "calcium-relevant" transcriptome. These data may aid interpretation of expression of results from future in vivo studies.

The antimalarial potential of 4-quinolinecarbinolamines may be limited due to neurotoxicity and cross-resistance in mefloquine-resistant Plasmodium falciparum strains.

The clinical potential of mefloquine has been compromised by reports of adverse neurological effects. A series of 4-quinolinecarbinolamines were compared in terms of neurotoxicity and antimalarial activity in an attempt to identify replacement drugs. Neurotoxicity (MTT [thiazolyl blue reduction] assay) was assessed by exposure of cultured embryonic rat neurons to graded concentrations of the drugs for 20 min. The 50% inhibitory concentration (IC(50)) of mefloquine was 25 microM, while those of the analogs were 19 to 200 microM. The relative (to mefloquine) therapeutic indices of the analogs were determined after using the tritiated hypoxanthine assay for assessment of the antimalarial activity of the analogs against mefloquine-sensitive (W2) and -resistant (D6 and TM91C235) Plasmodium falciparum strains. Five analogs, WR157801, WR073892, WR007930, WR007333, and WR226253, were less neurotoxic than mefloquine and exhibited higher relative therapeutic indices (RTIs) against TM91C235 (2.9 to 12.2). Conventional quinoline antimalarials were generally less neurotoxic (IC(50)s of 400, 600, and 900 for amodiaquine, chloroquine, and quinine) or had higher RTIs (e.g., 30 for halofantrine against TM91C235). The neurotoxicity data for the 4-quinolinecarbinolamines were used to develop a three-dimensional (3D), function-based pharmacophore. The crucial molecular features correlated with neurotoxicity were a hydrogen bond acceptor (lipid) function, an aliphatic hydrophobic function, and a ring aromatic function specifically distributed in the 3D surface of the molecule. Mapping of the 3D structures of a series of structurally diverse quinolines to the pharmacophore allowed accurate qualitative predictions of neurotoxicity (or not) to be made. Extension of this in silico screening approach may aid in the identification of less-neurotoxic quinoline analogs.

The acute neurotoxicity of mefloquine may be mediated through a disruption of calcium homeostasis and ER function in vitro.

BACKGROUND: There is no established biochemical basis for the neurotoxicity of mefloquine. We investigated the possibility that the acute in vitro neurotoxicity of mefloquine might be mediated through a disruptive effect of the drug on endoplasmic reticulum (ER) calcium homeostasis. METHODS: Laser scanning confocal microscopy was employed to monitor real-time changes in basal intracellular calcium concentrations in embryonic rat neurons in response to mefloquine and thapsigargin (a known inhibitor of the ER calcium pump) in the presence and absence of external calcium. Changes in the transcriptional regulation of known ER stress response genes in neurons by mefloquine were investigated using Affymetrix arrays. The MTT assay was employed to measure the acute neurotoxicity of mefloquine and its antagonisation by thapsigargin. RESULTS: At physiologically relevant concentrations mefloquine was found to mobilize neuronal ER calcium stores and antagonize the pharmacological action of thapsigargin, a specific inhibitor of the ER calcium pump. Mefloquine also induced a sustained influx of extra-neuronal calcium via an unknown mechanism. The transcription of key ER proteins including GADD153, PERK, GRP78, PDI, GRP94 and calreticulin were up-regulated by mefloquine, suggesting that the drug induced an ER stress response. These effects appear to be related, in terms of dose effect and kinetics of action, to the acute neurotoxicity of the drug in vitro. CONCLUSIONS: Mefloquine was found to disrupt neuronal calcium homeostasis and induce an ER stress response at physiologically relevant concentrations, effects that may contribute, at least in part, to the neurotoxicity of the drug in vitro.

Effect of sample size and P-value filtering techniques on the detection of transcriptional changes induced in rat neuroblastoma (NG108) cells by mefloquine.

BACKGROUND: There is no known biochemical basis for the adverse neurological events attributed to mefloquine. Identification of genes modulated by toxic agents using microarrays may provide sufficient information to generate hypotheses regarding their mode of action. However, this utility may be compromised if sample sizes are too low or the filtering methods used to identify differentially expressed genes are inappropriate. METHODS: The transcriptional changes induced in rat neuroblastoma cells by a physiological dose of mefloquine (10 micro-molar) were investigated using Affymetrix arrays. A large sample size was used (total of 16 arrays). Genes were ranked by P-value (t-test). RT-PCR was used to confirm (or reject) the expression changes of several of the genes with the lowest P-values. Different P-value filtering methods were compared in terms of their ability to detect these differentially expressed genes. A retrospective power analysis was then performed to determine whether the use of lower sample sizes might also have detected those genes with altered transcription. RESULTS: Based on RT-PCR, mefloquine upregulated cJun, IkappaB and GADD153. Reverse Holm-Bonferroni P-value filtering was superior to other methods in terms of maximizing detection of differentially expressed genes but not those with unaltered expression. Reduction of total microarray sample size (< 10) impaired the capacity to detect differentially expressed genes. CONCLUSIONS: Adequate sample sizes and appropriate selection of P-value filtering methods are essential for the reliable detection of differentially expressed genes. The changes in gene expression induced by mefloquine suggest that the ER might be a neuronal target of the drug.