Eiichi Fuse

Phase I Trial of 72-Hour Continuous Infusion UCN-01 in Patients With Refractory Neoplasms

PURPOSE To define the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of the novel protein kinase inhibitor, UCN-01 (7-hydroxystaurosporine), administered as a 72-hour continuous intravenous infusion (CIV). PATIENTS AND METHODS Forty-seven patients with refractory neoplasms received UCN-01 during this phase I trial. Total, free plasma, and salivary concentrations were determined; the latter were used to address the influence of plasma protein binding on peripheral tissue distribution. The phosphorylation state of the protein kinase C (PKC) substrate alpha-adducin and the abrogation of DNA damage checkpoint also were assessed. RESULTS The recommended phase II dose of UCN-01 as a 72-hour CIV is 42.5 mg/m(2)/d for 3 days. Avid plasma protein binding of UCN-01, as measured during the trial, dictated a change in dose escalation and administration schedules. Therefore, nine patients received drug on the initial 2-week schedule, and 38 received drug on the recommended 4-week schedule. DLTs at 53 mg/m(2)/d for 3 days included hyperglycemia with resultant metabolic acidosis, pulmonary dysfunction, nausea, vomiting, and hypotension. Pharmacokinetic determinations at the recommended dose of 42.5 mg/m(2)/d for 3 days included mean total plasma concentration of 36.4 microM (terminal elimination half-life range, 447 to 1176 hours), steady-state volume of distribution of 9.3 to 14.2 L, and clearances of 0.005 to 0.033 L/h. The mean total salivary concentration was 111 nmol/L of UCN-01. One partial response was observed in a patient with melanoma, and one protracted period ( > 2.5 years) of disease stability was observed in a patient with alk-positive anaplastic large-cell lymphoma. Preliminary evidence suggests UCN-01 modulation of both PKC substrate phosphorylation and the DNA damage-related G(2) checkpoint. CONCLUSION UCN-01 can be administered safely as an initial 72-hour CIV with subsequent monthly doses administered as 36-hour infusions.

Kinetic Analysis of the Cooperation of P-Glycoprotein (P-gp/Abcb1) and Breast Cancer Resistance Protein (Bcrp/Abcg2) in Limiting the Brain and Testis Penetration of Erlotinib, Flavopiridol, and Mitoxantrone

A synergistic effect of P-glycoprotein (P-gp)/Abcb1a and breast cancer resistance protein (Bcrp)/Abcg2 was reported to limit the brain penetration of their common substrates. This study investigated this based on pharmacokinetics using Mdr1a/1b(−/−), Bcrp(−/−), and Mdr1a/1b(−/−)/Bcrp(−/−) mice. Comparison of the brain- and testis-to-plasma ratios (Cbrain/Cplasma and Ctestis/Cplasma, respectively) of the reference compounds quinidine and dantrolene for P-gp and Bcrp, respectively, indicates that impairment of either P-gp and Bcrp did not cause any change in the efflux activities of Bcrp or P-gp, respectively, at both the blood-brain barrier (BBB) and blood-testis barrier (BTB). The Cbrain/Cplasma and Ctestis/Cplasma of the common substrates erlotinib, flavopiridol, and mitoxantrone were markedly increased in Mdr1a/1b(−/−)/Bcrp(−/−) mice even compared with Mdr1a/1b(−/−) and Bcrp(−/−) mice. Efflux activities by P-gp and Bcrp relative to passive diffusion at the BBB and BTB were separately evaluated based on the Cbrain/Cplasma and Ctestis/Cplasma in the knockout strains to the wild-type strain. P-gp made a larger contribution than Bcrp to the net efflux of the common substrates, but Bcrp activities were also significantly larger than passive diffusion. These parameters could reasonably account for the marked increase in Cbrain/Cplasma and Ctestis/Cplasma in the Mdr1a/1b(−/−)/Bcrp(−/−) mice. In conclusion, the synergistic effect of P-gp and Bcrp on Cbrain/Cplasma and Ctestis/Cplasma can be explained by their contribution to the net efflux at the BBB and BTB without any interaction between P-gp and Bcrp.

Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models.

UCN-01 (7-hydroxystaurosporine; NSC 638850) is a protein kinase antagonist selected for clinical trial based in part on evidence of efficacy in a preclinical renal carcinoma xenograft model. Schedule studies and in vitro studies suggested that a 72-h continuous infusion would be appropriate. In rats and dogs, maximum tolerated doses produced peak plasma concentrations of approximately 0.2–0.3 μM. However, concentrations 10-fold greater are well tolerated in humans, and the compound has a markedly prolonged T1/2. Specific binding to human α1-acidic glyco-protein has been demonstrated. These findings reinforce the need to consider actual clinical pharmacology data in “real time” with phase I studies.

Review of UCN‐01 Development: A Lesson in the Importance of Clinical Pharmacology

UCN‐01 is a protein kinase inhibitor under development as a novel anticancer drug. The initial pharmacologic features in patients were not predicted from preclinical experiments. The distribution volume and the systemic clearance were much lower than those in experimental animals (mice, rats, and dogs), and the elimination half‐life was unusually long (>200 hours). The unbound fraction in human plasma was also much smaller than that in dogs, rats and mice, as was the binding of UCN‐01 to human alpha‐1 acid glycoprotein much stronger than that to human serum albumin or human γ‐globulin. The association constants for alpha‐1 acid glycoprotein and human plasma were approximately 8 × 108 (mol/L)−1, indicating extremely high affinity. In this review article, the authors discuss the pharmacologic features of UCN‐01 across species and provide a perspective on how this information could be applied prospectively to the future development of this agent.

Quantitative Evaluation of the Impact of Active Efflux by P-Glycoprotein and Breast Cancer Resistance Protein at the Blood-Brain Barrier on the Predictability of the Unbound Concentrations of Drugs in the Brain Using Cerebrospinal Fluid Concentration as a Surrogate

This study investigated the impact of the active efflux mediated by P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) at the blood-brain barrier (BBB) on the predictability of the unbound brain concentration (Cu,brain) by the concentration in the cerebrospinal fluid (CSF) (Cu,CSF) in rats. Cu,brain is obtained as the product of the total brain concentration and unbound fraction in the brain (fu,brain) determined in vitro in brain slices. Twenty-five compounds, including P-gp and/or Bcrp substrates, were given a constant intravenous infusion, and their plasma, brain, and CSF concentrations were determined. P-gp and/or Bcrp substrates, such as verapamil, loperamide, flavopiridol, genistein, quinidine, dantrolene, daidzein, cimetidine, and pefloxacin, showed a higher CSF-to-brain unbound concentration ratio (Kp,uu,CSF/brain) compared with non-P-gp and non-Bcrp substrates. Kp,uu,CSF/brain values of P-gp-specific (quinidine and verapamil) and Bcrp-specific (daidzein and genistein) substrates were significantly decreased in Mdr1a/1b(−/−) and Bcrp(−/−) mice, respectively. Furthermore, consistent with the contribution of P-gp and Bcrp to the net efflux at the BBB, Kp,uu,CSF/brain values of the common substrates (flavopiridol and erlotinib) were markedly decreased in Mdr1a/1b(−/−)/Bcrp(−/−) mice, but only moderately or weakly in Mdr1a/1b(−/−) mice and negligibly in Bcrp(−/−) mice. In conclusion, predictability of Cu,brain by Cu,CSF decreases along with the net transport activities by P-gp and Bcrp at the BBB. Cu,CSF of non-P-gp and non-Bcrp substrates can be a reliable surrogate of Cu,brain for lipophilic compounds.

Molecular Cloning and Functional Analyses of OAT1 and OAT3 from Cynomolgus Monkey Kidney

No HeadingPurpose.The functional characterization of monkey OAT1 (SLC22A6) and OAT3 (SLC22A8) was carried out to elucidate species differences in the OAT1- and OAT3-mediated transport between monkey and human.Methods.The cDNAs of monkey OAT1 and OAT3 were isolated from monkey kidney, and their stable transfectants were established in HEK293 cells (mkOAT1- and mkOAT3-HEK). Transport studies were performed using cDNA transfectants, and kinetic parameters were compared among rat, monkey and human.Results.The amino acid sequences of mkOAT1 and mkOAT3 exhibit 97% and 96% identity to their corresponding human orthologues. For OAT1, there was no obvious species difference in the Km values and the relative transport activities of 11 substrates with regard to p-aminohippurate transport. For OAT3, there was no species difference in the Km values and in the relative transport activities of nine substrates with regard to benzylpenicillin transport between monkey and human. However, the relative transport activities of indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, and estrone-3-sulfate showed a difference between primates and rat and gave a poor correlation.Conclusions.These results suggest that monkey is a good predictor of the renal uptake of organic anions in the human.

INHIBITION OF OAT3-MEDIATED RENAL UPTAKE AS A MECHANISM FOR DRUG-DRUG INTERACTION BETWEEN FEXOFENADINE AND PROBENECID

Fexofenadine, a nonsedating antihistamine drug, is effective for the treatment of seasonal allergic rhinitis and chronic urticaria. Simultaneous administration of probenecid increases the plasma concentration of fexofenadine due to an inhibition of its renal elimination in healthy volunteers (Clin Pharmacol Ther 77:17–23, 2005). The purpose of the present study is to investigate the possibility that the drug-drug interaction between fexofenadine and probenecid involves the renal basolateral uptake process. The uptake of fexofenadine was determined in HEK293 cells expressing human organic anion transporter 1 (OAT1/SLC22A6), OAT2 (SLC22A7), OAT3 (SLC22A8), and organic cation transporter 2 (OCT2/SLC22A2). Only hOAT3-HEK showed a significantly greater accumulation of fexofenadine than that in vector-HEK, which was saturable with Km and Vmax values of 70.2 μM and 120 pmol/min/mg protein, respectively. Inhibition potency of probenecid for the uptake of fexofenadine was compared between hOAT3 and organic anion-transporting peptide 1B3 (hOATP1B3), a transporter responsible for the hepatic uptake of fexofenadine (Drug Metab Dispos 33:1477–1481, 2005). The Ki values were determined to be 1.30 and 130 μM for hOAT3 and hOATP1B3, respectively, with Hill coefficients of 0.76 and 0.64, respectively. The Ki value of probenecid for hOAT3, but not for hOATP1B3, was significantly lower than the maximum unbound plasma concentration of probenecid at clinical dosages. These results suggest that the renal drug-drug interaction between fexofenadine and probenecid is probably explained by an inhibition of the renal uptake of fexofenadine via hOAT3, at least in part.

Is the Monkey an Appropriate Animal Model to Examine Drug-Drug Interactions Involving Renal Clearance? Effect of Probenecid on the Renal Elimination of H2 Receptor Antagonists

The renal drug-drug interaction between famotidine (an H2 receptor antagonist) and probenecid has not been reproduced in rats. We have proposed that this is caused by a species difference in the transport activity by human/rat organic anion transporter (OAT) 3 and the expression of organic cation transporter (OCT) 1 in the rodent kidney. Since monkey OATs (mkOATs) exhibit similar transport activities to human orthologs, it is hypothesized that in vivo studies in monkeys will allow a more precise prediction of renal drug-drug interactions in humans. Famotidine and cimetidine were efficiently taken up by mkOAT3-expressing human embryonic kidney cells (Km, 154 and 71 μM, respectively), and their uptake was strongly inhibited by probenecid (Ki, 3.0–5.7 μM). Quantification of mkOCT1 and mkOCT2 mRNAs in the monkey kidney using real-time reverse transcription-polymerase chain reaction revealed their predominant expression in the liver and kidney, respectively. Crossover studies were conducted in cynomolgus monkeys. Famotidine was given by i.v. administration, with or without probenecid. Probenecid treatment caused a 65% reduction in the renal clearance (0.426 ± 0.079 versus 0.165 ± 0.027 l/h/kg) and a 90% reduction in the tubular secretion clearance (0.275 ± 0.075 versus 0.0230 ± 0.0217 l/h/kg), whereas it had no effect on the renal clearance of cimetidine. In contrast to the species-dependent effect of probenecid, allometric scaling using animal data (rat, dog, and monkey) successfully predicted the renal and tubular secretion clearance of famotidine in humans. These results suggest that monkeys are more appropriate animal species for predicting the renal drug-drug interactions in humans.

Physiological Modeling of Altered Pharmacokinetics of a Novel Anticancer Drug, UCN-01 (7-Hydroxystaurosporine), Caused by Slow Dissociation of UCN-01 from Human α1-Acid Glycoprotein

AbstractPurpose. The extremely low clearance and small distribution volumeof UCN-01 in humans could be partly due to the high degree of bindingto hAGP (1,2). The quantitative effects of hAGP on the pharmacokineticsof UCN-01 at several levels of hAGP and UCN-01 were estimatedin rats given an infusion of hAGP to mimic the clinical situation anda physiological model for analysis was developed. Methods. The plasma concentrations of UCN-01 (72.5–7250 nmol/kgiv) in rats given an infusion of hAGP, 15 or 150 nmol/h/kg, weremeasured by HPLC. Pharmacokinetic analysis under conditionsassuming rapid equilibrium of protein binding and incorporating thedissociation rate was conducted. Results. The Vdss and CLtot of UCN-01 (725 nmol/kg iv) in ratsgiven an infusion of hAGP, 150 nmol/h/kg, fell to about 1/250 and 1/700that in control rats. The Vdss and CLtot following 72.5–7250nmol/kg UCN-01 to rats given 150 nmol/h/kg hAGP were 63.9–688ml/kg and 3.18–32.9 ml/h/kg, respectively, indicating non-linearitydue to saturation of UCN-01 binding. The CLtot estimated by thephysiological model assuming rapid equilibrium of UCN-01 bindingto hAGP, was six times higher than the observed value while the CLtotestimated by the model incorporating koff, measured using DCC, wascomparable with the observed value. Conclusions. These results suggest that the slow dissociation ofUCN-01 from hAGP limits its disposition and elimination.

Pharmacokinetics and pharmacodynamics of a novel protein kinase inhibitor, UCN-01

Purpose: 7-Hydroxystaurosporine (UCN-01) is a potent protein kinase inhibitor and is being developed as a novel anticancer agent. We describe here its pharmacokinetics and pharmacodynamics in experimental animals. Methods: The pharmacokinetics of UCN-01 were studied following intravenous (i.v.) administration to mice, rats and dogs at doses of 1–9, 0.35–3.5 and 0.5 mg/kg, respectively. We also studied the pharmacodynamics of UCN-01 (9 mg/kg per day) during and after five consecutive i.v. administrations to nude mice bearing xenografted human pancreatic tumor cells (PSN-1). The concentrations of UCN-01 in plasma and tumor were measured by HPLC using a fluorescence detector. Results: UCN-01 in plasma after i.v. administration was eliminated biphasically in mice and rats, and triphasically in dogs. The elimination half-lives in mice, rats and dogs were 3.00–3.98, 4.02–4.46 and 11.6 h, respectively. The total clearance (Cltotal) values in mice, rats and dogs were high (1.93–2.64, 2.82–3.86 and 0.616 l/h per kg, respectively). The hepatic clearance (Clhepatic) in rats represented 54.0–81.3% of Cltotal. The volumes of distribution at steady-state in mice, rats and dogs were large (7.89–8.42, 13.0–16.9 and 6.09 l/kg, respectively). These pharmacokinetic parameters were dose-independent in mice and rats. UCN-01 produced significant inhibition of tumor growth during five consecutive i.v. administrations in mice bearing the xenografted PSN-1 cells, and the inhibitory effect continued for 3 days after the final administration. UCN-01 concentrations in tumor tissue were much higher than those in the plasma, and the ratio of tumor to plasma concentrations was about 500 at 24 h after five consecutive doses. Conclusions: The pharmacokinetic studies showed that UCN-01 has a high clearance and large distribution volume in various experimental animals, and its disposition is linear over the range of doses tested. The pharmacodynamic study showed that UCN-01 is distributed at much higher concentrations in tumor than those in plasma and that it significantly inhibits tumor growth. The high distribution of UCN-01 into tumor cells may contribute to the potent inhibition of tumor growth in vivo.