Pharmacokinetics-Pharmacodynamics Driven Approach for Lead Optimization in Anti-Mycobacterial and Anti-Malarial Drug Discovery

Ph.DDOCTOR OF PHILOSOPH

Artificial neural network analysis of pharmacokinetic and toxicity properties of lead molecules for Dengue fever, Tuberculosis and Malaria

Poor pharmacokinetic and toxicity profiles are major reasons for the low rate of advancing lead drug candidates into efficacy studies. The In-silico prediction of primary pharmacokinetic and toxicity properties in the drug discovery and development process can be used as guidance in the design of candidates. In-silico parameters can also be used to choose suitable compounds for in-vivo testing thereby reducing the number of animals used in experiments. At the Novartis Institute for Tropical Diseases, a data set has been curated from in-house measurements in the disease areas of Dengue, Tuberculosis and Malaria. Volume of distribution, half-life, total in-vivo clearance, in-vitro human plasma protein binding and in-vivo oral bioavailability have been measured for molecules in the lead optimization stage in each of these three disease areas. Data for the inhibition of the hERG channel using the radio ligand binding dofetilide assay was determined for a set of 300 molecules in these therapeutic areas. Based on this data, Artificial Neural Networks were used to construct In-silico models for each of the properties listed above that can be used to prioritize candidates for lead optimization and to assist in selecting promising molecules for in-vivo pharmacokinetic studies

Physico-chemical, in vitro potency, pharmaco-kinetic and dynamic properties of anti-TB drugs

Physico-chemical properties, in vitro potency, and in vitro and in vivo pharmacokinetic (PK) properties of new leads are critical determinants of in vivo efficacy. Hence, understanding how these parameters correlate is important for early drug discovery and preclinical development. In the current study we have characterized 36 first-line, second-line and other anti-TB drugs as well as clinical candidates. Although these data for most compounds have been reported, comparability of the existing data is limited as different methods and readouts were employed. Here we have generated a compendium of anti-TB drug characteristics using standardized methodology as a reference for the TB drug discovery community. Although the physico-chemical properties of anti-mycobacterials were diverse, they were proximal to gram negative anti-bacterials. Most of the compounds were bactericidal and had activity in a macrophage infection model. Majority of the anti-TB compounds showed good solubility, permeability and metabolic stability in standard in vitro PK assays. Improved solubility and Caco-2 permeability was noticed when compounds had molecular weight and cLog P < 500 and 5, respectively. Comprehensive data analysis using human PK parameters and their correlation with in silico and in vitro compounds properties showed that volume of distribution, clearance, plasma protein binding and oral bioavailability had significant relationship with cLogP. Not surprisingly, most compounds having moderate to high oral bioavailability complied with Lipinski’s rule of 5, and also had good solubility and high permeability

Pharmacokinetics-pharmacodynamics analysis of spiroindolone analogs and KAE609 in a murine malaria model

Limited information is available on pharmacokinetic and pharmacodynamic (PK-PD) parameters driving the efficacy of anti-malarial drugs. Our objective was to determine dose response relationship to a panel of related spiroindolone analogs and identify the PK-PD index that best correlate with the efficacy of KAE609, a selected class representative. Dose-response efficacy studies were conducted in the P. berghei murine malaria model and the relationship between dose and efficacy was examined. All spiroindolone analogs studied displayed maximum parasitemia reduction with ED90 values ranging between 6 and 38 mg/kg. Most of the analogs provided a maximum survival of 14 to 15 days for the highest dose administered, with complete cure (i.e. >30 days survival) achieved only with KAE609. Further, dose fractionation studies were conducted for KAE609 and the relationship between PK-PD indices and efficacy (i.e. parasitemia reduction) was examined. PK-PD indices were calculated using as threshold (TRE) the in vitro potency against P. berghei (2*IC99). The percentage of the time in which KAE609 plasma concentration remained above 2*IC99 for 48 hrs (%T>TRE) and the AUC0-48/TRE correlated well with parasite reduction (R2=0.96 and 0.95, respectively) followed by the Cmax/TRE (R2=0.88). We cannot clearly differentiate between concentration and time-dependent killing for KAE609 based on this experiment. Furthermore, the present results suggest that for KAE609 a dosing regimen covering T>TRE of 100%, AUC/TRE of 600 and Cmax/TRE of 35 for an observation period of 48h is likely to result in >99% parasitemia reduction in the mouse

PI4K is a prophylactic, but not radical curative target in Plasmodium vivax-type malaria parasites

Two Plasmodium PI4 kinase inhibitors, KDU691 and LMV599, were selected for in vivo testing as causal prophylactic and radical cure agents of P. cynomolgi sporozoite infected rhesus macaques, based on their in vitro activity against liver-stages. Animals were infected with P. cynomolgi sporozoites and compounds were dosed orally. Both KDU691 and LMV599 compounds were fully protective when administered prophylactically and the more potent compound LMV599 achieved protection as a single oral 25 mg/kg dose. In contrast, when tested for radical cure, five daily doses of 20 mg/kg of KDU691 or 25 mg/kg of LMV599 did not prevent relapse as all animals experienced a secondary infection due to the reactivation of hypnozoites in the liver. Pharmacokinetic data show that LMV599 achieved plasma exposure that were sufficient to achieve efficacy based on our in vitro data. These findings indicate that Plasmodium PI4K is a potential drug target for malaria prophylaxis but not radical cure. Longer in vitro culture systems will be required to assess compounds activity on established hypnozoites and predict radical cure in vivo

Design, synthesis and biological evaluation of indole-2-carboxamides, a promising class of anti-tuberculosis agents

Indole-2-carboxamides have been identified as a promising class of anti-tuberculosis agents from phenotypic whole cell high-throughput screening against mycobacteria. One of the hits KCD644, an N-cyclohexyl-4,6-dimethyl-1H-indole-2-carboxamide had low µM potency, high in vitro mouse liver microsomal clearance and low aqueous solubility. Structure activity relationship studies revealed that attaching further small alkyl groups to the cyclohexyl ring significantly improved activity against Mycobacterium tuberculosis (Mtb) but reduced aq solubility. Further, chloro, fluoro or cyano substitutions on the 4- and 6-positions of the indole ring and mono or dimethyl substitution on the 4-position of the cyclohexyl ring significantly improved both in vitro and in vivo metabolic stability. KDZ304 and KDZ349, the lead candidates from this study displayed improved in vitro activity compared to the current standard TB drugs. The low aq solubility of compounds in the series could not be mitigated due to the correlation of lipophilicity with potency against Mtb. However, both compounds displayed favorable in vivo pharmacokinetic properties in rodents when dosed in a microemulsion preconcentrate formulation (MEPC). Both KDZ304 and KDZ349 delivered good oral exposure and also demonstrated in vivo efficacy in a mouse model. Thus, indole-2-carboxamides represent a promising new class of anti-TB agents

Discovery of Tetrahydropyrazolopyrimidine Carboxamide De-rivatives as Potent and Orally Active Novel Anti-Tubercular Agents

ABSTRACT: Tetrahydropyrazolopyrimidine scaffold was identified as a hit series from a Mycobacterium tuberculosis (Mtb) whole cell high through-put screening (HTS) campaign. A series of derivatives of this class were synthesized to evaluate their structure-activity relationship (SAR) and structure-property relationship (SPR). Compound 9 showed potent bactericidal effect and activity against multi-drug resistant tuberculosis (MDR TB) strains. Furthermore compound 9 had a promising in vivo DMPK profile in mouse and exhibited potent in vivo activity in a mouse efficacy model, achieving a reduction of 3.5 log CFU of Mtb after oral administration to infected mice once a day at 100 mg/kg for 28 days. Thus the compound 9 is a potential candidate for inclusion in combination therapies for both drug-sensitive and drug-resistant TB

Indolcarboxamide, a promising pre-clinical candidate for the treatment of multi drug resistant tuberculosis

To combat the drug resistance in tuberculosis, new chemotherapeutics active against multi-drug resistant Mycobacterium tuberculosis are urgently needed. We have identified and characterized the indolcarboxamides as a new class of anti-tubercular bactericidal agents. Genetics and lipid profiling studies identified the molecular target of indolcarboxamides as mmpL3, a transporter of TMM, a promiscuous target essential for mycobacterial cell wall biosynthesis. Two lead candidates NITD-304 and NITD-349 showed a significantly potent anti-TB activity against both drug sensitive and multi-drug resistant clinical Mtb isolates. Both compounds displayed favorable pharmacokinetic properties after oral administration in pre-clinical species. NITD-304 and NITD-349 showed no apparent inhibition of major CYP enzymes and are highly efficacious in both acute and chronic TB mouse efficacy models. Furthermore, we have assessed in vitro and in vivo safety profile of both candidates including two weeks exploratory rat toxicology studies. NITD-304 and NITD-349 show the adequate therapeutic index to justify further development for inclusion in the combination therapies for drug sensitive as well as drug resistant TB

A Cryptosporidium PI(4)K inhibitor is a drug candidate for cryptosporidiosis

The apicomplexan parasite Cryptosporidium is a major cause of infectious diarrhea in children and there are no consistently effective therapies to treat this disease. The search for cryptosporidiosis therapeutics has been hindered by the dearth of models amenable to modern drug discovery approaches. We established a cryptosporidiosis drug discovery screening process built on scalable phenotypic assays and a novel mouse model. We identified pyrazolopyridines as selective ATP-competitive inhibitors of the Cryptosporidium lipid kinase PI(4)K. The pyrazolopyridine KDU731 inhibits Cryptosporidium growth in multiple in vitro assays. In addition, oral treatment with KDU731 results in potent reduction in oocyst shedding in Cryptosporidium infected immunocompromised mice as well as rapid resolution of diarrheal symptoms in the neonatal calf cryptosporidiosis model. Collectively, our results chemically validate the Cryptosporidium lipid kinase PI(4)K as a drug target for the treatment of cryptosporidiosis and support the progression of the drug candidate KDU731 to further preclinical characterization