Antiviral Nucleotide Incorporation by Recombinant Human Mitochondrial RNA Polymerase Is Predictive of Increased In Vivo Mitochondrial Toxicity Risk.

Nucleoside or nucleotide inhibitors are a highly successful class of antivirals due to selectivity, potency, broad coverage, and high barrier to resistance. Nucleosides are the backbone of combination treatments for HIV, hepatitis B virus, and, since the FDA approval of sofosbuvir in 2013, also for hepatitis C virus (HCV). However, many promising nucleotide inhibitors have advanced to clinical trials only to be terminated due to unexpected toxicity. Here we describe the in vitro pharmacology of compound 1, a monophosphate prodrug of a 2'-ethynyluridine developed for the treatment of HCV. Compound 1 inhibits multiple HCV genotypes in vitro (50% effective concentration [EC50], 0.05 to 0.1 µM) with a selectivity index of >300 (50% cytotoxic concentration [CC50], 30 µM in MT-4 cells). The active triphosphate metabolite of compound 1, compound 2, does not inhibit human α, ß, or γ DNA polymerases but was a substrate for incorporation by the human mitochondrial RNA polymerase (POLRMT). In dog, the oral administration of compound 1 resulted in elevated serum liver enzymes and microscopic changes in the liver. Transmission electron microscopy showed significant mitochondrial swelling and lipid accumulation in hepatocytes. Gene expression analysis revealed dose-proportional gene signature changes linked to loss of hepatic function and increased mitochondrial dysfunction. The potential of in vivo toxicity through mitochondrial polymerase incorporation by nucleoside analogs has been previously shown. This study shows that even moderate levels of nucleotide analog incorporation by POLRMT increase the risk of in vivo mitochondrial dysfunction. Based on these results, further development of compound 1 as an anti-HCV compound was terminated.

Naphthoquine-induced Central Nervous System and Hepatic Vasculocentric Toxicity in the Beagle Dog.

Naphthoquine phosphate (NP) was considered as a partner drug with a promising antimalarial drug candidate. Here we report unexpected adverse clinical signs and microscopic findings in a canine pilot toxicology study with NP. Male and female dogs were dosed daily by oral gavage with NP at 2, 10, or 50 mg/kg/day for a maximum of 14 days. NP was not tolerated at ≥10 mg/kg/day; several animals were sacrificed in moribund condition and marked neurological clinical signs were noted at 50 mg/kg/day. The main microscopic observation was central nervous system vasculocentric inflammation (mainly lymphocytes and macrophages) in the white and gray matter of various regions of the brain at ≥2 mg/kg/day and at lower incidence in the spinal cord at ≥10 mg/kg/day. Vasculocentric microscopic changes predominantly centered on the centrilobular vein were also observed in the liver at ≥2 mg/kg/day. Females were more sensitive than males with comparable NP plasma exposure. In conclusion, under the conditions of this study, the administration of NP to dogs via daily oral gavage for up to 2 weeks was not tolerated causing moribundity, marked neurological clinical signs, and vasculocentric microscopic changes in the central nervous system and the liver.

Vehicle Systems and Excipients Used in Minipig Drug Development Studies.

Minipigs have been used for dermal drug development studies for decades, and they are currently more frequently considered as the second nonrodent species for pivotal nonclinical studies, in lieu of the dog or nonhuman primate, for compounds delivered via standard systemic routes of administration. Little is known about the tolerability of different excipients in minipigs; sharing knowledge of excipient tolerability and compositions previously used in nonclinical studies may avoid testing of inadequate formulations, thereby contributing to reduced animal usage. This article reviews vehicles employed in the Göttingen(®)minipig based on the combined experience from a number of pharmaceutical companies and contract research organizations. The review includes vehicles tolerated for single or multiple dosing by the Göttingen minipig, some of which are not appropriate for administration to other common nonrodent species (e.g., dogs). By presenting these data for dermal, oral, subcutaneous, and intravenous routes of administration, studies to qualify these vehicles in minipigs can be minimized or avoided. Additionally, investigators may more frequently consider using the minipig in place of higher species if the tolerability of a vehicle in the minipig is known.

An adenosine nucleoside inhibitor of dengue virus.

Dengue virus (DENV), a mosquito-borne flavivirus, is a major public health threat. The virus poses risk to 2.5 billion people worldwide and causes 50 to 100 million human infections each year. Neither a vaccine nor an antiviral therapy is currently available for prevention and treatment of DENV infection. Here, we report a previously undescribed adenosine analog, NITD008, that potently inhibits DENV both in vitro and in vivo. In addition to the 4 serotypes of DENV, NITD008 inhibits other flaviviruses, including West Nile virus, yellow fever virus, and Powassan virus. The compound also suppresses hepatitis C virus, but it does not inhibit nonflaviviruses, such as Western equine encephalitis virus and vesicular stomatitis virus. A triphosphate form of NITD008 directly inhibits the RNA-dependent RNA polymerase activity of DENV, indicating that the compound functions as a chain terminator during viral RNA synthesis. NITD008 has good in vivo pharmacokinetic properties and is biologically available through oral administration. Treatment of DENV-infected mice with NITD008 suppressed peak viremia, reduced cytokine elevation, and completely prevented the infected mice from death. No observed adverse effect level (NOAEL) was achieved when rats were orally dosed with NITD008 at 50 mg/kg daily for 1 week. However, NOAEL could not be accomplished when rats and dogs were dosed daily for 2 weeks. Nevertheless, our results have proved the concept that a nucleoside inhibitor could be developed for potential treatment of flavivirus infections.

Inhibition of dengue virus by an ester prodrug of an adenosine analog.

Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen that infects humans. Neither a vaccine nor an antiviral therapy is currently available for DENV. Here, we report an adenosine nucleoside prodrug that potently inhibits DENV replication both in cell culture and in a DENV mouse model. NITD449 (2'-C-acetylene-7-deaza-7-carbamoyladenosine) was initially identified as a parental compound that inhibits all four serotypes of DENV with low cytotoxicity. However, in vivo pharmacokinetic studies indicated that NITD449 had a low level of exposure in plasma when dosed orally. To increase the oral bioavailability, we covalently linked isobutyric acids to the 3'- and 5'-hydroxyl groups of ribose via ester linkage to NITD449, leading to the prodrug NITD203 (3',5'-O-diisobutyryl-2'-C-acetylene-7-deaza-7-carbamoyl-adenosin). Pharmacokinetic analysis showed that upon oral dosing of the prodrug, NITD203 was readily converted to NITD449, resulting in improved exposure of the parental compound in plasma in both mouse and rat. In DENV-infected AG129 mice, oral dosing of the prodrug at 25 mg/kg of body weight reduced peak viremia by 30-fold. Antiviral spectrum analysis showed that NITD203 inhibited various flaviviruses (DENV, yellow fever virus, and West Nile virus) and hepatitis C virus but not Chikungunya virus (an alphavirus). Mode-of-action analysis, using a luciferase-reporting replicon, indicated that NITD203 inhibited DENV RNA synthesis. Although NITD203 exhibited potent in vitro and in vivo efficacies, the compound could not reach a satisfactory no-observable-adverse-effect level (NOAEL) in a 2-week in vivo toxicity study. Nevertheless, our results demonstrate that a prodrug approach using a nucleoside analog could potentially be developed for flavivirus antiviral therapy.

Preclinical evaluation of the antifolate QN254, 5-chloro- N'6'-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine, as an antimalarial drug candidate.

Drug resistance against dihydrofolate reductase (DHFR) inhibitors-such as pyrimethamine (PM)-has now spread to almost all regions where malaria is endemic, rendering antifolate-based malaria treatments highly ineffective. We have previously shown that the di-amino quinazoline QN254 [5-chloro-N'6'-(2,5-dimethoxy-benzyl)-quinazoline-2,4,6-triamine] is active against the highly PM-resistant Plasmodium falciparum V1S strain, suggesting that QN254 could be used to treat malaria in regions with a high prevalence of antifolate resistance. Here, we further demonstrate that QN254 is highly active against Plasmodium falciparum clinical isolates, displaying various levels of antifolate drug resistance, and we provide biochemical and structural evidence that QN254 binds and inhibits the function of both the wild-type and the quadruple-mutant (V1S) forms of the DHFR enzyme. In addition, we have assessed QN254 oral bioavailability, efficacy, and safety in vivo. The compound displays favorable pharmacokinetic properties after oral administration in rodents. The drug was remarkably efficacious against Plasmodium berghei and could fully cure infected mice with three daily oral doses of 30 mg/kg. In the course of these efficacy studies, we have uncovered some dose limiting toxicity at higher doses that was confirmed in rats. Thus, despite its relative in vitro selectivity toward the Plasmodium DHFR enzyme, QN254 does not show the adequate therapeutic index to justify its further development as a single agent.

Pharmacokinetics of lumiracoxib in plasma and synovial fluid.

BACKGROUND: Lumiracoxib is a new cyclo-oxygenase-2 (COX-2) selective inhibitor in development for the treatment of rheumatoid arthritis, osteoarthritis and acute pain. OBJECTIVE: To investigate the pharmacokinetics of lumiracoxib in plasma and knee joint synovial fluid from patients with rheumatoid arthritis. DESIGN: Open-label multiple-dose study evaluating the steady-state pharmacokinetics of lumiracoxib in plasma and synovial fluid after 7 days of treatment with lumiracoxib 400 mg once daily. PATIENT POPULATION: Males and females aged 18-75 years with rheumatoid arthritis, having moderate to significant synovial fluid effusion of the knee. OUTCOME MEASURES: Following a 7-day washout period for previous nonsteroidal anti-inflammatory drugs, 22 patients (17 female, 5 male) received lumiracoxib 400 mg once daily for seven consecutive days. On day 7, following an overnight fast, a final dose of lumiracoxib was administered and serial blood and synovial fluid samples were collected for up to 28 hours. Lumiracoxib and its metabolites (4'-hydroxy-lumiracoxib and 5-carboxy-4'-hydroxy-lumiracoxib) were measured by validated high performance liquid chromatography-mass spectrometry methods. The steady-state pharmacokinetics of lumiracoxib were evaluated in plasma and synovial fluid by both a population pharmacokinetic model and noncompartmental analysis. RESULTS: Lumiracoxib was rapidly absorbed (peak plasma concentration at 2 hours) and the terminal elimination half-life in plasma was short (6 hours). Lumiracoxib concentrations were initially higher in plasma than in synovial fluid; however, from 5 hours after administration until the end of the 28-hour assessment period, concentrations of lumiracoxib were higher in synovial fluid than in plasma. Peak drug concentration in synovial fluid occurred 3-4 hours later than the peak plasma concentration. The mean steady-state trough concentration of lumiracoxib in synovial fluid (454 microg/L) was approximately three times higher than the mean value in plasma (155 microg/L), and the area under the concentration-time curve from 12 to 24 hours after administration was 2.6-fold higher for synovial fluid than for plasma. Median lumiracoxib protein binding was similar in plasma and synovial fluid (range 97.9-98.3%). Concentrations of 4'-hydroxy-lumiracoxib, the active COX-2 selective metabolite, remained low in comparison with parent drug in both plasma and synovial fluid. The concentration of lumiracoxib in synovial fluid at 24 hours after administration would be expected to result in substantial inhibition of prostaglandin E(2) formation. CONCLUSION: The kinetics of distribution of lumiracoxib in synovial fluid are likely to extend the therapeutic action of the drug beyond that expected from plasma pharmacokinetics. These data support the use of lumiracoxib in a once-daily regimen for the treatment of rheumatoid arthritis.

In vivo and in vitro evaluation of three controlled release principles of 6-N-cyclohexyl-2'-O-methyladenosine.

6-N-Cyclohexyl-2'-O-methyladenosine was formulated into controlled release formulations exhibiting comparable in vitro release profiles using different formulation principles, i.e. osmotic pump tablets, membrane-coated pellets and hydrophilic matrix tablet. Dissolution behaviour of these formulations was evaluated in vitro under various testing conditions to assess the effect of pH and hydrodynamic conditions. It was found that osmotic tablets were not sensitive to dissolution media pH and hydrodynamics change, while drug release from monolithic hydrophilic matrix tablets were pH-dependent. When tested in vivo in dogs, it was found that metabolism of 6-N-Cyclohexyl-2'-O-methyladenosine was extensive and appeared to be saturable based on a pharmacokinetic study. Cumulative percent input in vivo (%dose) was obtained by numerical deconvolution, and compared to in vitro release profiles. A linear correlation between fraction absorbed (FRA) in vivo and fraction dissolved (FRD) in vitro was established for osmotic tablets--a true zero-order release formula, whereas only a nonlinear correlation was obtained for membrane-coated pellets. The difference in the in vivo behaviour of these formulations, despite their similar in vitro release characteristics, demonstrated the effect of different controlled release principles on their in vivo bioavailability. The curvature of fraction absorbed in vivo vs. fraction dissolved in vitro for membrane-coated pellets indicated that there was a time-scale difference between in vivo and in vitro testing. In conclusion, drug release from the osmotic system was independent of in vitro and in vivo conditions, where best sustained release effect was achieved, whereas the in vitro dissolution test employed for membrane-coated pellets and hydrophilic matrix tablets needed to be optimized to be biorelevant.

Indolcarboxamide is a preclinical candidate for treating multidrug-resistant tuberculosis.

New chemotherapeutic compounds against multidrug-resistant Mycobacterium tuberculosis (Mtb) are urgently needed to combat drug resistance in tuberculosis (TB). We have identified and characterized the indolcarboxamides as a new class of antitubercular bactericidal agent. Genetic and lipid profiling studies identified the likely molecular target of indolcarboxamides as MmpL3, a transporter of trehalose monomycolate that is essential for mycobacterial cell wall biosynthesis. Two lead candidates, NITD-304 and NITD-349, showed potent activity against both drug-sensitive and multidrug-resistant clinical isolates of Mtb. Promising pharmacokinetic profiles of both compounds after oral dosing in several species enabled further evaluation for efficacy and safety. NITD-304 and NITD-349 were efficacious in treating both acute and chronic Mtb infections in mouse efficacy models. Furthermore, dosing of NITD-304 and NITD-349 for 2 weeks in exploratory rat toxicology studies revealed a promising safety margin. Finally, neither compound inhibited the activity of major cytochrome P-450 enzymes or the hERG (human ether-a-go-go related gene) channel. These results suggest that NITD-304 and NITD-349 should undergo further development as a potential treatment for multidrug-resistant TB.

Importance of early characterization of physicochemical properties in developing high-dose intravenous infusion regimens for poorly water-soluble compounds.

TECHNICAL ABSTRACT: The aim of this work is to highlight the importance of the early characterization of physico-chemical properties to design an intravenous infusion regimen for a poorly water-soluble model compound. Compound X is a crystalline, poorly water-soluble weak base with a pH-dependent solubility profile. Due to its extremely low solubility above pH 6, the main challenge was to develop a suitable intravenous formulation for delivering high doses of compound X while minimizing the risk of precipitation upon injection into the blood (pH 7.4). A systematic approach, including formulation screening using minimum amounts of drug substance and evaluation of precipitation potential at the injection site, was employed in early discovery stages to identify a suitable infusion regimen for compound X in various preclinical models. More than 60 formulation variants were screened for solubility, pH, and precipitation potential using less than 150 mg of compound. Evaluation of precipitation potential upon injection was based on the calculation of the effective concentration at the injection site. Based on the results from formulation screening and the calculation of the effective concentration at the injection site, an intravenous infusion regimen was designed to overcome the precipitation upon injection risk associated with the poor physico-chemical properties of this compound. LAY ABSTRACT: This work highlights the impact of the physico-chemical properties of a poorly water-soluble compound on the design of high-dose intravenous infusion regimens for preclinical development. Crystalline compounds, especially weak bases, with poor aqueous solubility have a high tendency to precipitate upon injection into the blood (pH 7.4). The main challenge was to develop a suitable intravenous formulation for delivering high doses of such crystalline, poorly water-soluble, weakly basic compounds. A combination approach, including formulation screening using minimum amounts of drug substance and evaluation of precipitation potential at the injection site, was employed in early discovery stages to identify a suitable infusion regimen for various animal models. Based on the results from formulation screening and the calculation of the effective concentration at the injection site, a suitable intravenous infusion regimen was designed to overcome the precipitation upon injection risk associated with the poor physico-chemical properties of this compound.