Low Clearance Compound Research Articles

In Vitro Metabolism of Slowly Cleared G Protein-Coupled Receptor 139 Agonist TAK-041 Using Rat, Dog, Monkey, and Human Hepatocyte Models (HepatoPac): Correlation with In Vivo Metabolism.

Hepatic metabolism of low-clearance compound TAK-041 was studied in two different in vitro model systems using rat, dog, monkey, and human suspended cryopreserved hepatocytes and HepatoPac micropatterned coculture model primary hepatocytes. The aim of this work was to investigate the most appropriate system to assess the biotransformation of TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. TAK-041 exhibited very low turnover in suspended cryopreserved hepatocyte suspensions for all species, with no metabolites observed in human hepatocytes. However, incubations conducted for up to 14 days in the HepatoPac model resulted in more robust metabolic turnover. The major biotransformation pathways of TAK-041 proceed via hydroxylation on the benzene ring fused to the oxotriazine moiety and subsequent sulfate, glucuronide, and glutathione conjugation reactions. The glutathione conjugate of TAK-041 undergoes further downstream metabolism to produce the cysteine S-conjugate, which then undergoes N-acetylation to mercapturic acid and/or conversion to β-lyase-derived thiol metabolites. The minor biotransformation pathways include novel ring closure and hydrolysis, hydroxylation, oxidative N-dealkylation, and subsequent reduction. The HepatoPac model shows a notable species difference in the rate and in the extent of metabolic pathways of TAK-041, with dogs having the fastest metabolic clearance and humans the slowest. Furthermore, the model shows its suitability for establishing correlation with in vivo metabolism of low-turnover and extensively metabolized compounds such as TAK-041, displaying an extensive and unusual downstream sequential β-lyase-derived thiol metabolism in preclinical species and human. SIGNIFICANCE STATEMENT: This study investigated the most appropriate in vitro system to assess the biotransformation of the low-turnover and extensively metabolized compound TAK-041, determine any notable species difference in the rate and in the extent of its metabolic pathways, and establish correlation with in vivo metabolism. The HepatoPac model was identified and showed its suitability for species comparison and establishing correlation, with in vivo metabolism displaying an extensive and unusual downstream sequential β-lyase-derived thiol metabolism in preclinical species and human.

Metabolism and pharmacokinetics characterization of metarrestin in multiple species.

Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin. Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics. Metarrestin had an aqueous solubility of 150µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6mL/min/kg in monkeys to 48mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48h. Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appearsto be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.

Abstract 2755: NKTR-262: Prodrug pharmacokinetics in mice, rats, and dogs

Abstract Background: TLR7/8 agonists are currently under investigation in clinical trials to determine their anti-tumor effect. Systemic exposures to the TLR7/8 agonists have resulted in severe adverse effects, thereby preventing the TLR7/8 agonists from achieving therapeutic plasma concentrations. NKTR-262 is a novel polymer-modified TRL7/8 agonist prodrug which, upon intratumoral delivery, slowly liberates the TLR7/8 agonist to provide sustained intratumoral engagement of the TLR7/8 pathway to promote an immune stimulatory environment and tumor antigen release. Method: In vivo PK parameters were obtained in bilaterial tumor-bearing (CT26 and EMT6) and naïve Balb/c mice, SD rats, and Beagle dogs following a single dose (i.t., i.v. and s.c., respectively) of NKTR-262. Samples were analyzed by LC/MS/MS. Non-compartmental analysis was used to obtain PK parameters. In vitro studies with NKTR-262 and the corresponding non-polymer modified TLR7/8 agonist were performed to assess plasma release, agonist CLint in liver microsomes and hepatocytes, plasma protein binding, blood-to-plasma ratios and to identify CYP isoform(s) responsible for oxidative metabolism. PK/PD modeling was performed in NONMEM7 using a semi-mechanistic model described by Simeoni et al 2004. Results: NKTR-262 exhibited a low volume of distribution and low systemic clearance. NKTR-262 showed a prolonged elimination t½, with an order of dog &amp;gt; rat &amp;gt; mouse. NKTR-262 was eliminated renally (~50% total clearance) and by prodrug conversion. Release of the non-polymer modified TLR7/8 agonist from NKTR-262 was confirmed in vitro and in vivo in all pre-clinical species and in vitro in plasma from humans. The PK profiles of the released TLR7/8 agonist closely followed NKTR-262 PK profiles. The absolute bioavailability of NKTR-262 following i.t. and s.c. injection was low to moderate. I.t. administration of NKTR-262 generated high and sustained exposure to the TLR7/8 agonist in the treated tumor, which was two orders of magnitude greater than in plasma. Contralateral tumor and plasma exposures to the agonist were similar. The tumor type had no impact on plasma PK profiles. The agonist exhibited low plasma protein binding and distributed evenly between blood cells and plasma. The in vitro CLint of the agonist was low, and oxidative metabolism was mostly CYP3A4-mediated. The Simeoni model captured tumor volume change over time with an estimated EC50 1.34 ng/mL. Conclusion: The TLR7/8 agonist was released from NKTR-262 in vivo and in vitro in all pre-clinical species and in vitro in plasma from humans. Intratumoral administration of NKTR-262 resulted in pharmacologically active levels of the released TLR7/8 agonist in the tumor and reduced unwanted systemic exposure. Released agonist PK profiles followed the PK profiles of NKTR-262. NKTR-262 was cleared via prodrug conversion and renal clearance. The prodrug-generated TLR7/8 agonist is a low clearance compound with low protein binding. Citation Format: Myong Lee. NKTR-262: Prodrug pharmacokinetics in mice, rats, and dogs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2755.

Design of a Janus Kinase 3 (JAK3) Specific Inhibitor 1-((2S,5R)-5-((7H-Pyrrolo[2,3-d]pyrimidin-4-yl)amino)-2-methylpiperidin-1-yl)prop-2-en-1-one (PF-06651600) Allowing for the Interrogation of JAK3 Signaling in Humans.

Significant work has been dedicated to the discovery of JAK kinase inhibitors resulting in several compounds entering clinical development and two FDA approved NMEs. However, despite significant effort during the past 2 decades, identification of highly selective JAK3 inhibitors has eluded the scientific community. A significant effort within our research organization has resulted in the identification of the first orally active JAK3 specific inhibitor, which achieves JAK isoform specificity through covalent interaction with a unique JAK3 residue Cys-909. The relatively rapid resynthesis rate of the JAK3 enzyme presented a unique challenge in the design of covalent inhibitors with appropriate pharmacodynamics properties coupled with limited unwanted off-target reactivity. This effort resulted in the identification of 11 (PF-06651600), a potent and low clearance compound with demonstrated in vivo efficacy. The favorable efficacy and safety profile of this JAK3-specific inhibitor 11 led to its evaluation in several human clinical studies.

Discovery of BI 207524, an indole diamide NS5B thumb pocket 1 inhibitor with improved potency for the potential treatment of chronic hepatitis C virus infection.

The development of interferon-free regimens for the treatment of chronic HCV infection constitutes a preferred option that is expected in the future to provide patients with improved efficacy, better tolerability, and reduced risk for emergence of drug-resistant virus. We have pursued non-nucleoside NS5B polymerase allosteric inhibitors as combination partners with other direct acting antivirals (DAAs) having a complementary mechanism of action. Herein, we describe the discovery of a potent follow-up compound (BI 207524, 27) to the first thumb pocket 1 NS5B inhibitor to demonstrate antiviral activity in genotype 1 HCV infected patients, BILB 1941 (1). Cell-based replicon potency was significantly improved through electronic modulation of the pKa of the carboxylic acid function of the lead molecule. Subsequent ADME-PK optimization lead to 27, a predicted low clearance compound in man. The preclinical profile of inhibitor 27 is discussed, as well as the identification of a genotoxic metabolite that led to the discontinuation of the development of this compound.

Pharmacokinetics, metabolism and excretion of [14C]-lanicemine (AZD6765), a novel low-trapping N-methyl-d-aspartic acid receptor channel blocker, in healthy subjects

1. (1S)-1-phenyl-2-(pyridin-2-yl)ethanamine (lanicemine; AZD6765) is a low-trapping N-methyl-d-aspartate (NMDA) channel blocker that has been studied as an adjunctive treatment in major depressive disorder. The metabolism and disposition of lanicemine was determined in six healthy male subjects after a single intravenous infusion dose of 150 mg [14C]-lanicemine.2. Blood, urine and feces were collected from all subjects. The ratios of Cmax and AUC(0–∞) of lanicemine to plasma total radioactivity were 84 and 66%, respectively, indicating that lanicemine was the major circulating component with T1/2 at 16 h. The plasma clearance of lanicemine was 8.3 L/h, revealing that lanicemine is a low-clearance compound. The mean recovery of radioactivity from urine was 93.8% of radioactive dose.3. In urine samples, 10 metabolites of lanicemine were identified. Among which, an O-glucuronide conjugate (M1) was the most abundant metabolite (∼11% of the dose in excreta). In plasma, the circulatory metabolites were identified as a para-hydroxylated metabolite (M1), an O-glucuronide (M2), an N-carbamoyl glucuronide (M3) and an N-acetylated metabolite (M6). The average amount of each of metabolite was less than 4% of total radioactivity detected in plasma or urine.4. In conclusion, lanicemine is a low-clearance compound. The unchanged drug and metabolites are predominantly eliminated via urinary excretion.

Pharmacokinetics and elucidation of the rates and routes of N-glucuronidation of PF-592379, an oral dopamine 3 agonist in rat, dog, and human

PF-592379 is a potent, selective agonist of the dopamine 3 receptor, for the treatment of male erectile dysfunction and female sexual dysfunction.In vivo, PF-592379 has low-moderate clearance relative to liver blood flow of 6.3 and 8.5 ml/min/kg in dog and 44.8 and 58.2 ml/min/kg in rat. It has high permeability in Caco-2 cells and was completely absorbed in rat and dog pharmacokinetic studies with an oral bioavailability of 28% in both rats and 61 and 87% in the dogs. These data are consistent with the physicochemical properties of PF-592379, which indicate complete absorption by the transcellular route.Elimination of PF-592379 was predominantly metabolic in nature. In vitro routes of metabolism studies indicate that metabolism in the rat is a combination of P450 mechanisms and N-glucuronidation, whereas in dog and human, N-glucuronidation is the major route. NMR analysis indicates that N-glucuronidation is non-quaternary in nature and occurs on both the pyridyl amine and ring nitrogen. Rates of clearance via N-glucuronidation were predicted to be low in humans compared with acyl or phenolic glucuronidation. PF-592379 was predicted to have complete absorption from the gastrointestinal tract and an oral bioavailability of >60% in the clinic.Clinical data verified that PF-592379 is a low clearance compound in human, with a mean oral clearance of 6.5 ml/min/kg following a 200 mg oral dose. PF-592379 has ideal pharmacokinetic properties for an oral D3 agonist, intended for on demand dosing.

In Vitro Characterization of Sarizotan Metabolism: Hepatic Clearance, Identification and Characterization of Metabolites, Drug-Metabolizing Enzyme Identification, and Evaluation of Cytochrome P450 Inhibition

In vitro biotransformation studies of sarizotan using human liver microsomes (HLM) showed aromatic and aliphatic monohydroxylation and dealkylation. Recombinant cytochromes P450 (P450) together with P450-selective inhibitors in HLM/hepatocyte cultures were used to evaluate the relative contribution of different P450s and revealed major involvement of CYP3A4, CYP2C9, CYP2C8, and CYP1A2 in sarizotan metabolism. The apparent K(m, u) and V(max) of sarizotan clearance, as investigated in HLM, were 9 microM and 3280 pmol/mg/min, predicting in vivo hepatic clearance of 0.94 l/h, which indicates that sarizotan is a low-clearance compound in humans and suggests nonsaturable metabolism at the targeted plasma concentration (< or =1 microM). This finding is confirmed by the reported human clearance (CL/F of 3.6-4.4 l/h) and by the dose-linear area under the curve increase observed with doses up to 25 mg. The inhibitory effect of sarizotan toward six major P450s was evaluated using P450-specific marker reactions in pooled HLM. K(i, u) values of sarizotan against CYP2C8, CYP2C19, and CYP3A4 were >10 microM, whereas those against CYP2D6 and CYP1A2 were 0.43 and 8.7 microM, respectively. Based on the estimates of sarizotan concentrations at the enzyme active sites, no clinically significant drug-drug interactions (DDIs) due to P450 inhibition are expected. This result has been confirmed in human DDI studies in which no inhibition of five major P450s was observed in terms of marker metabolite formation.

Novel Tight-Binding Inhibitory Factor-κB Kinase (IKK-2) Inhibitors Demonstrate Target-Specific Anti-Inflammatory Activities in Cellular Assays and following Oral and Local Delivery in an in Vivo Model of Airway Inflammation

Nuclear factor-kappaB (NF-kappaB) is one of the major families of transcription factors activated during the inflammatory response in asthma and chronic obstructive pulmonary disease. Inhibitory factor-kappaB kinase 2 (IKK-2) has been shown to play a pivotal role in cytokine-induced NF-kappaB activation in airway epithelium and in disease-relevant cells. Nevertheless, the potential toxicity of specific IKK-2 inhibitors may be unacceptable for oral delivery in chronic obstructive pulmonary disease. Therefore, local delivery to the lungs is an attractive alternative that warrants further exploration. Here, we describe potent and selective small-molecule IKK-2 inhibitors [8-(5-chloro-2-(4-methylpiperazin-1-yl)isonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo[g]indazole-3-carboxamide (PHA-408) and 8-(2-(3,4-bis(hydroxymethyl)-3,4-dimethylpyrrolidin-1-yl)-5-chloroisonicotinamido)-1-(4-fluorophenyl)-4,5-dihydro-1H-benzo-[g]indazole-3-carboxamide (PF-184)] that are competitive for ATP have slow off-rates from IKK-2 and display broad in vitro anti-inflammatory activities resulting from NF-kappaB pathway inhibition. Notably, PF-184 has been designed to have high systemic clearance, which limits systemic exposure and maximizes the effects locally in the airways. We used an inhaled lipopolysaccharide-induced rat model of neutrophilia to address whether inhibiting NF-kappaB activation locally within the airways would show anti-inflammatory effects in the absence of systemic exposure. PHA-408, a low-clearance compound previously shown to be efficacious orally in a rodent model of arthritis, dose-dependently attenuated inhaled lipopolysaccharide-induced cell infiltration and cytokine production. Interestingly, PF-184 produced comparable dose-dependent anti-inflammatory activity by intratracheal administration and was as efficacious as intratracheally administered fluticasone propionate (fluticasone). Together, these results support the potential therapeutic utility of IKK-2 inhibition in inflammatory pulmonary diseases and demonstrate anti-inflammatory efficacy of an inhaled IKK-2 inhibitor in a rat airway model of neutrophilia.

Pharmacokinetics of Recombinant Factor VIIa in the Rat – A Comparison of Bio-, Immuno- and Isotope Assays

Recombinant human factor VIIa (rFVIIa) is an activated coagulation factor for intravenous use as a haemostatic agent in haemophiliacs who generate antibodies against factor VIII or IX. Plasma kinetic studies are important for the understanding of the action of rFVIIa which is exerted in the vascular compartment of the body, more specifically on the vessel walls at the site of injury. In the present study, rats were dosed 100 or 500 micrograms/kg 125I-rFVIIa i.v., without any side effects being observed, and the plasma profile of rFVIIa was studied by 3 different assays that were shown to correlate well at early times post-dose: trichloroacetic acid (TCA)-precipitable drug-related radioactivity, rFVIIa antigen determination by ELISA technique, and the assay of clot activity which is the only clinically applicable assay. The plasma concentration curve could be resolved into 1-3 exponentials, depending on the FVIIa detection principle that was employed. Initially, there was a short (ca. 10 min) phase of increasing concentrations before the attainment of Cmax. This was followed by a plasma recovery (Cmax x plasma volume/dose) in the vicinity of one half of the administered dose. The initial volume of distribution (V1) corresponded to the vascular compartment whereas the volume of distribution at steady state (Vss) was somewhat larger. Whole body clearance (CL-B) of rFVIIa was approx. 1 ml/min per kg, and mean residence time (MRT) and the half-life assumed to be associated with the loss of biological activity was approx. 1 h and 20-45 min, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

The disposition of pyrimethamine in the isolated perfused rat liver

We have investigated the disposition of pyrimethamine base in the isolated perfused rat liver (IPRL) preparation after the administration of pyrimethamine (0.5 mg, 5 μCi). In the first half hour of the study, pyrimethamine underwent marked hepatic uptake, thereafter perfusate plasma drug levels declined monoexponentially with a half life (t 1 2 ) of 3.0 ± 1.0 hr . Area under the perfusate plasma concentration/time curve (AUC) 0→ ∝ was 6.9 ± 1.9 μg/hr/ml. Pyrimethamine was found to be a low clearance compound (78.4 ± 25.3 ml/hr  8.6% of liver perfusate flow) with a large volume of distribution (267.5 ± 55.3 ml) in the IPRL. The combined AUCs (0→5hr) for pyrimethamine (AUC 4.8 ± 0.5 μg/hr/ml) and pyrimethamine 3-N-oxide (AUC 0→5hr 0.9 ± 0.6 μg/hr/ml) accounted for 57% of the total AUC 0→5hr of [ 14C] radioactivity (10.0 ± 2.6 μg/hr/ml). This indicates the presence of metabolites of pyrimethamine as yet unidentified in the perfusate. Biliary excretion of [ 14C] during the course of the IPRL preparations was extensive (29.0 ± 10.3%) though only a small proportion was due to pyrimethamine and the 3- N-oxide metabolite. The majority of radioactivity in the bile was attributable to highly polar, but unidentified metabolites of pyrimethamine. At the conclusion of each experiment (5 hr), a significant proportion of [ 14C] radioactivity was recovered from the livers (22.9 ± 5.3%). Subsequent HPLC analysis of the liver tissue indicated this to be unchanged pyrimethamine, with trace levels of the 3- N-oxide metabolite. Sub-cellular fractionation of the homogenized livers revealed the most pronounced localisation of pyrimethamine to be in the lipid rich 10,000 g pellet (13.0 ± 2.6%), the remainder being distributed equally between the 105,000 g pellet and supernatant. Neither pyrimethamine, [ 14C] radioactivity, nor pyrimethamine 3- N-oxide were extensively taken up by red cells throughout the study. Therefore, the large volume of distribution (267.5 ± 55.3 ml) underlines the extent of pyrimethamine localisation in the liver.

Pharmacokinetics of primaquine in man. I. Studies of the absolute bioavailability and effects of dose size.

The pharmacokinetics of primaquine have been examined in five healthy volunteers who received single oral doses of 15, 30 and 45 mg of the drug, on separate occasions. Each subject received an i.v. tracer dose of [14C]-primaquine (7.5 microCi), simultaneously with the 45 mg oral dose. Absorption of primaquine was virtually complete with a mean absolute bioavailability of 0.96 +/- 0.08. Elimination half-life, oral clearance and apparent volume of distribution for both primaquine and the carboxylic acid metabolite were unaffected by either dose size, or route of administration. The relationships between area under the curve and dose size were linear for both primaquine (r = 0.99, P less than or equal to 0.01) and its carboxylic acid metabolite (r = 0.99, p less than or equal to 0.01). The mean whole blood to plasma concentration ratios were determined for primaquine (0.81), and for the carboxylic acid metabolite of primaquine (0.84). Primaquine is a low clearance compound (CL = 24.2 +/- 7.4 l h-1), is extensively distributed into body tissues (V = 242.9 +/- 69.5 l) and is not subject to extensive first pass metabolism.

Correlation between the in vivo metabolism of hexobarbital and antipyrine in rats with a portacaval shunt.

To investigate how hepatic malfunction affects the disposition of hexobarbital (HB), an intermediate 'high-clearance' compound, and antipyrine (AP), a low-clearance compound, as well as the correlation between the rates of elimination of these drugs, their pharmacokinetics, were studied in control rats (n = 8) and in rats with a portacaval shunt (PCS; n = 9). Blood concentrations of parent drugs were measured, and urinary excretion of the following metabolites was determined: 3-hydroxymethylantipyrine (HMA), 4-hydroxyantipyrine and norantipyrine as primary metabolites of AP, and 3'-hydroxyhexobarbital (OH-HB) and 3'-ketohexobarbital (K-HB) as primary metabolites of HB. Blood elimination half-lives of AP and HB were more than four times longer in PCS rats than in control rats, increasing from 63.7 +/- 3.9 to 291 +/- 66 min, and from 20.1 +/- 1.8 to 84.2 +/- 7.6 min, respectively. Intrinsic clearance of HB (CLint, HB) was 167 +/- 19 ml/min/kg in controls and 27 +/- 4 ml/min/kg in PCS rats (CLpcs, HB). Intrinsic clearance of AP (CLint, AP) in control rats was 15.1 +/- 0.7 ml/min/kg and 5.9 +/- 0.7 ml/min/kg in PCS rats (CLpcs, AP). PCS reduced clearance for production of metabolites (CLMn) of AP by 50%, but CLMn of HB metabolites was decreased by more than 80%. The CLint, AP, CLint, HB CLpcs, HB, CLpcs, AP, and CLMn data were correlated. Total clearance correlated better in PCS rats than in control rats: r = 0.77 versus r = 0.10, respectively, thus suggesting a decrease in substrate selectivity under pathological conditions. CLOH-HB+K-HB, reflecting the major metabolic pathway of HB, correlated most closely with CLHMA in PCS (r = 0.91). Therefore, the underlying metabolic conversions of HB and AP may be mediated by the same or very similar forms of cytochrome P-450. Our results suggest that the predictive value of the model substrate approach is valid under pathological conditions.