Antitumor efficacy of CHMFL-KIT-110 solid dispersion in mouse xenograft models of human gastrointestinal stromal tumors.

PURPOSE: CHMFL-KIT-110, a selective c-KIT kinase inhibitor for gastrointestinal stromal tumors (GISTs), possesses a poorly water-soluble, limiting the further development of the drug. This study was to investigate the antitumor efficacy of CHMFL-KIT-110 and CHMFL-KIT-110 solid dispersion (laboratory code: HYGT-110 SD) in GIST tumor xenograft models and to explore the PK/PD relationship of HYGT-110 SD. METHODS: Plasma concentrations of HYGT-110 and HYGT-110 SD were determined by LC-MS/MS in KM mice. Antitumor activity was evaluated by measuring tumor volume and weight in c-KIT-dependent GIST xenograft models. PK/PD relationship was assessed by LC-MS/MS and Western Blot in the GIST-T1 xenografted mice. RESULTS: HYGT-110 exhibited a low oral bioavailability (10.91%) in KM mice. Compared with HYGT-110 treatment, the Cmax and AUC0-t of HYGT-110 SD in mice plasma were substantially increased by 18.81 and 6.76-fold, respectively. HYGT-110 SD (10, 30, and 100 mg/kg/day) also could dose-dependently decrease the tumor volume and weight in the GIST-882 cell-inoculated xenograft mouse models and show 86.35% tumor growth inhibition (TGI) at 28 days at a 25 mg/kg bid dosage in the GIST-T1 cell-inoculated xenograft mouse model. The free concentration of HYGT-110 in plasma was closely correlated with the inhibition of c-KIT phosphorylation levels in tumor tissues. CONCLUSIONS: In comparison with the HPMC formulation, both improved PK and PD characteristics of the solid dispersion formulation of CHMFL-KIT-110 were observed in in vivo animal experiments.

Preclinical characterization of itacitinib (INCB039110), a novel selective inhibitor of JAK1, for the treatment of inflammatory diseases.

Pharmacological modulation of the Janus kinase (JAK) family has achieved clinically meaningful therapeutic outcomes for the treatment of inflammatory and hematopoietic diseases. Several JAK1 selective compounds are being investigated clinically to determine their anti-inflammatory potential. We used recombinant enzymes and primary human lymphocytes to assess the JAK1 specificity of itacitinib (INCB039110) and study inhibition of signal transducers and activators of transcription (STAT) signaling. Rodent models of arthritis and inflammatory bowel disease were subsequently explored to elucidate the efficacy of orally administered itacitinib on inflammatory pathogenesis. Itacitinib is a potent and selective JAK1 inhibitor when profiled against the other JAK family members. Upon oral administration in rodents, itacitinib achieved dose-dependent pharmacokinetic exposures that highly correlated with STAT3 pharmacodynamic pathway inhibition. Itacitinib ameliorated symptoms and pathology of established experimentally-induced arthritis in a dose-dependent manner. Furthermore, itacitinib effectively delayed disease onset, reduced symptom severity, and accelerated recovery in three distinct mouse models of inflammatory bowel disease. Low dose itacitinib administered via cannula directly into the colon was highly efficacious in TNBS-induced colitis but with minimal systemic drug exposure, suggesting localized JAK1 inhibition is sufficient for disease amelioration. Itacitinib treatment in an acute graft-versus-host disease (GvHD) model rapidly reduced inflammatory markers within lymphocytes and target tissue, resulting in a marked improvement in disease symptoms. This is the first manuscript describing itacitinib as a potent and selective JAK1 inhibitor with anti-inflammatory activity across multiple preclinical disease models. These data support the scientific rationale for ongoing clinical trials studying itacitinib in select GvHD patient populations.

Population pharmacokinetic analysis of isoniazid, acetylisoniazid, and isonicotinic acid in healthy volunteers.

In this study, we aimed to quantify the effects of the N-acetyltransferase 2 (NAT2) phenotype on isoniazid (INH) metabolism in vivo and identify other sources of pharmacokinetic variability following single-dose administration in healthy Asian adults. The concentrations of INH and its metabolites acetylisoniazid (AcINH) and isonicotinic acid (INA) in plasma were evaluated in 33 healthy Asians who were also given efavirenz and rifampin. The pharmacokinetics of INH, AcINH, and INA were analyzed using nonlinear mixed-effects modeling (NONMEM) to estimate the population pharmacokinetic parameters and evaluate the relationships between the parameters and the elimination status (fast, intermediate, and slow acetylators), demographic status, and measures of renal and hepatic function. A two-compartment model with first-order absorption best described the INH pharmacokinetics. AcINH and INA data were best described by a two- and a one-compartment model, respectively, linked to the INH model. In the final model for INH, the derived metabolic phenotypes for NAT2 were identified as a significant covariate in the INH clearance, reducing its interindividual variability from 86% to 14%. The INH clearance in fast eliminators was 1.9- and 7.7-fold higher than in intermediate and slow eliminators, respectively (65 versus 35 and 8 liters/h). Creatinine clearance was confirmed as a significant covariate for AcINH clearance. Simulations suggested that the current dosing guidelines (200 mg for 30 to 45 kg and 300 mg for >45 kg) may be suboptimal (3 mg/liter ≤ Cmax ≤ 6 mg/liter) irrespective of the acetylator class. The analysis established a model that adequately characterizes INH, AcINH, and INA pharmacokinetics in healthy Asians. Our results refine the NAT2 phenotype-based predictions of the pharmacokinetics for INH.

Impact on creatinine renal clearance by the interplay of multiple renal transporters: a case study with INCB039110.

Serum creatinine is commonly used as a marker of renal function, but increases in serum creatinine might not represent changes in glomerular filtration rate (GFR). INCB039110 (2-(3-(4-(7H-pyrrolo[2,3-day]pyrimidin-4-yl)-1H-pyrazol-1-yl)-1-(1-(3-fluoro-2-(trifluoromethyl)isonicotinoyl)piperidin-4-yl)azetidin-3-yl)acetonitrile) is an inhibitor of the Janus kinases (JAKs) with selectivity for JAK1. In a phase 1 study, a modest and reversible increase in serum creatinine was observed after treatment with INCB039110. However, a dedicated renal function study with INCB039110, assessed by iohexol plasma clearance, conducted in healthy volunteers indicated no change in GFR. In vitro studies were therefore conducted to investigate the interaction of INCB039110 with five transporters that are likely involved in the renal clearance of creatinine. Cell systems expressing individual or multiple transporters were used, including a novel quintuple-transporter model OAT2/OCT2/OCT3/MATE1/MATE2-K. INCB039110 potently inhibited OCT2-mediated uptake of creatinine as well as MATE1-/MATE2-K-mediated efflux of creatinine. Given the interactions of INCB039110 with multiple transporters affecting creatinine uptake and efflux, an integrated system expressing all five transporters was sought; in that system, INCB039110 caused a dose-dependent decrease in transcellular transport of creatinine with weaker net inhibition compared with the effects on individual transporters. In summary, a molecular mechanism for the increase in serum creatinine by INCB039110 has been established. These studies also underline the limitations of using serum creatinine as a marker of renal function.

Noninvasive determination of 2-[18F]-fluoroisonicotinic acid hydrazide pharmacokinetics by positron emission tomography in Mycobacterium tuberculosis-infected mice.

Tuberculosis (TB) is a global pandemic requiring sustained therapy to facilitate curing and to prevent the emergence of drug resistance. There are few adequate tools to evaluate drug dynamics within infected tissues in vivo. In this report, we evaluated a fluorinated analog of isoniazid (INH), 2-[(18)F]fluoroisonicotinic acid hydrazide (2-[(18)F]-INH), as a probe for imaging Mycobacterium tuberculosis-infected mice by dynamic positron emission tomography (PET). We developed a tail vein catheter system to safely deliver drugs to M. tuberculosis aerosol-infected mice inside sealed biocontainment devices. Imaging was rapid and noninvasive, and it could simultaneously visualize multiple tissues. Dynamic PET imaging demonstrated that 2-[(18)F]-INH was extensively distributed and rapidly accumulated at the sites of infection, including necrotic pulmonary TB lesions. Compared to uninfected animals, M. tuberculosis-infected mice had a significantly higher PET signal within the lungs (P < 0.05) despite similar PET activity in the liver (P > 0.85), suggesting that 2-[(18)F]-INH accumulated at the site of the pulmonary infection. Furthermore, our data indicated that similar to INH, 2-[(18)F]-INH required specific activation and accumulated within the bacterium. Pathogen-specific metabolism makes positron-emitting INH analogs attractive candidates for development into imaging probes with the potential to both detect bacteria and yield pharmacokinetic data in situ. Since PET imaging is currently used clinically, this approach could be translated from preclinical studies to use in humans.

Combined use of pharmacokinetic modeling and a steady-state delivery approach allows early assessment of IkappaB kinase-2 (IKK-2) target safety and efficacy.

NF-kappaB activation is clearly linked to the pathogenesis of multiple inflammatory diseases including arthritis. The prominent role of IkappaB kinase-2 (IKK-2) in regulating NF-kappaB signaling in response to proinflammatory stimuli has made IKK-2 a primary anti-inflammation therapeutic target. PHA-408, a potent and selective IKK-2 inhibitor, was identified internally and used for our studies to assess this target. In early in vivo studies, PHA-408 demonstrated efficacy at high doses; however, the correlation between PHA-408 exposure and efficacy could not be established using standard dosing paradigms for the rat disease models. Similar concerns arose from early in vivo safety studies where appropriate NOAEL margins were not achieved. Following a full investigation of the physicochemical properties of the molecule and pharmacokinetic modeling, an oral steady-state delivery strategy was designed to administer PHA-408 to the rat for both efficacy and safety studies. Using this steady-state delivery, a clear dose-response relationship was established between plasma concentrations of PHA-408 and efficacy in the rat arthritis model. The same steady-state delivery approach was used to demonstrate the target safety. In summary, a combination of pharmacokinetic modeling with a steady-state delivery approach allowed us to establish confidence in both the mechanism and safety of the target.

Discovery of isonicotinamide derived beta-secretase inhibitors: in vivo reduction of beta-amyloid.

beta-Secretase inhibition offers an exciting opportunity for therapeutic intervention in the progression of Alzheimer's disease. A series of isonicotinamides derived from traditional aspartyl protease transition state isostere inhibitors has been optimized to yield low nanomolar inhibitors with sufficient penetration across the blood-brain barrier to demonstrate beta-amyloid lowering in a murine model.

N-isonicotinoyl-(L)-4-aminophenylalanine derivatives as tight binding VLA-4 antagonists.

A series of isonicotinoyl-(L)-aminophenylalanine derivatives was prepared and evaluated as VLA-4 antagonists. These compounds exhibit subnanomolar binding affinity to VLA-4 and significant off-rates. The interplay between off-rate, protein binding and pharmacokinetics is discussed.

Novel synthesis of 2-[18F]-fluoroisonicotinic acid hydrazide and initial biological evaluation.

2-[18F]-Fluoroisonicotinic acid hydrazide was synthesized by nucleophilic displacement reaction on ethyl-2- (trimethylammonium)-isonicotinate precursor in acetonitrile. Kryptofix 222 was used as the phase transfer catalyst. The intermediate fluorinated ethyl ester reacted with hydrazine hydrate to produce the hydrazide in excellent radiochemical yield. The overall radiochemical yield was greater than 70% with total synthesis time of approximately 60 minutes. Biological evaluation was performed in bacterial cells and biodistribution in normal CBA/J mice. It was found that the S. pneumoniae cells retained the radiotracer in an in vitro assay.

Metabolism and pharmacokinetics of the cardiotonic agent piroximone and of its major metabolite in dog.

1. Piroximone was administered orally (p.o.) and intravenously (i.v.) to male Beagle dog. In vitro, piroximone was incubated with dog liver microsomes. 2. Piroximone was metabolized in vivo to five metabolites (1-5) representing approximately 20% of the total administered dose. 3. The parent drug and its metabolites were totally eliminated in urine. 4. Reduced piroximone (piroximole), representing approximately 10% of the administered dose, was identified as the major metabolic product in vivo. 5. In vitro, piroximone was metabolized by dog liver microsomes to isonicotinic acid (1) and piroximole (4), with the same ratio as in vivo (1:4 = 0.2). The Michaelis-Menten parameters were determined for piroximole formation and were: Kmapp = 733 microM and Vmax app = 232 pmol/mg protein/min. 6. Comparison of the pharmacokinetics of piroximone and piroximole revealed that both compounds were very well absorbed (F = 93 +/- 7 and 89 +/- 8% respectively), slightly distributed (Vd app = 0.78 +/- 0.04 and 1.02 +/- 0.09 l/kg p.o., and 0.95 +/- 0.05 and 0.76 +/- 0.13 1/kg i.v. respectively) and excreted into urine to the same extent (UEx = 54.7 +/- 1.2 and 53.2 +/- 12.6% p.o., and 59.1 +/- 5.3 and 51.2 +/- 5.7% i.v. respectively), except that the clearance of piroximone was two-fold higher than that observed for piroximole (ClT = 7.77 +/- 1.35 and 4.12 +/- 0.44 ml/min/kg p.o., and 7.68 +/- 1.25 and 4.06 +/- 0.51 ml/min/kg i.v. respectively).

Drug-induced hepatic microgranulomatosis in cynomolgus monkeys.

This report discusses a unique drug-induced hepatotoxicity in cynomolgus monkeys treated orally with a novel potassium sparing experimental diuretic, [2,6-bis(4-chlorophenyl)-4-pyridinecarboxylic acid]. Groups of 6 adult male and female monkeys were treated orally with vehicle diluent, modified vehicle #122 or a suspension of the drug at 5.0, 12.5, or 32.0 mg/kg/day for 2 weeks. Another group of 5 monkeys were treated orally with 25.0 mg/kg/day of the drug for 2 weeks. Disposition of the drugs was evaluated in 2 monkeys in the later group that received 27.4 mg/kg of radiolabelled drug on the 1st and last day of dosing. Hepatic toxicity was characterized biochemically, light and electron microscopically, histochemically, immunocytochemically, and toxico-kinetically. Conjugated serum bilirubin, alanine transaminase, and aspartate transaminase levels were increased in monkeys treated with over 12.5 mg/kg/day of the diuretic. The periacinar hepatic plates of monkeys treated with 25.0 or 32.0 mg/kg/day were distorted by accumulation of PAS and oil red-O positive multinucleated Kupffer cells. The cytosol of these cells was expanded by phagolysosomes containing granular materials of varying electron densities. Granular electron dense materials were also in endothelial cells and bile canaliculi. Fatty change, cholestasis, and rare piecemeal hepatic necrosis were minimal. The drug was primarily excreted through urine. Plasma concentration and half life of the drug were increased with multiple dosing. The highest concentration of unexcreted parent drug was in the liver. Drug-induced noninflammatory hepatic microgranulomatosis, apparently caused by sequestered drug-lipid/mucopolysaccharide complex in the phagocytic cells of the liver, can occur in any species, including humans, if orally administered xenobiotics are presented to the liver in particulate form.

Synthesis of 2-[123I and 124I]-iodoisonicotinic acid hydrazide--potential radiotracers for tuberculosis.

The radiochemical syntheses of methyl 2-[123I]-iodoisonicotinate, 2-[123I]-iodoisonicotinic acid hydrazide and 2-[124I]-iodoisonicotinic acid hydrazide was accomplished. Iodine-123 was incorporated in the methyl ester molecule by an exchange reaction in glacial acetic acid. The average efficiency of iodine exchange reaction was (92.6 +/- 4.5)%. This radiotracer was extracted with ether and the solvent was evaporated. The residue was re-dissolved in anhydrous ethanol and treated with hydrazine under anhydrous conditions to obtain 2-[123I]-iodoisonicotinic acid hydrazide. The overall radiochemical yield was 69%. Biodistribution data of both radio-tracers in male Sprague-Dawley rats were collected. This is the first report of SPECT radiopharmaceuticals which may be useful for differential diagnosis of intracranial masses (tuberculoma vs glioma), and CNS tuberculosis in immunosuppressed subjects.

Studies on the metabolism of Inabenfide (a new plant growth regulator) in rats. I. Characterization of metabolites in rats.

1. The metabolism of 4'-chloro-2'-(alpha-hydroxybenzyl) isonicotinanilide (Inabenfide, IBF) was studied in the rat. After intraperitoneal administration of IBF to rats, eight metabolites were detected in urine by g.l.c.-mass spectrometry and a stable isotope technique. 2. The major metabolites were hydroxylated IBF, and minor metabolites were dihydrodiol IBF, methylated catechol IBF, IBF ketone, IBF N-oxide and an amine derivative. 3. Of these metabolites, IBF ketone was produced by oxidation of the carbinol between the benzene rings, which is an interesting metabolite since similar oxidation between benzene rings is not well known. 4. Metabolism involving an NIH shift of chlorine and an epoxide-diol pathway for IBF are presented.