An in vitro toolbox to accelerate anti-malarial drug discovery and development.

BACKGROUND: Modelling and simulation are being increasingly utilized to support the discovery and development of new anti-malarial drugs. These approaches require reliable in vitro data for physicochemical properties, permeability, binding, intrinsic clearance and cytochrome P450 inhibition. This work was conducted to generate an in vitro data toolbox using standardized methods for a set of 45 anti-malarial drugs and to assess changes in physicochemical properties in relation to changing target product and candidate profiles. METHODS: Ionization constants were determined by potentiometric titration and partition coefficients were measured using a shake-flask method. Solubility was assessed in biorelevant media and permeability coefficients and efflux ratios were determined using Caco-2 cell monolayers. Binding to plasma and media proteins was measured using either ultracentrifugation or rapid equilibrium dialysis. Metabolic stability and cytochrome P450 inhibition were assessed using human liver microsomes. Sample analysis was conducted by LC-MS/MS. RESULTS: Both solubility and fraction unbound decreased, and permeability and unbound intrinsic clearance increased, with increasing Log D7.4. In general, development compounds were somewhat more lipophilic than legacy drugs. For many compounds, permeability and protein binding were challenging to assess and both required the use of experimental conditions that minimized the impact of non-specific binding. Intrinsic clearance in human liver microsomes was varied across the data set and several compounds exhibited no measurable substrate loss under the conditions used. Inhibition of cytochrome P450 enzymes was minimal for most compounds. CONCLUSIONS: This is the first data set to describe in vitro properties for 45 legacy and development anti-malarial drugs. The studies identified several practical methodological issues common to many of the more lipophilic compounds and highlighted areas which require more work to customize experimental conditions for compounds being designed to meet the new target product profiles. The dataset will be a valuable tool for malaria researchers aiming to develop PBPK models for the prediction of human PK properties and/or drug-drug interactions. Furthermore, generation of this comprehensive data set within a single laboratory allows direct comparison of properties across a large dataset and evaluation of changing property trends that have occurred over time with changing target product and candidate profiles.

Antimalarial efficacy of MMV390048, an inhibitor of Plasmodium phosphatidylinositol 4-kinase.

As part of the global effort toward malaria eradication, phenotypic whole-cell screening revealed the 2-aminopyridine class of small molecules as a good starting point to develop new antimalarial drugs. Stemming from this series, we found that the derivative, MMV390048, lacked cross-resistance with current drugs used to treat malaria. This compound was efficacious against all Plasmodium life cycle stages, apart from late hypnozoites in the liver. Efficacy was shown in the humanized Plasmodium falciparum mouse model, and modest reductions in mouse-to-mouse transmission were achieved in the Plasmodium berghei mouse model. Experiments in monkeys revealed the ability of MMV390048 to be used for full chemoprotection. Although MMV390048 was not able to eliminate liver hypnozoites, it delayed relapse in a Plasmodium cynomolgi monkey model. Both genomic and chemoproteomic studies identified a kinase of the Plasmodium parasite, phosphatidylinositol 4-kinase, as the molecular target of MMV390048. The ability of MMV390048 to block all life cycle stages of the malaria parasite suggests that this compound should be further developed and may contribute to malaria control and eradication as part of a single-dose combination treatment.

Structure-Activity Relationship of the Antimalarial Ozonide Artefenomel (OZ439).

Building on insights gained from the discovery of the antimalarial ozonide arterolane (OZ277), we now describe the structure-activity relationship (SAR) of the antimalarial ozonide artefenomel (OZ439). Primary and secondary amino ozonides had higher metabolic stabilities than tertiary amino ozonides, consistent with their higher pKa and lower log D7.4 values. For primary amino ozonides, addition of polar functional groups decreased in vivo antimalarial efficacy. For secondary amino ozonides, additional functional groups had variable effects on metabolic stability and efficacy, but the most effective members of this series also had the highest log D7.4 values. For tertiary amino ozonides, addition of polar functional groups with H-bond donors increased metabolic stability but decreased in vivo antimalarial efficacy. Primary and tertiary amino ozonides with cycloalkyl and heterocycle substructures were superior to their acyclic counterparts. The high curative efficacy of these ozonides was most often associated with high and prolonged plasma exposure, but exposure on its own did not explain the presence or absence of either curative efficacy or in vivo toxicity.

Tetrahydro-2-naphthyl and 2-Indanyl Triazolopyrimidines Targeting Plasmodium falciparum Dihydroorotate Dehydrogenase Display Potent and Selective Antimalarial Activity.

Malaria persists as one of the most devastating global infectious diseases. The pyrimidine biosynthetic enzyme dihydroorotate dehydrogenase (DHODH) has been identified as a new malaria drug target, and a triazolopyrimidine-based DHODH inhibitor 1 (DSM265) is in clinical development. We sought to identify compounds with higher potency against Plasmodium DHODH while showing greater selectivity toward animal DHODHs. Herein we describe a series of novel triazolopyrimidines wherein the p-SF5-aniline was replaced with substituted 1,2,3,4-tetrahydro-2-naphthyl or 2-indanyl amines. These compounds showed strong species selectivity, and several highly potent tetrahydro-2-naphthyl derivatives were identified. Compounds with halogen substitutions displayed sustained plasma levels after oral dosing in rodents leading to efficacy in the P. falciparum SCID mouse malaria model. These data suggest that tetrahydro-2-naphthyl derivatives have the potential to be efficacious for the treatment of malaria, but due to higher metabolic clearance than 1, they most likely would need to be part of a multidose regimen.

Two analogues of fenarimol show curative activity in an experimental model of Chagas disease.

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi (T. cruzi), is an increasing threat to global health. Available medicines were introduced over 40 years ago, have undesirable side effects, and give equivocal results of cure in the chronic stage of the disease. We report the development of two compounds, 6 and (S)-7, with PCR-confirmed curative activity in a mouse model of established T. cruzi infection after once daily oral dosing for 20 days at 20 mg/kg 6 and 10 mg/kg (S)-7. Compounds 6 and (S)-7 have potent in vitro activity, are noncytotoxic, show no adverse effects in vivo following repeat dosing, are prepared by a short synthetic route, and have druglike properties suitable for preclinical development.

A prodrug approach toward cancer-related carbonic anhydrase inhibition.

The selective inhibition of cancer-associated human carbonic anhydrase (CA) enzymes, specifically CA IX and XII, has been validated as a mechanistically novel approach toward personalized cancer management. Herein we report the design and synthesis of a panel of 24 novel glycoconjugate primary sulfonamides that bind to the extracellular catalytic domain of CA IX and XII. These compounds were synthesized from variably acylated glycopyranosyl azides and either 3- or 4-ethynyl benzene sulfonamide using Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC). The CA enzyme inhibition profile for all compounds was determined, while in vitro metabolic stability, plasma stability, and plasma protein binding for a representative set of compounds was measured. Our findings demonstrate the influence of the differing acyl groups on these key biopharmaceutical properties, confirming that acyl group protected carbohydrate-based sulfonamides have potential as prodrugs for selectively targeting the extracellular cancer-associated CA enzymes.

In vitro and in vivo characterization of the antimalarial lead compound SSJ-183 in Plasmodium models.

The objective of this work was to characterize the in vitro (Plasmodium falciparum) and in vivo (Plasmodium berghei) activity profile of the recently discovered lead compound SSJ-183. The molecule showed in vitro a fast and strong inhibitory effect on growth of all P. falciparum blood stages, with a tendency to a more pronounced stage-specific action on ring forms at low concentrations. Furthermore, the compound appeared to be equally efficacious on drug-resistant and drug-sensitive parasite strains. In vivo, SSJ-183 showed a rapid onset of action, comparable to that seen for the antimalarial drug artesunate. SSJ-183 exhibited a half-life of about 10 hours and no significant differences in absorption or exposure between noninfected and infected mice. SSJ-183 appears to be a promising new lead compound with an attractive antimalarial profile.

3',4'-Bis-difluoromethoxycinnamoylanthranilate (FT061): an orally-active antifibrotic agent that reduces albuminuria in a rat model of progressive diabetic nephropathy.

Cinnamoylanthranilates including tranilast have been identified as promising antifibrotics that can reduce fibrosis occurring in the kidney during diabetes, thereby delaying and/or preventing kidney dysfunction. Structure-activity relationships aimed at improving potency and metabolic stability have led to the discovery of FT061. This compound, which bears a bis-difluoromethoxy catechol, attenuates TGF-ß-stimulated production of collagen in cultured renal mesangial cells (approx 50% at 3 µM). When dosed orally at 20mg/kg to male Sprague Dawley rats, FT061 exhibited a high bioavailability (73%), Cmax of 200 µM and Tmax of 150 min, and a half-life of 5.4h. FT061 reduced albuminuria when orally dosed in rats at 200 mg kg/day in a late intervention study of a rat model of progressive diabetic nephropathy.

Discovery and structure-activity relationships of pyrrolone antimalarials.

In the pursuit of new antimalarial leads, a phenotypic screening of various commercially sourced compound libraries was undertaken by the World Health Organisation Programme for Research and Training in Tropical Diseases (WHO-TDR). We report here the detailed characterization of one of the hits from this process, TDR32750 (8a), which showed potent activity against Plasmodium falciparum K1 (EC(50) ~ 9 nM), good selectivity (>2000-fold) compared to a mammalian cell line (L6), and significant activity against a rodent model of malaria when administered intraperitoneally. Structure-activity relationship studies have indicated ways in which the molecule could be optimized. This compound represents an exciting start point for a drug discovery program for the development of a novel antimalarial.

Comparative antimalarial activities and ADME profiles of ozonides (1,2,4-trioxolanes) OZ277, OZ439, and their 1,2-dioxolane, 1,2,4-trioxane, and 1,2,4,5-tetraoxane isosteres.

To ascertain the structure-activity relationship of the core 1,2,4-trioxolane substructure of dispiro ozonides OZ277 and OZ439, we compared the antimalarial activities and ADME profiles of the 1,2-dioxolane, 1,2,4-trioxane, and 1,2,4,5-tetraoxane isosteres. Consistent with previous data, both dioxolanes had very weak antimalarial properties. For the OZ277 series, the trioxane isostere had the best ADME profile, but its overall antimalarial efficacy was not superior to that of the trioxolane or tetraoxane isosteres. For the OZ439 series, there was a good correlation between the antimalarial efficacy and ADME profiles in the rank order trioxolane > trioxane > tetraoxane. As we have previously observed for OZ439 versus OZ277, the OZ439 series peroxides had superior exposure and efficacy in mice compared to the corresponding OZ277 series peroxides.

Synthesis of acylated glycoconjugates as templates to investigate in vitro biopharmaceutical properties.

A series of novel glycopyranosyl azides were synthesised wherein the carbohydrate moiety was peracylated with four acetyl, propionyl, butanoyl, pentanoyl (valeryl) or 3-methylbutanoyl (isovaleryl) ester linked groups. A panel of glycoconjugates was synthesised from these glycopyranosyl azides using copper-catalysed azide-alkyne cycloaddition. The in vitro metabolic stability, plasma stability and plasma protein binding was then measured to establish the impact of the different acyl group when presented on a common scaffold. The acetyl, propionyl and butanoyl esters exhibited metabolism consistent with esterase processing, and various mono-, di- and tri-acylated hydrolysis products as well as the fully hydrolysed compound were detected. In contrast, the pentanoyl and 3-methylbutanoyl esters were stable.

Metallocene-based inhibitors of cancer-associated carbonic anhydrase enzymes IX and XII.

In this study, 20 metallocene-based compounds comprising extensive structural diversity were synthesized and evaluated as carbonic anhydrase (CA, EC 4.2.1.1) inhibitors. These compounds proved moderate to good CA inhibitors in vitro, with several compounds displaying selectivity for cancer-associated isozymes CA IX and CA XII compared to off-target CA I and CA II. Compound 6 was the most potent ferrocene-based inhibitor with K(i)s of 5.9 and 6.8 nM at CA IX and XII, respectively. A selection of key drug-like parameters comprising Log P, Log D, solubility, and in vitro metabolic stability and permeability were measured for two of the ferrocene-based compounds, regioisomers 1 and 5. Compounds 1 and 5 were found to have characteristics consistent with lipophilic compounds, however, our findings show that the lipophilicity of the ferrocene moiety is not well modeled by replacement with either a naphthyl or a phenyl moiety in software prediction tools.

Probing a potential in vivo drug-excipient interaction: temporal effects of a modified ß-cyclodextrin on the intravenous pharmacokinetics of a model high-affinity drug ligand.

The investigational synthetic ozonide, OZ209, has previously been shown to have high binding affinity for sulfobutylether(7)-ß-cyclodextrin [(SBE)(7)-ß-CD] resulting in altered pharmacokinetics when administered intravenously to rats in a (SBE)(7)-ß-CD aqueous formulation. In the present study, OZ209 and (SBE)(7)-ß-CD have been used to probe whether a modified ß-CD excipient, on systemic administration, can bind to and alter the pharmacokinetics of a coadministered drug. When (SBE)(7)-ß-CD was administered 60 min after OZ209, a spike in the concentration of OZ209 in blood and plasma was detected within 2 min of the (SBE)(7)-ß-CD infusion, and this was accompanied by a temporary decrease in the whole blood-to-plasma partitioning ratio of OZ209, the duration of which was dependent upon the dose of (SBE)(7)-ß-CD. Administration of (SBE)(7)-ß-CD also resulted in increased urinary excretion of OZ209. By contrast, administration of (SBE)(7)-ß-CD 4 h prior to OZ209 had no pronounced effect on the blood or plasma pharmacokinetics of OZ209, consistent with the (SBE)(7)-ß-CD having been largely eliminated prior to the administration of OZ209. This study is the first to demonstrate an in vivo drug-excipient interaction between a modified ß-CD and a coadministered drug, and also demonstrates that such an interaction can be avoided through appropriate consideration of CD pharmacokinetics.

Analogues of fenarimol are potent inhibitors of Trypanosoma cruzi and are efficacious in a murine model of Chagas disease.

We report the discovery of nontoxic fungicide fenarimol (1) as an inhibitor of Trypanosoma cruzi ( T. cruzi ), the causative agent of Chagas disease, and the results of structure-activity investigations leading to potent analogues with low nM IC(50)s in a T. cruzi whole cell in vitro assay. Lead compounds suppressed blood parasitemia to virtually undetectable levels after once daily oral dosing in mouse models of T. cruzi infection. Compounds are chemically tractable, allowing rapid optimization of target biological activity and drug characteristics. Chemical and biological studies undertaken in the development of the fenarimol series toward the goal of delivering a new drug candidate for Chagas disease are reported.

3,5-Diaryl-2-aminopyridines as a novel class of orally active antimalarials demonstrating single dose cure in mice and clinical candidate potential.

A novel class of orally active antimalarial 3,5-diaryl-2-aminopyridines has been identified from phenotypic whole cell high-throughput screening of a commercially available SoftFocus kinase library. The compounds were evaluated in vitro for their antiplasmodial activity against K1 (chloroquine and drug-resistant strain) and NF54 (chloroquine-susceptible strain) as well as for their cytotoxicity. Synthesis and structure-activity studies identified a number of promising compounds with selective antiplasmodial activity. One of these frontrunner compounds, 15, was equipotent across the two strains (K1 = 25.0 nM, NF54 = 28.0 nM) and superior to chloroquine in the K1 strain (chloroquine IC(50) K1 = 194.0 nM). Compound 15 completely cured Plasmodium berghei-infected mice with a single oral dose of 30 mg/kg. Dose-response studies generated ED(50) and ED(90) values of 0.83 and 1.74 mg/kg for 15 in the standard four-dose Peters test. Pharmacokinetic studies in the rat indicated that this compound has good oral bioavailability (51% at 20 mg/kg) and a reasonable half-life (t(1/2) ∼ 7-8 h).

The involvement of a Na⁺- and Cl⁻-dependent transporter in the brain uptake of amantadine and rimantadine.

Despite their structural similarity, the two anti-influenza adamantane compounds amantadine (AMA) and rimantadine (RIM) exhibit strikingly different rates of blood-brain barrier (BBB) transport. However, the molecular mechanisms facilitating the higher rate of in situ BBB transport of RIM, relative to AMA, remain unclear. The aim of this study, therefore, was to determine whether differences in the extent of brain uptake between these two adamantanes also occurred in vivo, and elucidate the potential carrier protein facilitating their BBB transport using immortalized human brain endothelial cells (hCMEC/D3). Following oral administration to Swiss Outbred mice, RIM exhibited 2.4-3.0-fold higher brain-to-plasma exposure compared to AMA, which was not attributable to differences in the degree of plasma protein binding. At concentrations representative of those obtained in vivo, the hCMEC/D3 cell uptake of RIM was 4.5-15.7-fold higher than that of AMA, with Michaelis-Menten constants 6.3 and 238.4 µM, respectively. The hCMEC/D3 cellular uptake of both AMA and RIM was inhibited by various cationic transporter inhibitors (cimetidine, choline, quinine, and tetraethylammonium) and was dependent on extracellular pH, membrane depolarization and Na⁺ and Cl⁻ ions. Such findings indicated the involvement of the neutral and cationic amino acid transporter B°,⁺ (ATB°,⁺) in the uptake of AMA and RIM, which was demonstrated to be expressed (at the protein level) in the hCMEC/D3 cells. Indeed, AMA and RIM appeared to interact with this transporter, as shown by a 53-70% reduction in the hCMEC/D3 uptake of the specific ATB°,⁺ substrate ³H-glycine in their presence. These studies suggest the involvement of ATB°,⁺ in the disposition of these cationic drugs across the BBB, a transporter with the potential to be exploited for targeted drug delivery to the brain.

Diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)) protects against peroxynitrite-induced nitrosative damage and prolongs survival in amyotrophic lateral sclerosis mouse model.

Amyotrophic lateral sclerosis (ALS) is a progressive paralyzing disease characterized by tissue oxidative damage and motor neuron degeneration. This study investigated the in vivo effect of diacetylbis(N(4)-methylthiosemicarbazonato) copper(II) (CuII(atsm)), which is an orally bioavailable, blood-brain barrier-permeable complex. In vitro the compound inhibits the action of peroxynitrite on Cu,Zn-superoxide dismutase (SOD1) and subsequent nitration of cellular proteins. Oral treatment of transgenic SOD1G93A mice with CuII(atsm) at presymptomatic and symptomatic ages was performed. The mice were examined for improvement in lifespan and motor function, as well as histological and biochemical changes to key disease markers. Systemic treatment of SOD1G93A mice significantly delayed onset of paralysis and prolonged lifespan, even when administered to symptomatic animals. Consistent with the properties of this compound, treated mice had reduced protein nitration and carbonylation, as well as increased antioxidant activity in spinal cord. Treatment also significantly preserved motor neurons and attenuated astrocyte and microglial activation in mice. Furthermore, CuII(atsm) prevented the accumulation of abnormally phosphorylated and fragmented TAR DNA-binding protein-43 (TDP-43) in spinal cord, a protein pivotal to the development of ALS. CuII(atsm) therefore represents a potential new class of neuroprotective agents targeting multiple major disease pathways of motor neurons with therapeutic potential for ALS.

Discovery of 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105), a tubulin polymerization inhibitor with potent antiproliferative and tumor vascular disrupting properties.

A structure-activity relationship (SAR) guided design of novel tubulin polymerization inhibitors has resulted in a series of benzo[b]furans with exceptional potency toward cancer cells and activated endothelial cells. The potency of early lead compounds has been substantially improved through the synergistic effect of introducing a conformational bias and additional hydrogen bond donor to the pharmacophore. Screening of a focused library of potent tubulin polymerization inhibitors for selectivity against cancer cells and activated endothelial cells over quiescent endothelial cells has afforded 7-hydroxy-6-methoxy-2-methyl-3-(3,4,5-trimethoxybenzoyl)benzo[b]furan (BNC105, 8) as a potent and selective antiproliferative. Because of poor solubility, 8 is administered as its disodium phosphate ester prodrug 9 (BNC105P), which is rapidly cleaved in vivo to return the active 8. 9 exhibits both superior vascular disrupting and tumor growth inhibitory properties compared with the benchmark agent combretastatin A-4 disodium phosphate 5 (CA4P).

Synthetic ozonide drug candidate OZ439 offers new hope for a single-dose cure of uncomplicated malaria.

Ozonide OZ439 is a synthetic peroxide antimalarial drug candidate designed to provide a single-dose oral cure in humans. OZ439 has successfully completed Phase I clinical trials, where it was shown to be safe at doses up to 1,600 mg and is currently undergoing Phase IIa trials in malaria patients. Herein, we describe the discovery of OZ439 and the exceptional antimalarial and pharmacokinetic properties that led to its selection as a clinical drug development candidate. In vitro, OZ439 is fast-acting against all asexual erythrocytic Plasmodium falciparum stages with IC(50) values comparable to those for the clinically used artemisinin derivatives. Unlike all other synthetic peroxides and semisynthetic artemisinin derivatives, OZ439 completely cures Plasmodium berghei-infected mice with a single oral dose of 20 mg/kg and exhibits prophylactic activity superior to that of the benchmark chemoprophylactic agent, mefloquine. Compared with other peroxide-containing antimalarial agents, such as the artemisinin derivatives and the first-generation ozonide OZ277, OZ439 exhibits a substantial increase in the pharmacokinetic half-life and blood concentration versus time profile in three preclinical species. The outstanding efficacy and prolonged blood concentrations of OZ439 are the result of a design strategy that stabilizes the intrinsically unstable pharmacophoric peroxide bond, thereby reducing clearance yet maintaining the necessary Fe(II)-reactivity to elicit parasite death.

Preclinical comparison of intravenous melphalan pharmacokinetics administered in formulations containing either (SBE)7 m-ß-cyclodextrin or a co-solvent system.

The aim of this work was to evaluate the impact of sulfobutyl ether ß-cyclodextrin ((SBE)(7 m)-ß-CD; Captisol(®)) on the in vivo pharmacokinetics of melphalan in rats. Melphalan is a chemically unstable antineoplastic drug which in the current commercial formulation (Alkeran(®) for Injection) has some limitations with regard to solubility, stability and biocompatibility. Melphalan formulations containing (SBE)(7 m)-ß-CD have previously been evaluated in vitro and shown to significantly reduce the rate of degradation and to simplify the reconstitution procedure for lyophilised melphalan. In this study, melphalan was administered intravenously in rats in formulations that either contain (SBE)(7 m)-ß-CD or a co-solvent system (i.e. the commercial formulation). Pharmacokinetic parameters, including half-life, volume of distribution, clearance and extent of renal elimination of melphalan were essentially unchanged between the two formulations. These findings indicate that the pharmacokinetics of melphalan are not altered in the presence of (SBE)(7 m)-ß-CD consistent with a rapid shift in the equilibrium to the fully dissociated drug from the fraction associated with the cyclodextrin host molecule upon intravenous administration.