We evaluated the nonlinear (dose-dependent) gastrointestinal absorption of 5-fluorouracil (5-FU), cefatrizine and L-carnitine in rats in vivo by physiological mechanism-based approach to clarify their absorption characteristics. For every drug, the intestinal absorption rate constant (ka), which was estimated by analysis of gastrointestinal disposition after oral administration, decreased with dose, consistent with the concentration-dependent intestinal membrane transport in situ by the involvement of carrier-mediated transport. Excellent in vitro (in situ)-in vivo correlations were observed in intestinal transport, including data for passively absorbed drugs from our previous reports, and apparent absorption rate constant (ka') and fraction absorbed (Fa) from plasma concentration data were successfully related to ka by the equations of 1 /ka'= 1 /ka + 1/kg and Fa = 1 - exp(-ka·Tsi), where kg and Tsi are gastric emptying rate constant and intestinal transit time, respectively. These relations enable us to predict oral drug absorption from in vitro (in situ) membrane permeability, using physiological parameters to define absorption. This should be helpful for optimizing oral drug therapy and developing rational oral drug delivery strategies.

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In this report, we have determined the uptake of salicylic acid (SA) by liposomes and proposed the pH-dependent but not carrier-mediated transport mechanisms of SA through the lipid bilayer. The permeability of various drugs to the liposomal membrane, estimated from their inhibitory effects on SA uptake, correlated well with their permeability to rat intestinal membrane in vivo, and that to Caco-2 monolayer in vitro. In addition, the high permeability of SA or benzoic acid to the lipid bilayer was confirmed by means of molecular mechanics calculations in which the polar surface area and the electrical potential of drug molecules were used as the parameters of their permeability. Our results could be expected to describe the mechanisms of the intestinal absorption of monocarboxylic acid drugs.

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We investigated the effect of cationic drugs on carrier mediated transport using in vitro and in situ techniques. The initial uptake of glucose, alanine and glutamic acid by rat intestinal brush border membrane vesicles(BBMV) was distinctly inhibited by imipramine in the presence of Na⁺ gradient, but not inhibited in the absence of Na⁺ gradient. This result indicates that imipramine inhibited carrier mediated transport. Imipramine exhibited competitive inhibition with respect to Na⁺ concentration, while exhibited non-competitive inhibition with respect to glutamic acid concentration. This finding suggests that imipramine affected carrier mediated transport by competitive inhibition to Na⁺ binding site of the carrier. On the other hand, in situ single pass perfusion method showed that absorption of alanine was also inhibited by imipramine and other cationic drugs in the presence of Na⁺, but not inhibited in the absence of Na⁺. This result is agreement with that of BBMV study. The inhibition of carrier mediated alanine transport by cationic drugs in BBMV corresponded to that in situ. There was a relatively good correlationship between the BBMV study and in situ perfusion study in the inhibitory effects.

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The mechanism of renal excretion of polyamine drugs has been investigated in vivo and in vitro. Trientine clearance (CLtri) in the rat was significantly faster than creatinine clearance (CLcr). However, CLtri decreased to almost the same level as the CLcr when trientine and the same number of moles of copper ions were administered simultaneously to the rat. To clarify this active excretion system for trientine, the uptake of trientine and spermine, a physiological polyamine, was investigated using the rat renal proximal tubular brush-border membrane vesicles (BBMV). An outwardly directed Na⁺ gradient remarkably enhanced the uptake of these polyamine compounds, however, it did not affect the uptake of trientine-Cu complex. The Na⁺-dependent uptake of [³H]spermine was completely inhibited by spermine, trientine and tetraethylenepentamine. On the contrary, physiological polyamines (putrescine and spermidine) which have 2 or 3 amino-groups, and aminoglycoside antibiotics which have 4 or 5 cationic amines, did not affect the uptake of spermine in the presence of an outward Na⁺ gradient. These results suggest that the mechanism contribute to the renal tubular secretion of spermine is a Na⁺/spermine antiport system, and this transporter recognizes the straight-chain polyamine drug which has more than 4 amino-groups in its molecule.

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We demonstrated the presence of an efflux transport system for tolbutamide, a sulfonylurea, from brain to blood based on an in vivo and in vitro transport study. Brain to unbound plasma concentration ratios of [¹⁴C]tolbutamide increased in the presence of high concentrations of cold tolbutamide and sulfonamide in vivo. Based on a transcellular transport study by mouse brain capillary endothelial cell (MBEC4 cell), which could be functionally involved in the blood-brain barrier (BBB), we found that the abluminal to luminal flux of [¹⁴C]tolbutamide was faster than the reverse directional flux and that both directions of transport were saturable. By using uptake study, the uptake of ['4C]tolbutamide is pH-dependent. For the therapeutic plasma concentration of tolbutamide (1-16.9 μM), the efflux transport rate would be faster than the influx rate. The net efflux phenomenon was consistent with the fact that the brain distribution of tolbutamide is very low in vivo. These findings suggested that BBB transport of sulfonylurea was regulated by two transport systems which are influx and efflux transport systems.

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MRP and cMOAT are the organic anion transporters involved in the efflux of organic anions from cells. We have already found two novel cDNA sequences (MLP-1 and -2: MRP like protein) which show high homology with MRP and cMOAT. Liver expression of MLP-2 was observed only in mutant rats (EHBR) whose cMOAT is hereditarily defective, suggesting that MLP-2 is an inducible transporter. MLP-2 is induced in normal rat liver by injection of phenobarbital, ANIT and bile duct ligation. In normal rats, MLP-2 is expressed mainly in the duodenum and jejunum. To clarify the nature of the organic anion transport mechanism in the intestine, we used Caco-2 cells as an intestinal model. DNP-SG and estradiol 17 β-D-glucuronide were taken up by BBMV prepared from Caco-2 in an ATP-dependent manner. Northern blot analysis suggested high expression of human cMOAT and MLP-2 and low expression of MRP in cultured Caco-2 cells.

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PEPT1 and PEPT2 are H⁺-coupled peptide transporters expressed preferentially in the intestine and kidney, respectively, which mediate uphill transport of oligopeptides and peptide-like drugs such as β-lactam antibiotics. In the present study, we have compared the recognition of β-lactam antibiotics by LLC-PK, cells stably transfected with rat PEPT1 or PEPT2 cDNA. Among the examined drugs, cyclacillin (aminopenicillin) and cefadroxil (aminocephalosporin) showed the most potent inhibitory effects on glycylsarcosine uptake in PEPT1- and PEPT2-expressing cells, respectively. Comparison of the Ki values of β-lactam antibiotics between PEPT1 and PEPT2-expressing cells suggested that PEPT2 had a much higher affinity for β-lactam antibiotics with an α-amino group. We have examined interactions of β-lactam antibiotics with rat renal brush-border membrane vesicles, in which PEPT1 and PEPT2 appeared to be coexpressed. The apparent Ki values of the antibiotics for glycylsarcosine transport were closely correlated with those in the PEPT2-expressing cells, suggesting that β-lactam antibiotics interact predominantly with PEPT2 rather than PEPT1 in renal brush-border membranes. In conclusion, 1) PEPTI and PEPT2 show different recognitions of β-lactam antibiotics, and 2) β-lactam antibiotics predominantly interact with renal PEPT2 at pharmacological concentrations.

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The enhancing effects by sodium caprate in the colon can be elucidated by the increase in intracellular calcium level which activates contraction of perijunctional actomyosin and widens the lateral intercellular space. Also for the effects of acylcarnitines and organic acids, the paracellular effects were found and the reduction of intracellular ATP level which was induced by acidosis was presented as a new enhancing mechnism. The above effects on the membrane barrier function returned to the control values within 3 hr after removal of enhancers, but the membrane dysfunction which was obtained as reduction of short current (Isc) continued for several hours. The membrane damage was found due to the decrease in the secretion of chloride ion which might induce diarrhea. For the practical use of enhancers, their physiological or pathlogical effcects should be further examined in more detail.

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The potency of surface coating liposomes with some materials was investigated for oral delivery of peptide drugs by using insuin as a model peptide. When insulin solution was orally administered to rats, no hypoglycemic effect was observed. In contrast, mucin-coated liposome(Mucin-LIP) and PEG-coated liposome(PEG-Lip) prolonged the decrease in the glucose level after administration. Surface coating liposomes showed the slow release of insulin in the bile salt solution, whereas non-coated liposome was burst by the bile salts. Furthermore, insulin encapsulated in surface coating liposomes was stable even in the intestinal fluid. From the results of the study for the intestinal transit of liposomes, it was clarified that surface coating liposomes, especially PEG-Lip, much delayed the mean transit time of insulin. In conclusion, both the improvement of insulin stability in GI tract and the slow transit by the encapsulation of insulin in surface coating liposomes achieved the longer dulation of oral hypoglycemic activity of insulin. The surface coating should be the potential way to add desirable functions to the liposome for oral drug delivery.

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The objective of this study was to estimate colon-specific delivery of insulin or anti-ulcerative colitis drugs with chitosan capsules. When insulin was orally co-administered with sodium glycocholate using chitosan capsules, a marked increase in plasma insulin concentrations and a corresponding decrease in plasma glucose levels were observed. On the other hand, when 5-ASA was orally administered using chitosan capsules in TNBS-induced colitis rats, we found better therapeutic effects of 5-ASA than those using 5-ASA CMC suspension. Furthermore, the mucosal tissue concentrations of 5-ASA in the large intestine after oral administration using chitosan capsules was higher than those in CMC suspension. Thus, the chitosan capsules may be useful carriers for the colon-specific delivery of peptides as well as anti-inflammatory drugs.

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To estimate quantitative information of antitumor effect, simulations were performed by the physiological model linked with a cell kill kinetic model. Antitumor effect of liposomal DOX was increased with the increase of blood circulating time. There was the optimum rate of drug release (krel) from long circulating liposomes. To examine this model, we prepared liposomes with different krel, which can be made by changing lipid composition such as HEPC, EPC and CHO. Antitumor effect of liposomal DOX with different krel was evaluated by survival experiments. Liposomes with the lowest krel showed the highest antitumor effect. This result was different from our predicitori based on the computer simulations, which regured some corrections of our model.

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In cancer therapy, intratumoral injection is an effective way to maximize the action of injected substances at the tumor site, however there is little information on the disposition characteristics of the injected materials after intratumoral injection. To clarify the effect of molecular size and surface charge of various drugs and carriers in the tumor at an organ level, their pharmacokinetic properties after intratumoral injection were studied in the perfusion experiments employing the tissue-isolated tumor preparation. Pharmacokinetic analysis of their venous outflow patterns revealed that alteration of the physicochemical properties of drugs and carriers dominantly changed the rate of transfer from the poorly-perfused region to the well-perfused region, suggesting that this process is the determinant factor which alters the intratumoral behavior of drugs and carriers after direct injection.

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PepT1, which is localized in the small intestinal and renal epithelial brush border membranes, takes up native di and tripeptide as well as peptidemimetic drugs such as β-lactam antibiotics and bestatin. The limited tissue distribution and broad specificity of PepT1 may be advantageous to deliver peptidemimetic drugs to the tissues which need to be supplied of nutrients for continuous growth. Accordingly, we investigated the substrate specificity of human PepT1 and measured the oligopeptide transport activities in tumor cells using human fibrosarcoma cell line, HT-1080 as the model tumor cells. Although it has been unclear whether cefdinir and cefaclor are transported by PepT1 or not, the present study clearly showed that they are transported by PepT1. The affinity of zwitterionic antibiotics to PepT1 is maximal around pH 6, but in anionic ones, higher activity was observed at more acidic pH. These results indicate that PepT1 has a broad specificity and its transport activity is influenced by ionization state of substrates. To explore the feasibility of targeting human tumor cells via the peptide transport system, oligopeptide uptake was studied in HT-1080 and compared with that in normal the human fibroblast cell line IMR-90 by measuring the uptake of radiolabeled Gly-Sar. The uptake of Gly-Sar by HT-1080 was concentration and pH dependent. The initial rate of Gly-Sar uptake had a Km of 11.4 mM and Vmax of 26.8 nmol/15 min/mg protein and was inhibited by various di and tripeptides, but not by amino acids, tetra or pentapeptides. On the other hand, IMR-90 showed negligible peptide transport activity. The present finding could be the basis of a novel strategy for the specific delivery of oligopeptide-mimetic anticancer drugs into tumor cells.

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Arg-vasopressin (AVP) derivatives modified at the N-terminal amine with various sugars via an octamethylene were synthesized and tissue uptake was evaluated after i.v. injection in rats. Glucosyl, mannosyl and 2-deoxyglucosyl derivatives showed significantly higher tissue uptake clearance by the kidney than galactosyl and α-mannosyl derivatives. In vivo tissue uptake analysis of the glucosyl derivatives showed that only the kidneys had a saturable uptake mechanism via the antiluminal membrane. The derivatives which showed high renal uptake clearance showed specific binding to the kidney membrane in vitro. These results suggested that there was a novel sugar transport mechanism in the kidneys which could be utilized as a delivery method for glycosylated peptides.

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A new in situ experimental method was designed to well predict and understand the muscle targeting and skin disposition of a topically applied drug. A disk shaped agar gel (3.85 cm in diameter and 0.5 cm in thickness) was firstly inserted between the hypodermis and the muscle layer in hairless rats. Flurbiprofen (FP), a model drug, was administered topically (using 3% HPC gel containing 1% drug) and intravenously into the rats. Drug disposition following topical application could be satisfactorily described by a pharmacokinetic model including cutaneous transport. The amounts of FP migrated from the formulation to the agar gel and the systemic circulation over 10 h were 12.4 and 1.92μg, respectively; namely most drug was absorbed into the systemic circulation. A ratio of FP into the agar gel from the formulation/the systemic circulation was found to 97.2/2.8. Moreover, the addition of several enhancers or vasoconstrictors affected a ratio of FP migration to the agar gel. This technique can be utilized as a new tool to evaluate the skin disposition of topically applied drugs.

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The purpose of this study is to characterize the ocular membrane permeability and absorption of ophthalmic drug. Male albino rabbits were used throughout the study.
The in vitro penetrations of beta-blockers were measured across the isolated corneal and conjunctival membranes using a two-chamber glass diffusion cell. The lipophilic character of the corneal barrier was determined by the partition parameter of beta-blockers, not by the diffusion parameter. Ocular permeability coefficients of peptide drugs and hydrophilic macromolecules were extremely lower than those of beta-blockers. Ocular membranes have been also characterized by in situ absorption of beta-blockers using a cylindrical cell. As the results, the corneal route was a dominant route of access to the aqueous humor. A pharmacokinetic model including the diffusion process for ocular drug delivery was demonstrated. Distribution volume and elimination rate of tilisolol and timolol in tear fluid and aqueous humor were estimated by instillation of drug and injection of drug into the anterior chamber. The in vivo permeability parameters for tilisolol and timolol were estimated from the model.

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Tissue distribution and local targeting kinetics of PSC 833 (PSC), a multidrug resistance (MDR) modifier, were extensively studied in vitro, with human originated tumor cells, and in vivo, with tumor bearing mice. In vitro studies demonstrated that the membrane transfer and interaction with flux-carrier-protein are relatively rapid for PSC, and estimated an inhibitory constant (Ki) of 0.02 μg/ml using daunorubicin as substrate. In vivo distribution study also revealed the rapid drug distribution to normal and neoplastic tissues. These tissue distribution profiles were modeled by taking into account the time and dose-dependent plasma protein binding which was studied separately with an ex vivo technique. A physiologically-based pharmacokinetic (PBPK) model developed accordingly not only described globally the drug disposition in the whole body but also quantitatively characterized the extent of P-glycoprotein suppression efficacy at tumor site as well as brain exposure which increases sensitively to the increase in the blood concentration, resulting potential neurotoxicity. The present approach based on the PBPK model is considered helpful to optimize the safe and efficient use of the MDR modifier.

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Correlation between in vivo and in vitro activities of aldehyde oxidase was examined in several mammalian species. The in vitro activity was determined by measuring oxidation of N¹-methylnicotinamide to the corresponding 2-pyridone and 4-pyridone derivatives by liver cytosols. On the other hand, the in vivo activity of the enzyme was determined by measuring above pyridone derivatives as urinary metabolites of nicotinamide. Marked strain differences were observed when aldehyde oxidase activity of liver cytosols was examined in five strains of rats (Sea:SD, Jcl:SD, Std:Wistar/ST, Crj:CD and WKA/Sea). The in vitro activity was well correlated with the in vivo activity of the enzyme in these strains. Significant species differences were observed when aldehyde oxidase activity was examined in rabbits, guinea pigs, hamsters, monkeys and dogs. The in vitro activity was also correlated with the in vivo activity of the enzyme in these species. Human liver cytosols exhibited relatively high aldehyde oxidase activity which was variable individually. Such variabilityof aldehyde oxidase activity in human was also observed in the in vivo studies.

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7α- and 7β-OH-Δ⁸-THC MALCO activities in mouse liver were significantly enhanced by dexamethasone and phenobarbital. A cytochrome P450, named P450MDX-B, was purified from hepatic microsomes of dexamethasone-pretreated mice. Anti-P45OMDX-B antibody inhibited 7-OH-Δ⁸-THC MALCO activity in mouse liver. A cDNA clone encoding a P450 isozyme was isolated from a C57BL/6 mouse liver cDNA library. The nucleotide sequence of coding region of this cDNA was identical with that of the known mouse Cyp3al 1 and the deduced amino acid sequence had an N-terminal sequence identical to that of P45OMDX-B. Expression plasmid of Cyp3al1 was transformed into exponentially growing COS-7 cells by the method of electroporation. Immunologically related protein with P45OMDX-B which had the same molecular mass was expressed in the COS cells microsomes (COS-3a11). The COS-3a11 catalyzed the oxidation of 7-OH-Δ⁸-THC to 7-oxo-Δ⁸-THC. Oxygen-18 (¹⁸O) derived from atmospheric oxygen was incorporated into 7-oxo-Δ⁸-THC from 7α-OH-Δ⁸-THC by hepatic microsomes, P45OMDX-B and COS3a11. On the other hand, incorporation of the stable isotope into the oxidized metabolite from 7β-OH-Δ⁸-THC was negligible. However, when 7β-OH-Δ⁸-THC was incubated with mouse hepatic microsomes using cumene hydroperoxide instead of NADPH under ¹⁸O₂, ¹⁸O was not incorporated into 7-oxo-Δ⁸-THC.
These results suggested that 7α-OH-Δ⁸-THC may be oxidized to the corresponding ketone by Cyp3all via a gemdiol pathway. On the other hand, 7β-OH-Δ⁸-THC may be converted to the ketone through a stereo selective dehydration of an enzyme-bound gem-diol rather than through a direct hydrogen extraction as a peroxy form of the enzyme.

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An enzymatic study was performed to clarify the mechanism of 18 acute deaths in patients who had received the new oral antiviral drug, sorivudine (SRV), during anticancer chemotherapy with 5-fluorouracil (5-FU) prodrugs. Human dihydropyrimidine dehydrogenase (hDPD), playing a key role in the liver as the rate-limiting enzyme in catabolism of 5-FU, was expressed in E. coli, purified, and incubated in the presence of NADPH with SRV or (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of SRV. hDPD was rapidly and irreversibly inactivated by BVU, but not by SRV. Radioactivity of [¹⁴C]BVU was incorporated into hDPD in the presence of NADPH in a manner reciprocal to the enzyme inactivation. In the absence of NADPH, hDPD was not inactivated by BVU, nor radiolabeled with [¹⁴C]BVU. Thus, as we demonstrated previously with studies using the rat, the acute deaths were strongly suggested to be attributable to markedly elevated tissue 5-FU levels, which were responsible for irreversible inhibition of hDPD by covalent binding of a reduced form of BVU as a suicide inactivator. A significant linear correlation (r=0.58, p=0.005) was observed between liver-DPD activity and levels of the immuno-detectable protein with anti-hDPD-anti-serum, indicating that the antibody could be a valuable tool for the DPD deficiency screening.

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The stereoselectivity was evaluated for the metabolism and disposition of bunitrolol (BTL) in the rabbit. The enantioselectivity [(-)>(+)] was observed in liver microsomal 4-hydroxylation of BTL racemate (20 μM), but not in that of enantiomer (each 10 μM). No significant selectivity was shown in microsomal glucuronidation of BTL and 4-OH-BTL, while liver cytosolic sulfation was of negligible. After oral administration of BTL racemate (20 mg/kg), blood levels of BTL enantiomers as conjugate or nonconjugate (C or NC) were almost same, but those of 4-OH-BTL as C or NC showed the selectivity of [(-)>(+)]. Most of BTL and 4-OH-BTL in urine and bile were glucuronides and the selectivity of [(-)<(+)]was observed for BTL, whereas a reveresed one [(-)>(+)] was for 4-OH-BTL. These results suggest that the blood and urine levels of 4-OH-BTL as C or NC reflects the stereoselectivity of [(-)>(+)] in the BTL 4-hydroxylation, but the reversed selectivity [(-) >(+)] may exist for the excretion of BTL as C or NC to urine and bile, resulting in the same levels of BTL enantiomers in the blood following the oral administration of racemic BTL to the rabbit.

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Guinea pig liver cytochromes P450 capable of being inducible with phenobarbital (PB) was studied. Immunoblot analysis of liver microsomes showed that guinea pig CYP2C subfamily is increased markedly by PB treatment while CYP2B 18 and a CYP3A isozyme are increased only slightly. To clarify the difference in inducubility between rat and guinea pig CYP2B subfamily, we performed cloning of guinea pig genomic DNA coding the CYP2B. A clone of 19.1 kb was obtained, and sequenced by Shotgun method. The result showed that our clone contains regions corresponding to exon 5, 6, 7 and 8 of rat CYP2B2 gene. The sequencing has not yet been completed, and we wish to present the sequence data in more detail than described above.

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The mRNAs or proteins of CYP2D isoforms have been demonstrated to be expressed not only in liver but also in several extrahepatic tissues. However, the functions and properties of CYP2D isoforms in extrahepatic tissues have yet to be clarified. The tissue distributions and the catalytic properties of the isoforms give valuable insights into their physiological and pharmacological functions. In this study, the tissue distributions of four isoforms (CYP2D1/5, 2D2, 2D3 and 2D4/18) in rat CYP2D subfamily were investigated. The expression of CYP2D mRNA in twelve kinds of tissues was detected by RT-PCR. mRNA of CYP2D1/5 was expressed in all tissues except the brain. mRNAs of CYP2D2 and CYP2D3 were mainly expressed in liver, kidney and small intestine mucosa. In brain, only mRNA of CYP2D4/18 was expressed. Furthermore, we cloned four cDNAs (CYP2D1, 2D2, 2D3 and 2D4) belonging to the CYP2D subfamily. Expression plasmids carrying CYP2D cDNAs were transformed intoSaccharomyces cerevisiae. A catalytic study of these CYP2D isoforms was done with debrisoquine, bufuralol and lidocaine. CYP2D2 had the highest debrisoquine 4-hydroxylation activity. Both CYP2D3 and CYP2D2 exhibited high lidocaine 3-hydroxylation activity. The activity of CYP2D1 was relatively low toward the three substrates. These findings indicate that CYP2D2 and CYP2D3 play major roles in the metabolism of xenobiotics in rat liver, kidney and small intestine mucosa which are exposed to xenobiotics such as drugs, food components and environmental contamination and that it is possible that CYP2D4 has endogenous substrates other than dugs.

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Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Omeprazole 5-hydroxylation by liver microsomes of a human sample which contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, while a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive towards fluvoxamine and anti-CYP2C9 antibodies. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal P450 enzymes. When recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 μM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver. Different contributions of CYP2C19 and CYP3A4 In the oxidation of testosterone and progesterone by human liver microsomes are also reported.

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Simultaneous intake of grapefruit juice, but not other fruit beverages, is now known to alter pharmacokinetics and efficacies of various drugs.. Although drugs described above do not relate structurally with each other, a form of cytochrome P450 (CYP) 3A4, is shown to be responsible for the metabolisms of these drugs. The exact mechanism of this interaction remains yet unclear, but the suppression of drug metabolisms has been suggested as a major mechanism. We have isolated four components from grapefruit juice that inhibit human CYP3Amediated drug oxidation. The structures of these components were identified as furocoumarin derivatives by absorption spectra, APCI-LC/MS/MS and NMR after their purification by a reversed-phase HPLC. These furocoumarins are strong candidates for causative agents of grapefruit juice-mediated drug interaction, because of their inhibition equal or stronger than did a typical CYP3A4 inhibitor, ketoconazole, on microsomal testosterone 6β-hydroxylation in human livers.

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The species difference for disposition of propranolol (PL) drivatives between rat and dog was investigated. In the intravenous administration, the total clearances of butyryl PL were 1.5 fold and 30 fold greater than hepatic blood flow in rat and dog, respectively. In vitro hydrolysis experiments showed that hepatic hydrolytic clearances of rat and dog were equivalent to hepatic blood flow, indicating that butyryl PL was mainly hydrolyzed in liver for rat. On the other hand, dog showed markedly high hydrolytic activity in lung, and it was demonstrated from an artery injection experiment that butyryl PL recieved the first-pass metabolism in lung. In the oral administration, we found that ester derivatives of PL improved the oral bioavailability of PL in dogs according to the following mechanism; increases of absorption rate and hepatic uptake rate and rapid hepatic hydrolysis lead to metabolic saturation of PL with increase of hepatic PL concentration. However, unfortunately, ester derivatives did not improve oral bioavailability of PL in rats due to almost complete hydrolysis of ester derivatives in small intestine.

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N-acetyltransferase 2 (NAT2) and cytochrome P450 2C19 enzymes are known to exhibit a hereditarily determined polymorphism. The characterization of genetic variation at the DNA level for these enzymes has made it possible to determine an individual genotype. The common method is a polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) method using blood samples. We have developed a novel method for determining the NAT2 and CYP2CI9 genotypes using genomic DNA extracted from single hairs, buccal cells and fmgernail. The N-acetylation activity for procainamide and isoniazid was well correlated with NAT2 genotypes. Furthermore, we have first found that the anti-H. pylori efficacy of omeprazole can be related to the CYP2C19 genotype, that is, the eradication effect of omeprazole with amoxicillin was highly efficient in CYP2C19 poor metabolizers, suggesting that CAM or metronidazole needs not to be used for this group on the first line therapy. Genotyping by PCR-RFLP, which is a simple in vitro test, can provide a new strategy to choose an optimal regimen based upon the individual genotype.

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Effects of genetic polymorphisms of CYP2C9/19 and metabolic inhibition by coadministered drugs on the inter and intra-individual variations in the warfarin-induced anticoagulation were studied using in vivo pharmacokinetic data obtained from cardiac patients and in vitro enzyme kinetics obtained from human liver microsomes and recombinant CYP2C9 wild type and Leu₃₅₉ variant. Results indicated that both heterozygous 2C9 Leu₃₅₉ mutation and the two concomitantly given drugs (i.e., benzbromarone and bucolome) possessing potent inhibitory effect on the in vitro S-warfarin metabolism would be associated with a clinically relevant reduction in the in vivo CLpo, u of pharmacologically more potent S-warfarin in humans. Estimation for changes in in vivo CLpo, u based upon in vitro enzyme kinetic data was considered feasible for warfarin.

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In vitro-in vivo scaling works well for metabolic enzyme inhibition, but is less successful with enzyme induction. In this paper, we are attempt to establish the prediction method for enzyme induction in vivo from cultured hepatocytes studies. Phenobarbital (PB) was used as a model inducer for CYP2B and the enzyme activities were assessed by pentoxyresorufin O-depentylation (PROD). In vivo induction was determined in liver microsomes isolated from rats after administration of PB. In vitro induction was assessed in cultured rat hepatocytes treated with PB. To compare in vitro induction with in vivo induction, plasma unbound concentration of PB has been used as a method of approximating unbound hepatocellular PB concentration in rats. Maximum induction (6 fold) and EC50 values (15-20 μM) after single administration of PB were well agreed between in vitro and in vivo studies. Similar results were obtained in repeated administration of PB. In conclusion, although there still remain some problems for quantitative prediction of the enzyme induction, we showed the possibility and effectiveness of the prediction of inducibility in vivo from cultured hepatocytes.

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The in vitro intrinsic clearances (CLint, in vitro) of P450-mediated reactions obtained by using human liver microsomes were compared with the in vivo intrinsic clearances (CLint, in vivo) which were calculated from pharmacokinetic data in literature. Although CLint, in vitro and CLint, in vivo agreed well with each other for most of the drugs investigated, more than 10-fold differences were observed for some drugs, indicating the possible involvement of the first-pass metabolism in the gut and/or interindividual variability in the hepatic metabolism. The AUCoral and bioavailability of YM796, a compound being developed for the treatment of dementia, were well predicted from in vitro metabolic studies taking the non-linear first-pass metabolism into consideration. Furthermore, we have tried to predict in vivo drug-drug interactions from in vitro data on drug metabolism obtained from the literature. Assuming the same unbound concentration of the inhibitor in the liver and in plasma, the degree of increase in the AUC caused by the metabolic inhibition was underestimated for some of the drug combinations. The transport study using isolated rat hepatocytes indicated that the contribution of the active transport of the inhibitors such as quinidine, erythromycin etc. into the hepatocytes is not so large. The drug-drug interactions involving the gastrointestinal absorption process may have to be considered for the more precise prediction.

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(1) We tried to predict the increase rate (F) of plasma concentration or miaazolam (MDZ) in the presence of inhibitors in rats, using the following equation. F=1+Ci/Ki (Ci:concentration of inhibitor, Ki:inhibitory constant). The predicted F in the presence of itraconazole, ketoconazole, cimetidine, and nizatidine using unbound concentrations of inhibitors in the liver were close to the observed values, suggesting the concentrative uptake of inhibitors into the liver must be taken into consideration to predict F. (2) There was a good correlation between the cell-to-medium concentration ratio (C/M) and the liver-to-unbound plasma concentration ratio (Kpf) in rats, suggesting drug concentration in the liver can be predicted from C/M and unbound plasma concentration. (3) We attemptted to predict F of plasma concentration of MDZ in the presence of inhibitors in human from absorption constant and Kpf in rats. The predicted F in the presence of cimetidine corresponded to the observed value.

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