Search

We previously showed that naproxen induced the oxidative stress in the liver microsomes and the isolated hepatocytes of rats. In this study, the in situ effect of naproxen on the rat liver tissue was investigated, using the isolated perfused liver from the view-point of the naproxen-induced hepatotoxicity. The leakage of glutamic-oxaloacetic transaminase (GOT) from the perfused liver and appearance of thiobarbituric acid reactive substances (TBARS) in the perfusate increased with the progress of perfusion after a lag time of about 1 h. The naproxen-perfusion of the liver decreased the biliary excretion of glutathione (GSH) and oxidized glutathione, glutathione disulfide (GSSG) prior to TBARS production and GOT leakage. GSSG content in the naproxen-perfused liver was significantly higher than in the control. TBARS appeared in the perfusate of the naproxen-perfused liver for 30 min, but not in the control. The biliary excretion clearance (CLbile) of indocyanine green (ICG), a reagent for testing the liver function, in the liver perfused with naproxen decreased to a half of that in the liver perfused without naproxen. Thus, the naproxen-induced oxidative stress in the liver was shown to affect the physiological function of liver through the impairment of biliary excretion, which is recognized as a detoxification system.

The effect of dietary polyphenols on the intestinal glucuronidation and absorption of a model phenolic drug, α-naphthol (α-NA), was studied in isolated rat small intestine. (−)-Epicatechin significantly inhibited the glucuronidation of α-NA. Chrysin, (−)-epigallocatechin galleate (EGCG), and quercetin decreased the rate of glucuronidation, although not significantly. Baicalin did not affect the glucuronidation. The rate of absorption of α-NA in the presence of these polyphenols also varied. The absorption clearance ( CL abs ) and the metabolic clearance ( CL met ) were inversely correlated, and this relationship was well explained in the metabolic inhibition model with kinetic parameters (knowledge-based prediction) which characterizes the relationship between the CL abs and CL met of α-NA (Biochim Biophys Acta 1998, 1425, 398.). These results indicate that the concomitant intake of some polyphenols can increase the absorption of a phenolic drug, and the effect is predictable. (−)-Epicatechin and chrysin are effective for the inhibition of glucuronidation and promotion of intestinal drug absorption.

We have studied intestinal metabolism and transport, which is considered to be sequential (1) or competitive (2) process in absorption (Scheme 1). (1) Disaccharide (maltose, cellobiose, lactose) conjugates of p-nitrophenol were hydrolyzed to p-nitrophenyl β-glucosides (p-NPβglc) on the mucosal side. p-NPβglc was transported by Na+/glucose cotransporter (SGLT1). Transport clearance of p-NPβglc formed from cellobiose and lactose conjugates of p-NP were higher than that from maltose or of p-NPβglc itself. These results suggest that SGLT1 is cooperatively coupled with lactase/phloridzin hydrolase catalyzing hydrolysis of cellobiose and lactose conjugates. There might be cooperative relationship between peptidase and H+/oligopeptide cotransporter or amino acid transporter as well. (2) Kyotorphin (KTP) was too unstable in intestine to be absorbed. KTP appeared on the serosal side in the presence of peptidase inhibitors. Meanwhile, cyclic KTP was stable in intestine to be absorbed. Absorption clearance of cyclic KTP was higher than the overall transport clearance of KTP, which was calculated according to the metabolic inhibition model. Competitive process was observed in intestinal absorption of α-naphthol as well. These results indicate that metabolism degradation and membrane transport are competitive. Unless drug is stabilized against metabolic enzyme, intestinal absorption of drug can not be improved even if membrane transport is increased.

Intestinal transport and metabolism of modified kyotorphin (KTP) were studied in rats. Modified KTPs studied were C-terminally modified KTP with p-aminophenyl-beta-D-glucoside (KTP-pAPbeta glc), N-terminally modified KTP-pAPbeta glc with t-butyloxycarbonyl group (Boc-KTP-pAPbeta glc) and the N- and C-terminally modified KTP by cyclization (cyclic KTP). KTP-pAPbeta glc was metabolized at a similar rate to that of KTP, and did not appear on the serosal side. Although Boc-KTP-pAPbeta glc was also metabolized, it was more stable than KTP and appeared on the serosal side. Cyclic KTP was also quite stable and appeared on the serosal side. The modified KTPs were evaluated kinetically for absorption consisting of membrane transport and metabolism. Absorption clearance (CL(abs)) of cyclic KTP, Boc-KTP-pAPbeta glc and Boc-KTP was higher than that of KTP (0.247 microl/min/cm) (Mizuma et al., Biochim. Biophys. Acta 1335 (1997) 111-119), which is the theoretical maximum by complete inhibition of peptidase activity, indicating that derivatization of KTP increases the membrane permeability. Furthermore, the data clearly showed that the greater the metabolic clearance (CL(met)) of KTP and the KTP derivatives, the lower the absorption clearance (CL(abs)). These results and further simulation study led to the conclusion that metabolic degradation in the intestinal tissues is more critical than membrane permeability (transport) for oral delivery of peptide drugs. Based on the stability of cyclic KTP in serum, this appears to be a good candidate analgesic peptide drug.

We examined the relation ship between the charge of the amino or carboxylic function of a substrate and the substrate recognition by the large neutral amino acid (LNAA) transport carrier, using the in situ brain perfusion technique. Glucose-coupled L-tyrosine (GcpY), which has a free carboxylic function, and 2-(L-tyrosylamide)-2-deoxy-D-glucose (Y-2DG), which has a free amino function were synthesized. The inhibitory effect of GcpY on [3 H]L-tyrosine uptake was larger than that of N-methly-L-phenylalanine or N-acetyl-L-phenylalanine, whereas Y-2DG did not affect it. These results indicate that a free amino group is not required for recognition, provided that the modified amino group is able to take a positive charge. Steric factors appeared to be relatively unimportant.

In order to study the proper use of urate-lowering drugs, we searched and analyzed a recently established database of 151, 804 community pharmacy records for the one-year period ending in August 1997. A total of 5, 312 prescriptions for typical urate-lowering drugs, allopurinol and benzbromarone, were searched. The number of male patients was found to be approximately 6.5 times that of females (578 and 89, respectively). The average age was 59.8 years (range 15 to 90 years). Of the 5, 312 prescriptions examined 81.4% were for allopurinol, 17.1% for benzbromarone and 1.5% for both. The number of prescriptions for allopurinol was approximately 4.5 times greater than that for benzbromarone. Hyperuricemia is classified into three types. The first type, includes patients who under-excrete uric acid, occurs in approximately 55% of patients. Uricosuric drugs such as benzbromarone are considered to be the drugs of choice. The second type, consists of over-producers of uric acid, occurs in approximately 10% of patients. The third type, comprising a mixed category, accounts for the remaining 30%. Allopurinol is recommended for the latter group of both under-excreters and over-producers. Given this classification, benzbromarone should thus be prescribed on a relatively frequent basis.The average number of drugs per prescription was 5.9 (range 1 to 25 drugs) thus indicating that patients with hyperuricemia tend to be prescribed multiple-drug regimens. From the analysis of concomitant drugs, it was found that the most frequently prescribed drug was furosemide (22.4%), followed by nifedipine sustained-release preparations (15.7%), pravastatin sodium (11.7%) and aspirin diaulminate for children (10.7%). The most frequently prescribed drug group was vasodilators (60.2%) including calcium antagonists and nitrates, followed by peptic ulcer agents (44.4%), anti-arteriosclerotic agents (27.1%), anticoagulants (25.1%), diuretics (24.2%) and ACE inhibitors (20.7%). These finding suggest that patients with hyperuricamia tend to have multiple complications such as hypertension, hypercholesterolemia, and ischemic cardiopathies.Inappropriate drug use and combinations are as follows: Alkalinizing agents, which prevent urate stone formation and are recommended for hyperuricemia, appear to be under-utilized (only 11.8%). Diuretics, trichlormethiazide (2.5%) and furosemide (22.4%) commonly cause hyperuricemia and thus should be avoided. When allopurinol and captopril are administered concomitantly (2.9%), a dangerous hypersensitivity reaction can also sometimes occur.These results indicate that doctors and pharmacists need to obtain a greater awareness of inappropriate drug use and dangerous drug combinations.

The methotrexate (MTX) administration to rats causes the damage of small intestine. The small intestinal damage was evaluated by measuring the intestinal permeability of the poorly absorbable compound, fluorescein isothiocyanate (FITC)-labeled dextran (average molecular weight, 4,400) (FD-4) using the in vitro everted intestine technique and by determining the FD-4 that appeared in plasma using the in situ closed loop intestine technique. The MTX administration to rats fed with the standard laboratory diet increased the small intestinal permeability of FD-4 due to the damage of the small intestine. Interestingly, the permeability of FD-4, when MTX was administered to rats fed with the aged garlic extract containing diet, was depressed almost to the level of control rats without the MTX treatment. The present study showed that the aged garlic extract protected the small intestine from the damage induced by the action of MTX on the crypt cells.

Transport and recognition of aminopeptidase-resistant cellobiose-coupled tyrosylglycylglycine (CcpTGG) by intestinal Na+/glucose cotransporter (SGLT1) was examined in rat small intestine. Inhibitory effect of phloridzin (SGLT1 inhibitor) on the CcpTGG transport was extremely low. Concentration dependence of the CcpTGG transport was observed, but the primary component of transport was passive diffusion. However, CcpTGG significantly inhibited SGLT1-mediated transport, indicating its recognition by SGLT1. Other glucose-conjugates also inhibited SGLT1-mediated transport. These results indicate that recognition of sugar conjugates by SGLT1 is much less restricted than transport, and that it should be relatively easier to design SGLT1 inhibitors than SGLT1-transportable sugar conjugates.

To examine whether the retention of imidazole analogues in the organ rich with elastic connective tissue can be of toxicological significance, the effect of 14C labeled 2-methylimidazole (2MI) as a model compound on the mechanical, biochemical, and morphological properties of the rat aorta, together with its irreversible interaction with the tissue macromolecules, was investigated after chronic treatment during maturation (10 and 100 μmol/kg/day for 28 days, from 4 to 8 weeks of age, subcutaneous injection). 1. Aortic extensibility was significantly decreased in 2MI-dose-dependent manner, whereas tensile strength showed an increasing trend. 2. Aortic elastin content was decreased dose-dependently, while collagen content was increased. 3. The low dose of 2MI scarcely altered histological picture of the aortic wall compared to the control group (isotonic saline dosing), but the high dose caused focal disorganization and fragmentation of the medial elastic laminae. Thickness of the aortic media showed a dose-dependent increasing trend. 4. The level of irreversibly bound 2MI-equivalent in elastin fraction was considerably high as compared with that in non-elastin fraction, and was increased dose-dependently. The aortic microautoradiograph showed that [14C]2MI-derived radioactivity occurred in the media, the region rich with elastic fiber. These findings indicate that 2MI affects maturation of the elastic organ materially, and that the effect can be closely linked with the binding of 2MI to elastin. It is possible that the same holds true for some other imidazole-based compounds that are retained in large quantities in the elastic connective tissue.

Purpose. To characterize the intestinal absorption of a β-glucose conjugate of acyclovir (9-[(2-hydroxyethoxy) methyl] guanine, ACV) and compare it to ACV and its analogues in terms of stability and transport by Na+/glucose cotransporter (SGLT1).

The intestinal transport of glucose- and galactose-conjugated acetaminophen (APAP glycoside) by Na + /glucose cotransporter (SGLT1) was studied. SGLT1-mediated transport of APAP glycosides preferred glucoside>galactoside and β-anomer>α-anomer. These preferences agree with previous studies. NMR spectroscopic and molecular modeling studies indicated that the conformation of the glucose ring of α- and β-glucosides of APAP, as well as glycosides in previous studies, is in the 4 C 1 chair form, the same form as glucose itself. Molecular dynamics analysis also indicated that the glucose ring was in the 4 C 1 chair form, and that there are differences between the rotational spaces of aglycones and hydroxy groups of glucose moieties between anomers. Therefore, we conclude that the β-anomeric preference of glucose conjugate transport by SGLT1 is not due to the conformation of the glucose ring, but to the configuration of the aglycone at C-1 of the monosaccharide moiety.

The intestinal absorption of analgesic peptides (leucine enkephalin and kyotorphin) and modified peptides in rat were studied. Although these peptides were not absorbed, the absorbability (absorption clearance) of these peptides were increased in the presence of peptidase inhibitors. In order to kinetically analyze these phenomena, we proposed the metabolic inhibition model, which incorporated the metabolic clearance (metabolizability) with the absorption clearance. Metabolic activity was determined with intestinal homogenates. The higher the metabolic clearance was, the lower was the absorption clearance. The relationships between the absorption clearance and the metabolic clearance of the experimental data as well as of the theoretical values were hyperbolic. This model predicted the maximum absorption clearances of cellobiose-coupled leucine enkephalin (0.654 &mgr;l/min/cm) and kyotorphin (0.247 &mgr;l/min/cm). Details of the experimental methods are described.

Spontaneous chemiluminescence associated with the cell injury was observed in the isolated rat hepatocyte suspension during acetaminophen (APAP) metabolism, indicating the occurrence of oxidative stress. APAP apparently affected the hepatocytes in various manners. APAP, at low concentrations (1-2 mM), damaged the hepatocytes due to lipid peroxidation provoked during APAP metabolism, while at high concentrations (5-50 mM), APAP protected the hepatocytes due to a chemical antioxidant effect of the unmetabolized APAP that remained in the medium because of the saturation of APAP metabolism. The covalent binding of APAP to the hepatocytes increased with APAP concentration up to 50 mM without loss of cell viability. When an overdose of APAP was administered to rats, the APAP plasma concentration was around 1-3 mM, which corresponded to the concentration range where lipid peroxidation occurred in the isolated hepatocytes. Thus, it seems likely that lipid peroxidation contributes to the APAP-induced hepatotoxicity in the early stage of the toxic process.

Intestinal absorption of four cyclic dipeptides was studied in the everted small intestine of the rat. Cyclic seryltyrosine (cyclo(Ser-Tyr)) was stable enough to be transported whereas linear seryltyrosine was not. The absorption clearance of cyclo(Ser-Tyr) was concentration-dependent, and for cyclo(Ser-Tyr) at 125 μM decreased in the presence of glycylsarcosine (10 mM) or cephalexin (10 mM), which were reported to be absorbed by oligopeptide transporter. The absorption clearance was also reduced at 4°C and in the presence of 1 mM dinitrophenol. Kinetic analysis of cyclo(Ser-Tyr) absorption showed that Km and Vmax were 19.8 μM and 0.295 nmol min−1 cm−1, respectively. It was also suggested that cyclic aspartylphenylalanine and cyclic histidylphenylalanine were absorbed by oligopeptide transporters, but cyclic histidylproline was not. The absorption clearance of cyclo(Ser-Tyr) in the control was much higher than the value of the correlation line representing a plot of passive transport (which was obtained from the absorption clearance of cyclic peptides in the presence of glycylsarcosine (10 mM)) against hydrophobicity (oil-water partition coefficient). These results indicate that cyclo(Ser-Tyr) is absorbed by the oligopeptide transporter.

Intestinal absorption of beta-disaccharide (cellobiose, maltose and lactose) conjugates of p-nitrophenol (p-nitrophenyl beta-disaccharide) were examined in terms of the hydrolysis of disaccharide conjugate to monosaccharide conjugate and the transport of monosaccharide conjugate by Na+/glucose transport carrier (SGLT1). beta-Cellobioside, beta-maltoside and beta-lactoside of p-nitrophenol (p-NP) were hydrolyzed to p-nitrophenyl beta-glucoside (p-NPbeta glc) on the mucosal side, and p-NPbeta glc appeared on the serosal side. Although p-NP beta-disaccharide, p-NP and p-NP glucuronide also appeared on the serosal side, their amounts were much lower than that of p-NPbeta glc. The amount of p-NPbeta glc transported to the serosal side was decreased in the presence of phloridzin (transport inhibitor of SGLT1) and in the absence of Na+ (a cosubstrate of SGLT1), indicating that p-NPbeta glc was formed from p-NP beta-disaccharide on the mucosal side and transported to the serosal side by SGLT1. Furthermore, the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-cellobioside and p-NP beta-lactoside by lactase-phloridzin hydrolase (LPH), was much higher than that of p-NPbeta glc itself, although the absorption clearance of p-NPbeta glc, which was formed from p-NP beta-maltoside by maltase was similar to that of p-NPbeta glc itself. These results indicated that p-NPbeta glc was transported by the vectorial cooperation of SGLT1 with LPH from mucosal p-NP beta-cellobioside or p-NP beta-lactoside.

We examined the enhancing action of sodium caprate and palmitoylcarnitine on the permeability of fluorescein isothiocyanate dextran 4000 as a paracellular permeant compound in isolated rat and human colon samples using the Ussing-type chamber method. In the absence of an enhancer, the permeation clearance of fluorescein isothiocyanate dextran 4000 was not significantly different in the rat and human colons, but the electric membrane resistance was smaller in the rat colon than in the human colon. Sodium caprate and palmitoylcarnitine increased permeation clearance and decreased electric membrane resistance in both types of colonic membrane, showing that the rat colon can be used as a model of the human colon for studies of enhancer effects. A calmodulin antagonist significantly inhibited the action of sodium caprate in both colonic membranes. However, it tended to promote the effects of palmitoylcarnitine on permeation clearance and electric membrane resistance. These results suggest that sodium caprate induces the contraction of the perijunctional actomyosin ring to widen the tight junction and that the mechanism of palmitoylcarnitine is different from that of sodium caprate in the human colon, as reported previously for Caco-2 cell monolayers.

The two permeation pathways for colorectal drug absorption, the transcellular and paracellular pathways, were examined. The transcellular pathway is generally a principal route for drugs with some degree of lipophilicity. The contribution of the paracellular pathway to drug absorption is significant when absorption enhancers such as capric acid (C10) and decanoylcarnitine (DC) are used. The action mechanism of C10 on the transcellular pathway was examined by membrane perturbation. The following in vitro effects of C10 on the paracellular pathway were observed: (i) an increase in the equivalent pore radius; (ii) an increase in the permeabilities of water-soluble non-electrolytes and ionic drugs; (iii) a decrease in the junctional resistance and an increase in the membrane capacitance. The action mechanism of C10 in the paracellular pathway was especially elucidated by stimulation to the contraction of the perijunctional actomyosin ring. Finally, an in vivo C10 effect was also observed, as an enhancement of rectal drug absorption from suppositories in rats.

The transport of bovine serum albumin (BSA) across Peyer's patches (PP) was increased by conjugate synthesis with lectin concanavalin A (Con A). The transport of the intact form was increased by the conjugation and significantly reduced by colchicine. The increase is thought to be due to the endocytosis activity of PP in response to lectin Con A. The transport of the conjugate across the jejunal epithelium (JE) was also increased, and it was concluded that the increase was due to the increase in stability against enzymatic degradation. These results suggest that conjugate synthesis with lectin is an effective method to improve the poor absorption of polypeptides and protein drugs in the intestine.

The effect of anionic compounds on the hepatic uptake of paracetamol sulphate, a conjugative metabolite of paracetamol, has been studied. Hepatic uptake of paracetamol sulphate by isolated hepatocytes was inhibited by bromosulphophthalein, dibromosulphophthalein and p-nitrophenyl sulphate, but not by probenecid or cholic acid. Bromosulphophthalein and dibromosulphophthalein also inhibited the uptake of paracetamol sulphate in the rat isolated perfused liver. Saturable uptake of paracetamol sulphate was also observed in the absence of inorganic sulphate. The uptake of paracetamol sulphate was reduced by 1 and 10 mm inorganic sulphate. Bromosulphophthalein and p-nitrophenyl sulphate inhibited the uptake of paracetamol sulphate in the absence of inorganic sulphate. These results indicate that paracetamol sulphate shares a transporter with bromosulphophthalein, dibromosulphophthalein and p-nitrophenyl sulphate, all of which contain the sulphate or sulphonate group. Therefore, the sulphate or sulphonate moiety might be crucial for interaction with the transporter, and mutual inhibition of hepatic uptake among these compounds is likely.

A chemiluminescence(CL)-high performance liquid chromatography (HPLC) system, where CL detector was equipped with normal phase HPLC, was used in order to specifically detect lipid hydroperoxide in the heart of doxorubicin (DXR) administered mice. Luminol and cytochrome c were used as the luminescence reagents specific for the hydroperoxide. Phosphatidylcholine hydroperoxide was detected in the heart, indicating participation of lipid peroxidation in the DXR-induced cardiotoxicity. This system is of great use to study the DXR-induced lipid peroxidation in vivo.

Intestinal transport and metabolism of kyotorphin (KTP) were studied in rat everted small intestine. KTP on the mucosal side was metabolized completely within 60 min, and any amounts of KTP were not detected on the serosal side. On the other hand, [D-Arg2]-KTP (D-KTP) was stable on the mucosal side to appear on the serosal side. However, N-t-butoxycarbonyl-KTP (Boc-KTP), which was metabolized on the mucosal side faster than KTP, appeared on the serosal side. In intestinal homogenate, KTP was metabolized, and the metabolic clearance (CL(met)) was decreased by peptidase inhibitors, bestatin, o-phenanthrolin and tryptophan hydroxamate. In the presence of these peptidase inhibitors, the absorption clearance (CL(abs)) of KTP was increased. The less the CL(met) of KTP was, the more the CL(abs) of KTP was. Meanwhile, Boc-KTP in intestinal homogenate was stable even in the absence of peptidase inhibitors. The CL(abs) of Boc-KTP was constant irrespective of the stability on the mucosal side. Kinetic analysis by the metabolic inhibition model indicated that the stabilization of KTP in the intestinal tissue could increase the CL(abs) up to 0.247 microl/min per cm, which was as much as the CL(abs) of stable D-KTP. These results led to the conclusion that rate-limiting process in intestinal absorption of KTP is metabolic degradation in intestinal tissue during the absorption.

Aminopeptidase-degradable tyrosylglycylglycine (TGG), a part of leucine enkephalin, was coupled with lactonolactone to conjugate galactose with TGG by way of amide bond linkage. Lactonolactone-coupling improved the intestinal absorption of TGG as well as the stability in intestine and in serum, like galactose conjugation by way of CH2-NH linkage (lactose-coupling). However, lactonolactone-coupling also provided TGG with peptidase-susceptible property different from that of TGG.

We studied the plasma pharmacokinetics of the lactone form and the carboxylate form of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), after intravenous bolus administrations of each form of SN-38 and of CPT-11 to rats. After the SN-38 lactone injection, SN-38 lactone was predominant at first, and then the lactone to the carboxylate concentration ratio (LC ratio) was maintained from 30 to 90 min after the injection. The carboxylate was predominant throughout the period after the carboxylate dosing. Model-dependent analyses showed that the SN-38 lactone had greater plasma clearance and a greater distribution volume than the carboxylate. The CPT-11 administration resulted in a predominant plasma SN-38 lactone concentration. The contribution of the SN-38 lactone AUC to the total SN-38 AUC (57%) was independent of the dose of CPT-11. These results suggest that it is possible to estimate the SN-38 lactone concentration and AUC from the total SN-38 concentration without separate determination of the lactone and carboxylate. Our results showed that both SN-38 lactone and CPT-11 administration gave the predominant SN-38 lactone in plasma; however, only CPT-11 could sustain the lactone concentration at a high level, which is necessary for antitumor activity.

The mechanism of paracellular expansion by absorption enhancers, e.g., EDTA, sodium caprate (C10), and decanoylcarnitine (DC), was studied, the focus being on the process of actin microfilament contraction in the tight junction. The effects of various inhibitors such as KN-62 (a specific inhibitor of Ca2+/calmodulin dependent protein kinase), H7 (a protein kinase C (PKC) inhibitor), and W7 (a calmodulin antagonist) were examined on the paracellular expansion by the enhancers in Caco-2 cells. From the experimental results, the following mechanisms were suggested. EDTA activates PKC by depletion of extracellular calcium via chelation resulting in expansion of the paracellular route. C10 increases the intracellular calcium level by an interaction with the cell membrane independent of cell polarity resulting in contraction with actin microfilament. DC interacts specifically with the apical membrane to increase the intracellular calcium level, but the mechanistic details subsequent to the increase of calcium are not clear.

The absorption enhancers improve drug absorption affecting both of transcellular and paracellular routes, and the effect of the enhancers is larger in the paracellular route opening the tight junction. Theses findings are based on the effects on depolarization of fluorescence in the brush border membrane, Solomon's equation, voltage clamp method and impedance analysis of the colonic membrane. The mechanism of tight junction opening by caprate (C10) that is one of representative enhancers is revealed that C10 releses Ca ion from the intracellular store and that the released Ca ion contracts the actin filament through the activation of myosin light chain kinase. But for other representative enhancers such EDTA and palmitoyl carnitine, other different mechanism is needed but not cleared yet. Glucose transporter is found to work for the glycoside absorption, and sugar coupling with some oligo peptides enhance the absorption not only by transporter use but also by stabilization of peptides. Vitamin A protects the damage of small intestine by methotrexate and improves the absorption of nutrients. Fluorescence spectra and protein aggregates which are produced during lipid peroxidation in the membrane such as microsome are measured directly from the membrane. They are used as indexes of the extent and site of lipid proxidation. The lipid peroxidation of heart mitochondria by doxorubicin (DXR) is demonstrated with these indexes. Chemiluminescence that is weak but measured very sensitively is also used as an index of lipid peroxidation. The chemiluminescence spectrum shows the production of the activated carbonyl and single oxygen during DXR oxidation. Naproxan which is known comparatively safe is revealed by the above indeces to give lipid peroxidation in liver during its metabolism. It is discussed that the lipid peroxidation in liver is a probable cause of liver damage which is occasionally reported for naproxen treatment.

Naproxen, a non-steroidal anti-inflammatory drug, induced lipid peroxidation in isolated hepatocytes of rats. The viability of the hepatocytes decreased upon lipid peroxidation, and this effect was accompanied by the formation of high molecular weight protein aggregates in the hepatocytes. Protein aggregation occurred slowly compared with the formation of thiobarbituric acid reactive substances (TBARS). The increase of TBARS was strongly correlated with the decrease of intracellular glutathione. Chemiluminescence was produced from the hepatocyte suspension during naproxen metabolism, and was correlated with the formation of TBARS. These results indicate that lipid peroxidation in the hepatocytes was provoked by reactive oxygens produced in the process of naproxen metabolism.

Promptness of medicine preparation is one of the important tasks the pharmacy has to tackle. A new automated dispensing system has been developed in order to adopt parallel preparation of the prescription. The system consists of a large LAN system which is connected to a host-computer, control-computer, automatic preparation machines and conveyer lines. The prescription data issued by each physician are first audited by the host computer and then used as the date for preparation. Prepared data checked by pharmacists are delivered to the manual preparation station (tablets, powder, topical drugs, and solutions) as a preparation instruction sheet and transmitted directly to the automatic preparation machines (e.g. medicine bag printing machines and automatic tablet dispensing and packaging machines). In collecting the prepared medicines, a controlled conveyer line was established. The waiting time decreased significantly after the system was introduced. This system not only reduces actual medicine preparation time but also improves the progress of the dispensing operation efficiency.

An attempt was made to elucidate the mechanism for accumulation and retention of imidazole derivatives in the connective tissue by using the radio-labeled imidazoles, [2-14C]imidazole and 2-methyl[2-14C]imidazole. Distribution studies in rats revealed that these compounds were retained in the connective tissue, probably with the irreversible binding to elastin. The binding formation could not be ascribed to spontaneously occurring noncatalytic reaction or to P450-mediated activation. Although pretreatment of rats with a xanthine oxidase (XOD) inhibitor allopurinol (ALP) resulted in decreased levels of tissue-bound aortic radioactivity from the imidazoles, in vitro binding to aortic tissue was scarcely observed in the presence of purified XOD. On the other hand, certain oxidation products generated in copper(II)/ascorbate system (Cu/VC) were shown to bind firmly to aortic tissue. Under this condition, addition of ALP decreased the tissue binding. ALP can form the complex with both Cu and VC in vitro as reported previously. In view of this information, in vivo ALP-treatment effect described above may be well explained by inhibition of Cu/VC-catalyzed oxidation. Moreover, from LC/MS/MS analysis of the oxidation products in Cu/VC, it was conceivable that imidazolones bind to α-aminoadipic-δ-semialdehyde (allysine) residues in elastin or collagen. Based on these findings, the nonenzymatic oxidation process is considered to be one of the major causative elements in the metabolic activation of imidazole derivatives. Further studies using LEC rat, an animal model of Wilson's disease, are currently being carried out.

The permeability of peptides across rabbit jejunal epithelium (JE) and Peyer’s patches (PP) was compared. Kyotorphin (L-tyrosyl-L-arginine) was almost completely hydrolyzed during its membrane transport in both PP and JE, but [D-Arg2] Kyotorphin (L-tyrosyl-D-arginine) was less hydrolyzed in PP than in JE. Since the permeability of intact [D-Arg2] Kyotorphin was almost equal in PP and JE, no superiority of PP to JE was found for dipeptide transport. More intact fluorescein isothiocyanate (FITC)-labeled bovine serum albumin (FITC-BSA) and concanavalin A (FITC-Con A) were transported in PP than in JE. At both absorption sites, the transport of the intact FITC-Con A was superior to that of the intact FITC-BSA. Colchicine significantly reduced the total transport of the intact and degradation forms of both peptides and the reduction ratio was greater in PP than in JE. Accordingly, it was suggested that PP can be used as prominent absorption sites for polypeptides since they have lower peptidase activity and higher endocytosis activity than JE.

This study was undertaken to ascertain the total indexes of adverse drug reaction and with these indexes to clarify the characteristics of adverse reaction to individual antibiotic. The frequencies of adverse reaction in 14 sites were calculated by the accumulative data of 22 antibiotics obtained from clinical trials and post-marketing surveillance which were reported by the Ministry of Health and Welfare. They were examined by principal component analysis.The results were as follows: 1. There were strong intercorrelations in the frequencies of adverse reactions which affect the whole body in general inclusive of the hepatobiliary system and blood. 2. The first and second principal components were suggested to reveal the frequencies of adverse drug reaction affecting the whole body, and affecting local regions such as the ear, application site and urinary system, respectively. Their cumulative proportion was 61.4%. 3. The first principal component score tended to be larger in parenteral cephems, whereas the second principal component score tended to be larger in aminoglycosides. 4. The first principal component score of ceftizoxime sodium constituted the largest extreme. 5. The frequency of adverse reaction to fosfomycin which affected the whole body was obserbed to decrease by changing Na salt to Ca salt.

For our objectives to find ways of improving poor drug absorption from intestine, the active absorption of glucose and galactose by the glucose transport system in the intestne was so attractive that an effect of conjugating sugar to the drugs on its intestinal absorption was studied. After absorption of 2-naphthyl α-glucoside (2-NAαglc), the major costituents appearing on the serosal side were 2-naphthol (2-NA) and its glucuronide. Meanwhile, after absorption of 2-naphthyl β-glucoside (2-NAβglc), the major constituents appearing on the serosal side were 2-NAβglc and 2-NA glucuronide. The total (glycoside + aglycone + glucuronide) absorption of 2-NAβglc and 2-NAαglc was significantly (P

Nasal absorption of recombinant human granulocyte colony-stimulating factor (rhG-CSF) was examined in the rat. The relative bioavailability of rhG-CSF for subcutaneous administration was approximately 2%, as evaluated from the immunologically active rhG-CSF concentration in rat plasma and the area under the curve (AUC) of the plasma rhG-CSF concentration versus time for 8 hr. Pharmacological availability relative to subcutaneous administration was determined from the increase in total blood leukocyte numbers. The pharmacological availability was 5-10%, determined from the AUC for the increased ratio of total leukocyte numbers versus time for 48 hr; it was slightly dependent on the pH and the osmotic pressure of the dosing solution. Accordingly, the plasma concentration of rhG-CSF did not always reflect its pharmacological effects. Relative bioavailability and pharmacological availability were increased about 23 times and 3 times, respectively, by polyoxyethylene 9-lauryl ether (Laureth-9), but no increase in availability occurred with sodium glycocholate. The increase in total leukocyte numbers was maintained during multiple rhG-CSF dosing, and the addition of Laureth-9 further increased the pharmacological effects of this agent. This study indicates that nasal administration of rhG-CSF is an effective parenteral administration route.

Vitamin A (VA) protects the small intestine from the methotrexate (MTX)-induced damage. The in vivo effects of MTX and/or VA on crypt cells of small intestine were investigated using rats orally administered MTX (15 mg/kg body weight) and/or VA (5,000 IU/kg body weight). The thymidine kinase activity of crypt cells separated from villus cells of small intestine of MTX plus VA-treated rats was lower than that from control rats but higher than that from MTX-treated rats. VA-treated rats showed almost the same activity of thymidine kinase as control rats. The amounts of the end products, adenosine monophosphate (AMP) and guanosine monophosphate (GMP), contained in crypt cells of treated rats were determined to investigate the in vivo effects of MTX and/or VA on de novo purine synthesis. MTX treatment significantly decreased the amounts of both AMP and GMP but both amounts were not affected by MTX plus VA treatment. Treatment with VA alone appeared to increase their amounts slightly. Thus, although the oral administration of MTX to rats inhibited DNA synthesis in crypt cells, VA coadministration protected the salvage pathway of pyrimidine synthesis and the de novo purine synthesis in crypt cells.

Oil violet in a fat emulsion was taken up into the parenchymal cells of rat liver in vitro. The uptake was greater at 37 degrees C than 25 degrees C or 4 degrees C, and it was increased by addition of postheparin plasma including lipoprotein lipase activity into the reaction medium. The uptake from the emulsion with smaller particles was greater than that from the emulsion with larger particles. The hepatic uptake of 14C-cholesteryl oleate in the emulsion by recirculating perfusion of the liver in situ was also increased by postheparin plasma in the perfusion medium. Its enhancing effect was found for distribution in the parenchymal cells but not in the Kupffer cells. The previous perfusion of Intralipos of the commercial fat emulsion reduced the hepatic uptake of 14C-cholesteryl oleate in the emulsion. The emulsion particle sizes were reduced by postheparin plasma both in vitro and in situ. Consequently, it was suggested that a lipid-soluble compound entrapped in the fat emulsion is taken up into the parenchymal cells by receptor-mediated process via the reduced particle sizes emulsion (remnant), which is a mechanism similar to the dietary fat metabolism.

The permeability of fosfomycin (FOM), which is a low molecular weight and water-soluble antibiotic, across rat and rabbit jejunal membrane was investigated on a paracellular route and a transcellular route separated by voltage-clamp method. It was determined that FOM permeates through these two routes in both animals. Under a physiologically normal condition, the transmucosal electrical potential difference (about 2 mV), the contribution of the transcellular route to FOM membrane permeability was greater than that of the paracellular route. The transcellular permeability was significantly reduced in the presence of inorganic phosphate. The inhibitory effects of the inorganic phosphate coincided with our reports on FOM uptake by rat jejunal brush-border membrane vesicles in vitro and on FOM absorption by rat jejunal single-pass perfusion in situ.

The enhancing effects of 0.25% sodium caprate (C10) and sodium caprylate (C8) on the paracellular permeation of seven water-soluble nonelectrolytes (inulin, polyethylene glycol 900, mannitol, erythritol, glycerol, thiourea, and urea) across the isolated rat colonic epithelium were examined using the Ussing-type chamber technique. The paracellular changes were also measured by impedance analysis. In both the presence and the absence of enhancers, the permeation clearances (Pm) for inulin (12–15 A in molecular radius) to erythritol (3.2 A) increased linearly with the increase in their free diffusion coefficients (Dfr), showing the existence of a paracellular shunt pathway unrestricted to any molecular size. Glycerol (2.9 A), thiourea (2.6 A), and urea (2.3 A) had higher clearances than the expected linear values, showing the existence of a restricted paracellular or transcellular pathway. Both C10 and C8 increased the permeabilities in the two pathways, but C10 was more effective than C8. The increase in the permeabilities via the shunt pathway caused by the enhancers was greater than that via the restricted pathway, and thus, the two-phase pattern in the relationship of Pm and Dfr was similar to that in the absence of enhancers. The transcellular permeabilities for urea and thiourea, which were obtained from the efflux experiments, were increased by the enhancers. However, the relative increase caused by C10 was smaller than that of the paracellular-restricted permeabilities. The paracellular changes probably were due to the increase in pore area per unit diffusive path length. A decrease in the resistance of the intercellular junctions involving a simultaneous increase of membrane capacitance was observed in the presence of C10, corresponding to an increase of pore area per unit path length. The effect of C10 on the paracellular permeability was reversible, and the junctional resistance, membrane capacitance, and Pm of mannitol returned to the control level following the removal of C10.

Detection of the time-resolved decay of the fluorescence emitted from lipid peroxidized membranes succeeded using peroxidized microsomes of rat liver. Analysis of the time resolved decay showed that three components were most adequate to fit the observed decay. This suggests the heterogeneity of the fluorescent substances related to the species of fluorophores, their forms and/or their environment. Measurements of the fluorescence lifetimes of the fluorescent substances will be of great use to further characterize the fluorescent substances.

Three physiologically based pharmacokinetic models A--C, incorporating enterohepatic recycling, were developed to predict glycyrrhizin (GLZ) disposition in rat plasma and tissues, and human serum. Model A, which included fourteen compartments (artery, vein, tissues except brain, and gut lumen) with the assumption of direct excretion of GLZ from the liver into the gut lumen gave fairly good agreement between the observed and predicted disposition profiles in rat, but was unsuitable in man, where elimination is very rapid. Models B and C for man were obtained by adding a gallbladder compartment (drug storage organ) for the excretion from the liver into the gut lumen and by assuming continuous transfer from the storage compartment or instantaneous emptying from it during meal ingestion as the excretion process from the gallbladder into the gut lumen, respectively. The agreement between the observed and predicted serum concentration time-course profiles was better with model C than model B, especially in the terminal elimination phase, where secondary peaks appeared. However, it was thought that the observed serum disposition can be sufficiently well predicted by model B. In conclusion, prediction in rat was successful in all compartments except the brain, which shows a negligible distribution. Scale-up of the disposition kinetics of GLZ from rat to man was also successful.

In order to obtain an insight into the retention mechanism of drugs with imidazole moiety in the connective tissue, the in vitro characteristics of the interaction between 14C-labeled imidazoles (imidazole and its 2-methyl derivative) and slices of dog aorta were studied. We found that cupro-ascorbate-catalyzed oxidative reactions for the imidazoles led to their irreversible binding with connective tissue, and that, from a study using protein modifiers, the aldehydic function intrinsic to the tissue protein was involved in the binding formation. This characteristic in vitro was observed under physiological conditions, hence it can be extrapolated to the in vivo situation and could also give a clue to the nature of the retention of imidazole-containing drugs in connective tissue.

Intestinal absorption of cyclic phenylalanylserine (cyclo(Phe-Ser)), a precursor of gliotoxin, was studied in isolated rat small intestine as a model cyclic dipeptide. Absorption clearance (CLabs) decreased in the presence of glycylsarcosine, cephalexin or cephradine, substrates for H+/oligopeptide cotransporter (PEPT1). CLabs of cyclo(Phe-Ser) also decreased at 4 degrees C. These indicate that cyclo(Phe-Ser) is in part transported by PEPT1. However, Eadie-Hofstee plot of absorption revealed an atypical profile at lower concentrations of cyclo(Phe-Ser) (around 0.1 mM). Moreover, comparative experiments of absorptive and excretive transport showed that excretive transport from the serosal to mucosal side of isolated intestinal tissue at a 0.1 mM cyclo(Phe-Ser) was superior to absorptive transport from the mucosal side to the serosal side, and vise versa at a 1 mM cyclo(Phe-Ser). These results as well as the results of kinetic analysis indicate that intestinal absorption consists of passive transport, carrier-mediated absorptive transport by PEPT1 and carrier-mediated excretive transport, resulting in atypical absorption. Although cyclic dipeptides have potentials for drug, their intestinal absorption may be complex. The results of this study lead us conclude that absorptive and excretive transport by the small intestine acts as an interface between the body and ingested compounds.

Intestinal absorption of peptides in linear form has been studied extensively, but there is little knowledge of peptides in a cyclic form. In this report, intestinal absorption of cyclic phenylalanylserine (cyclo(Phe-Ser)), a precursor of gliotoxin, was studied in isolated rat small intestine as a model cyclic dipeptide. Absorption clearance (CLabs) decreased in the presence of glycylsarcosine, cephalexin or cephradine, substrates for H+/oligopeptide cotransporter (PEPT1). CLabs of cyclo(Phe-Ser) also decreased at 4 degrees C, thus indicating that cyclo(Phe-Ser) is in part transported by PEPT1. However, the Eadie-Hofstee plot of absorption revealed an atypical profile at lower concentrations of cyclo(Phe-Ser) (around 0.1 mM). Moreover, comparative experiments of absorptive and excretive transport showed that excretive transport from the serosal to mucosal side of isolated intestinal tissue at a 0.1 mM cyclo(Phe-Ser) was superior to absorptive transport from the mucosal side to the serosal side, and vice versa at a 1 mM cyclo(Phe-Ser). A kinetic model was constructed, in which cyclo(Phe-Ser) concentration for excretive transport was assumed to be at the binding site of excretive transporter, but not the unbound cytoplasmic concentration. These results as well as the results of kinetic analysis indicate that intestinal absorption consists of passive transport, carrier-mediated absorptive transport by PEPT1 and carrier-mediated excretive transport, resulting in atypical absorption. Although cyclic dipeptides have potentials as drugs, their intestinal absorption may be complex. The results of this study lead us to conclude that absorptive and excretive transport by the small intestine acts as an interface between the body and ingested compounds.

The effects of sodium caprate (C10) and disodium ethylenediaminetetraacetate (EDTA) on the epithelial permeability of polyethylene glycol 4000 (PEG4000) were examined in Caco-2 cell monolayers on the polycarbonate membrane (filter) of a Transwell cluster dish. Under serosal Ca(2+)-free conditions irrespective of the presence or absence of mucosal Ca2+, the permeability of PEG4000 was remarkably enhanced, even in the absence of enhancer. For control experimental conditions with a permeation enhancer in Caco-2 cells, Ca2+ was added to both the mucosal and serosal sides. Under the control conditions, EDTA increased the permeability of PEG4000 to 14 times the control and C10 by 3.5 times. Mucosal Ca(2+)-free conditions further increased the EDTA-enhancing effect, to 29 times the control value. In contrast to the EDTA effect, however, the C10 effect was unchanged in the absence of mucosal Ca2+. These results suggest that the EDTA effect is induced by chelation with Ca2+ but that C10 has some other action in addition to chelation.

Uptake of cyclic dipeptides by H+/oligopeptide cotransporter (PEPT1) was studied in monolayers of the human intestinal cell line, Caco-2. The cyclic dipeptides studied were cyclic glycylphenylalanine (cyclo(Gly-Phe)), cyclic phenylalanylserine (cyclo(Phe-Ser)), cyclic seryltyrosine (cyclo(Ser-Tyr)) and cyclic glycyltyrosine (cyclo(Gly-Tyr)). These molecules have both peptide bonds and aromatic rings, and are similar in structure to cephalexin and cephadroxil, which are transported by PEPT1. Cellular uptake of these cyclic dipeptides was pH dependent, and was inhibited by the addition of PEPT1 substrates such as glycylsarcosine, indicating PEPT1-mediated transport. Michaelis constants (Km) for these cyclic dipeptides were cyclo(Ser-Tyr) < cyclo(Phe-Ser), and cyclo(Gly-Tyr) < cyclo(Gly-Phe), indicating that tyrosine possessing phenol moiety has higher affinity for PEPT1 than phenylalanine possessing benzen moiety. The Km for cephadroxil possessing phenol moiety was reportedly lower than that for cephalexin possessing benzen moiety. Therefore, it was concluded that the phenolic hydroxyl group of the substrate may enhance affinity for PEPT1.

Antitumour drugs such as methotrexate (MTX) and 5-fluorouracil (5-FU) induce intestinal damage. This is a serious side effect of cancer chemotherapy. The present studies examined whether or not aged garlic extract (AGE) protects against damage from these antitumor drugs. Both drugs were administered orally for 4 or 5 d to rats fed a standard laboratory diet with and without 2% AGE. The small intestinal absorption of the poorly absorbable compound, fluorescein isothiocyanate--labeled dextran (FD-4; average molecular weight, 4400) was used to evaluate the damage to the intestine using the in vitro everted intestine technique and the in situ intestinal loop technique. FD-4 absorption increased in the antitumour drug-treated rats fed the diet without garlic. Interestingly, FD-4 absorption was depressed in rats fed the diet containing AGE. These results suggest that AGE may protect the small intestine of rats from antitumour drug-induced damage.

Paracetamol (200 and 500 mg kg−1) was given intraperitoneally to rats pretreated with 3-methylcholanthrene for 3 days. Glutamic oxalacetic acid transaminase (GOT) activity in plasma increased in rats receiving 500 mg kg−1 paracetamol. Plasma GOT activity was low at the dose of 200 mg kg−1, but the same dose to diethyl maleate pretreated rats increased the GOT activity. High mol. wt protein aggregates were found to be formed in liver homogenates and microsomes of rats which showed high plasma GOT activity, accompanied by depletion of hepatic glutathione. The formation of protein aggregates in the liver of rats following large doses of paracetamol suggests a contribution of lipid peroxidation to paracetamol-induced hepatotoxicity.