The fully synthetic and novel 3-hydroxy-3-methylglutaryl-coenzyme A (HMG—CoA) reductase inhibitor, cerivastatin, was found to show highly liver-selective distribution in rats. To elucidate the mechanism on such organ-selective distribution, the possibility of the carrier-mediated uptake of cerivastatin was investigated using primary cultured rat hepatocytes (cultured for 4 h).
Cerivastatin was found to be taken up into hepatocytes in a saturated manner, and kinetic evaluation revealed that the Michaelis-Menten constant (K_m) and the maximum uptake velocity (V_max) were 5.86μM and 260 pmol/min/mg protein, respectively. Almost comparable kinetic parameters were obtained in the absence of Na⁺ (K_m=4.68 μM, V_max=268 pmol/min/mg protein), indicating that the cerivastatin uptake is Na⁺-independent. Kinetic estimation also revealed that the specific uptake clearance accounted for approximately 70 ?? 80% of the total uptake clearance. Based on such a high specific uptake clearance, an extremely high cell-to-medium concentration ratio was observed at equilibrium after 60-min incubation.
A metabolic inhibitor, 2, 4-dinitrophenol, diminished the total uptake clearance of cerivastatin to about 41% of the control value, demonstrating an ATP-dependent uptake. Bromosulfophtalein, an organic anion taken up via Na⁺-independent multispecific anion transporters, exhibited the concentration-dependent inhibition toward the cerivastatin uptake. Cholate and taurocholate, substrates for organic anion transporting polypeptides (oatp) 1, inhibited the cerivastatin uptake into isolated rat hepatocytes. In addition, pravastatin, substrate for oatp2, also reduced the total uptake clearance of cerivastatin.
In conclusion, cerivastatin undergoes the hepatocellular uptake via Na⁺-independent multispecific anion transporters, causing a highly liver-selective distribution. Inhibition studies also suggested that oatp 1 and oatp 2 would be responsible for the active hepatocellular uptake of cerivastatin.

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Pharmacokinetic drug-drug interactions (PDDI), in which the pharmacokinetic clearance of one drug is altered by a co-administered drug, can be divided mechanistically into two general categories: 1. inhibitory PDDI, the inhibition of the metabolic clearance of one drug by a co-administered drug; and 2. inductive PDDI, the enhancement of the metabolic clearance of one drug by a co-administered drug. It is believed that drug-drug interaction potential can be defined using a mechanism-based approach which includes the following sets of studies. 1: Identification of the major drug metabolizing enzymes. 2: Evaluation of the inhibitory potential for drug-metabolizing enzymes. 3. Evaluation of the induction potential for drug-metabolizing enzymes. Human liver-derived experimental systems: hepatocytes, liver microsomes, and cDNA-expressed microsomes, are useful experimental systems for this mechanistic approach to the evaluation of drug-drug interaction potential preclinical.

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When the metabolism of a drug is competitively or noncompetitively inhibited by another drug, the degree of in vivo interaction can be evaluated from the Iu/Ki ratio, where Iu is the unbound concentration around the enzyme and Ki is the in vitro inhibition constant of the inhibitor. In the case of tolbutamide/sulfaphenazole interaction, the increase in tolbutamide AUC predicted using an estimated maximum value of Iu was comparable with the predictions taking the concentration profile of the inhibitor into consideration and also with the in vivo observation.
We have also evaluated the metabolic inhibition potential of a number of drugs known to be inhibitors or substrates of cytochrome P450 (CYP) by estimating their Iu/Ki ratio using literature data. Using the maximum unbound concentration of the inhibitor at the inlet to the liver as Iu, the possibility of drugs causing in vivo drug-drug interactions could be predicted based on their Iu/Ki ratios.
On the other hand, in the case of interactions involving a mechanism-based inhibition, it is necessary to consider the exposure time of the enzyme to the inhibitor and enzyme turnover in predicting from in vitro data. A case of 5-fluorouracil/sorivudine interaction has been predicted based on a physiologically-based pharmacokinetic model.
Although it can be assumed for most inhibitors that the value of Iu is equal to the unbound concentration in the liver capillary, this assumption is not valid if the inhibitor is actively taken up by the liver. In the in vitro uptake studies using isolated rat hepatocytes and ATP depletors, none of the investigated inhibitors was found to be highly concentrated in the liver, and the predicted in vivo interaction was not greatly affected by taking account of active transport of the inhibitor.
We have also attempted to establish a rational methodology for predicting the drug interactions via hepatic transporters responsible for drug uptake and subsequent excretion. Both isolated hepatocytes and canalicular membrane vesicles were used to determine the intrinsic potential for the interaction via hepatic uptake and biliary excretion at the sinusoidal and canalicular membranes, respectively. We have demonstrated that the degree of inhibition of each membrane transport process can be accurately estimated also by considering the Iu/Ki ratios. We have also established a rational methodology to predict the degree of inhibition of net biliary excretion (from blood to bile) which can also be applied to clinical situations and prevent researchers from making false negative predictions.

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Information concerning drug interactions is essential for physicians and patients when attempting to limit the number of serious adverse events. This information, however, is generally the result of research initiated by pharmaceutical companies and regulatory authorities, and as such not aimed at the needs of either physician or patient. This article carefully assesses these needs and intends to provide guidance in order to improve the current situation. To this end a number of practical examples of drug interaction are re-evaluated in terms of their informative utility.

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The Drug Interaction Working Group consisting of 31 Japanese pharmaceutical companies was organized to generate a drug interaction database by the Human and Animal Bridge Discussion Group. Protocols for the assay methods of 10 isoforms of human liver cytochrome P450 (CYP1A1 & 2, CYP2A6, CYP2B6, CYP2C8 & 9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4) were provided, and the collection of the data on inhibition of these isoforms (Ki) by drugs already on the market has been started. A common, human liver microsome pool, which was prepared from 10 liver samples supplied by the National Disease Research Interchange in the USA, is being used for this measurement. In the case of no inhibition, the highest concentration of the test article examined is to be reported. As well, information on plasma concentration, plasma protein binding ratios in humans, the main metabolic pathway and the drug-metabolizing enzymes for the drug in question are being collected. As a result, the database will be able to automatically calculate, using the above-described parameters, a theoretical estimate of the magnitude of the drug interaction caused by the enzyme inhibition.

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In current clinical practice, concomitant multiple drug administration regimens involving the simultaneous use of two or more kinds of drugs are extensively applied. To ensure the proper use of the drug and their safety, drug-drug interaction is a critically important factor. This issue also has aroused great social interest in view of the well-known sorivudin event.
The majority of adverse drug-drug interactions are due to pharmacokinetic interactions, and around three quarters of these are associated with drug metabolism.
Most of the interactions occurred as results of inhibition or induction to metabolic enzymes are clinically significant.
The FDA of the U. S. A. and the CPMP of the E. U. have successively published drug-drug interaction guidance in 1997. In Japan, the guideline/guidance for drug-drug interaction is under discussion.
Our JPMA focused on the theme of drug-drug interaction and established a working group in May 1997 to study drug-drug interaction, with dedicated efforts being made to prepare a database on drug-drug interaction. It is the very important mission for pharmaceutical manufacturers to provide pharmacokinetic/pharmacodynamic information of drugs for safe use.
In July 1998, JPMA member companies were requested to complete questionnaires. The inquiry has elicited a roughly 80% response on 65 drugs until the present. We will persevere in our task to establish a database that will be of real practical value, on the basis of the most helpfull data provided by the companies concerned. We would also like to refer to our future plan adding the background and results of the activities that are being carried out.

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In 1993, eighteen acute deaths occurred in Japanese patients who received a clinical dose of the new oral antiviral drug, sorivudine (SRV: 1-β-D-arabinofuranosyl-(E)-5-(2-bromovinyl) uracil), for herpes zoster. That was only within 40 days after SRV was approved by the Japanese government for clinical use. All patients who died had been receiving one of 5-fluorouracil (5-FU) prodrugs for anticancer chemotherapy. None of patients who received SRV alone or SRV and anti-cancer drugs other than 5-FU or its prodrugs, died or suffered from toxic symptoms. A toxicokinetic study using rats orally given simultaneously with SRV and the 5-FU prodrug, tegafur (FT: 1-(2-tetrahydrofuryl)-5-fluorouracil), once a daily, strongly suggested that the patients died from a marked increase in tissue 5-FU levels by the mechanismbased inhibition of hepatic dihydropyrimidine dehydrogenase (DPD), with (E)-2-(5-bromovinyl) uracil (BVU), a metabolite of SRV by gut flora. DPD is a rate-limiting enzyme in the catabolism of 5-FU in the rat and the human. All rats showed a marked increase in the 5-FU level in plasma and tissues such as intestines and bone marrow from the first day of the experiment and died within 10 days after the repeated administration of SRV and FT. Before the death, the rats showed severe diarrhea with bloody flux and marked decreases in white blood cell and platelet counts as had been reported for the patients.
In the presence of NADPH, DPD isolated from the untreated rat liver and human recombinant DPD were completely inactivated and radiolabeled by [¹⁴C] BVU. Two radioactive peptides were isolated by HPLC from a tryptic digest of the radiolabeled human DPD. Amino acid sequencing indicated that the two peptides were derived from a common regional sequence of the DPD, which was in the potential pyrimidine-binding domain located at positions 665-686 of the enzyme subunit. Only the Cys residue at a position of 671 in the DPD was found to be radiolabeled and unidentified by the amino acid sequencing of the tryptic fragments. MALDI-TOF MS analysis indicated that the SH of Cys671 formed a sulfide bond by the reaction with the allyl bromide type of reactive metabolite, 5-(2-bromovinylydeny) uracil, yielded in the DPD subunit. The sulfide bond formation appears to make it impossible for the DPD to interact with the 5-FU through the substrate-binding domain. Therefore, the patients who died from the drug-drug interactions were most likely to become extremely poor metabolizers for 5-FU by the mechanism-based inhibition of DPD with BVU from SRV.

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Charcoal hemoperfusion is an effective extracorporeal treatment in cases of drug overdose. It is generally accepted that charcoal hemoperfusion cannot be remove drugs with a relatively large volume of distribution and high protein binding. The volume of distribution of a drug is the most important factor that limits the efficiency of charcoal hemoperfusion. It has be reported that drugs with small volume of distribution (< 50 L) are more efficiently removed by this treatment, based on single compartment kinetics. In contrast, there are no guidelines on the effect of protein binding properties of a drug on its removal by the treatment. So, we attempted to define a guideline based on the protein binding percentage of the drug for use in charcoal hemoperfusion treatment of cases of drug overdose. We measured plasma concentrations of the 20 different (a total of 32) drugs in 12 overdosed patients hemoperfused by an activated charcoal column. The extraction efficiency of a charcoal column, (A-V) /A, immediately after the beginning of hemoperfusion from the concentration in the blood entering (A) and that leaving the column (V), were then determined. From the relationship between extraction efficiency and protein binding percentage of the drugs, we found that drugs with a binding percentage as high as 95% can be removed by charcoal adsorption. Therefore, at binding percentages below 95% the clinical decision of whether to initiate hemoperfusion or not should be made only by consideration of the volume of distribution

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Among a wide variety of biological functions of vanadium, the insulin-like effect of vanadium is the most interesting and important. Vanadyl ion (+4 oxidation state of vanadium) and its complexes have been shown to normalize the blood glucose levels of streptozotocin-induced diabetic rats (STZ-rats). During our investigations on vanadyl complexes with several types of coordination mode, vanadyl-6methylpicolinate complex (VO (6MPA) ₂) was found to exhibit an excellent insulin-mimetic activity without toxicity as evaluated by both in vitro and in vivo experiments. Electron spin resonance (ESR) is available to measure the paramagnetic species in biological samples. We have developed the in vivo blood circulation monitoring-ESR (BCM-ESR) method to analyze the ESR signals due to paramagnetic metal ions and stable organic radicals in real time. In the present study, we applied this method to analyze the disposition of paramagnetic vanadyl species in the circulating blood in rats in order to know the relationship between the blood glucose normalizing effect and global disposition of vanadium. ESR spectra due to vanadyl species were detected in the circulating blood of rats, and their pharmacokinetic parameters were estimated using compartment models. The results indicated that vanadyl species were distributed to the peripheral tissues and eliminated from the body through the urine. The exposure of vanadyl species in the blood was enhanced by VO (6MPA)₂ treatment. In addition, the distribution of vanadium in several organs was examined by neutron activation analysis (NAA). Vanadium accumulation in the bone was suggested to relate with the long-term insulin-mimetic effectiveness of VO(6MPA)₂. Based on these results, we concluded that the metallokinetic character of vanadyl complexes is closely related with the structure and insulin-mimetic activity of the complexes.

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