MIB-1 index is unlikely to predict relapse-free survival in patients who underwent R0-esophagectomy for esophageal squamous cell carcinoma.

MIB-1 is a cell proliferation marker and has previously been investigated as a diagnostic or prognostic indicator of malignancy. Previous studies have investigated MIB-1 index and clinicopathological factors in relation to prognosis of patients with esophageal cancer, with conflicting results. The aim of this study is to assess the prognostic significance of MIB-1 index in patients with thoracic esophageal squamous cell carcinoma. A total of 78 patients who underwent R0-esophagectomy for thoracic esophageal squamous cell carcinoma were enrolled in this study. Preoperatively, 29 patients underwent chemotherapy, six underwent chemoradiotherapy, and the remaining did not undergo any preoperative therapy. The MIB-1 labeling index was reported by counting 500 tumor cells in the hot spots of nuclear labeling. Correlations between MIB-1 index, clinicopathological factors, and relapse-free survival (RFS) were investigated. The mean MIB-1 index was 39.3 ± 21.0 (range: 0-91.3). There was no significant correlation between clinicopathological factors and MIB-1 index in the study patients, irrespective of whether they underwent preoperative therapy. Univariate analysis revealed no significant association between MIB-1 index and RFS. However, depth of tumor invasion, lymph node metastasis and stage, all showed a significant correlation to RFS. Multivariate analysis of RFS revealed that stage was the only significant factor. Conversely, MIB-1 index was not significantly related to RFS (p = 0.41). In conclusion, MIB-1 index is unlikely to be a significant prognostic indicator for esophageal cancer.

Analysis of hepatic metabolism affecting pharmacokinetics of propranolol in humans.

We examined the metabolic kinetics of propranolol, constructed from saturable and non-saturable components, using liver microsomes. The metabolic activity in rat microsomes was much higher than that in human microsomes within the clinically observed plasma range. Using the physiologically based pharmacokinetic (PBPK) model incorporating the obtained metabolic parameters, the plasma kinetics of propranolol was well correlated with reported values, and then used to analyze the effect of hepatic first-pass metabolism on propranolol plasma pharmacokinetics in clinical doses. The simulated plasma concentrations and AUC values of propranolol increased proportionally to its dose; these levels were almost equivalent to intrinsic clearance (CLint1), presumed to be non-saturable. When Michaelis-Menten parameters were decreased to one twentieth, plasma concentrations slightly increased after 160 mg dosing. A similar result was obtained with steady-state plasma levels after repeated administration. On the other hand, the first-order absorption rate constant of propranolol did not affect AUC values. The dose-normalized AUC value started to increase about 10(3)mg dosing. When the dose exceed 10(6)mg dose, the CLint1 component hardly contributed to propranolol pharmacokinetics. Accordingly, under the conditions of the PBPK model, propranolol pharmacokinetics was considered to be dose-independent within the clinical dose range.

Physiologically based pharmacokinetics of KNI-272, a tripeptide HIV-1 protease inhibitor.

The effects of dose on the pharmacokinetic characteristics of KNI-272 were evaluated in rats after intravenous (iv) administration. The plasma kinetics of KNI-272 were dose-independent within a dose range of 1.0 to 10.0 mg/kg. However, when the dose was increased to 50.0 mg/kg, the area under the plasma concentration-time curve (AUC)/dose significantly increased and the total plasma clearance (Cl(tot)) significantly decreased, possibly due to saturation of hepatic metabolism. On the other hand, the terminal elimination half-life (t(1/2,lambda(z))) was independent of dose. Using biochemical and physiological parameters obtained from in vitro and in vivo studies, we developed a physiologically based pharmacokinetic (PBPK) model for KNI-272 in rats in which concentration-dependent nonlinear hepatic metabolism (Michaelis-Menten type metabolism) was considered. Using this PBPK model, plasma KNI-272 concentration-time profiles were simulated. From these profiles it was demonstrated that the terminal elimination phase was proportional to the dose at lower doses. However, as the dose increased to 50.0 mg/kg, the simulated plasma concentrations at the terminal elimination phase increased more than the increase of dose in the same way as the observed data. Accordingly, the dose-dependent plasma kinetics observed after a 50.0 mg/kg dose was considered to be attributable in part to concentration-dependent hepatic metabolism in rats.

Metabolic characterization of a tripeptide human immunodeficiency virus type 1 protease inhibitor, KNI-272, in rat liver microsomes.

KNI-272 is a tripeptide protease inhibitor for treating human immunodeficiency virus type 1 (HIV-1). In in vitro stability studies using rat tissue homogenates, KNI-272 concentrations in the liver, kidney, and brain decreased significantly with time. Moreover, in tissue distribution studies, KNI-272 distributed highly to the liver, kidney, and small intestine in vivo. From these results and reported physiological parameters such as the tissue volume and tissue blood flow rate, we considered the liver to be the main organ which takes part in the metabolic elimination of KNI-272. Then the hepatic metabolism of KNI-272 was more thoroughly investigated by using rat liver microsomes. KNI-272 was metabolized in the rat liver microsomes, and five metabolites were found. The initial metabolic rate constant (kmetabolism) tended to decrease when the KNI-272 concentration in microsomal suspensions increased. The calculated Michaelis-Menten constant (K(m)) and the maximum velocity of KNI-272 metabolism (Vmax), after correction for the unbound drug concentration, were 1.12 +/- 0.09 micrograms/ml (1.68 +/- 0.13 microM) and 0.372 +/- 0.008 microgram/mg of protein/min (0.558 +/- 0.012 nmol/mg of protein per min), respectively. The metabolic clearance (CLint,metabo), calculated as Vmax/K(m), was 0.332 ml/mg of protein per min. Moreover, by using selective cytochrome P-450 inhibitors and recombinant human CYP3A4 fractions, KNI-272 was determined to be metabolized mainly by the CYP3A isoform. In addition, ketoconazole, a representative CYP3A inhibitor, inhibited KNI-272 metabolism competitively, and the inhibition constant (Ki) was 4.32 microM.

Binding characteristics of KNI-272 to plasma proteins, a new potent tripeptide HIV protease inhibitor.

The binding characteristics of KNI-272, a potent and selective human immunodeficiency virus (HIV) protease inhibitor, were evaluated in rat and human plasma, and in solutions of human alpha 1-acid glycoprotein (AAG) and human serum albumin (HSA). The unbound fractions (Fu) of KNI-272 were 12.13 and 2.24% in rat and human plasma, respectively, at the drug concentration of 1.0 microgram mL-1. Although KNI-272 binds to both AAG and HSA, the Fu of KNI-272 in AAG solution was 1.83%, and only one-quarter of that in HSA solution (Fu = 6.78%). Binding displacing agents, such as disopyramide, warfarin, diazepam, and digitoxin, were used to determine the binding site of KNI-272 on these plasma proteins. The Fu of KNI-272 in AAG solution increased 14-fold when disopyramide was added to the AAG solution. In addition, warfarin, diazepam, and digitoxin were added to HSA solution as representative drugs bound to distinct binding sites on HSA, namely sites I, II, and III, respectively. The Fu values of KNI-272 in HSA solution significantly increased when warfarin and diazepam were added. In particular, with the addition of warfarin to HSA solution, the Fu of KNI-272 increased to 16%. The modified Scatchard plots of KNI-272 binding to AAG and HSA both showed biphasic curves, and the KNI-272 binding sites at low concentration range on AAG and HSA disappeared with the addition of disopyramide and warfarin, respectively. Therefore, it is considered that KNI-272 binds to the identical site as disopyramide on AAG and site I on HSA in the low KNI-272 concentration range. By comparing the KNI-272 binding parameters obtained in human plasma and these protein solutions, we can assume that KNI-272 binding at low concentration in human plasma is mainly concerned with the binding on AAG. As KNI-272 concentration in plasma increases, HSA becomes concerned with KNI-272 binding.

The bioavailability of oral dosage forms of a new HIV-1 protease inhibitor, KNI-272, in beagle dogs.

The bioavailability (BA) of a tripeptide protease inhibitor, KNI-272, which has a strong pharmacological potential for treating human immunodeficiency virus type 1 (HIV-1), has been studied in beagle dogs by administering several oral dosage forms. The tested dosage forms were form 1, plain gelatin capsules; forms 2 and 3, gelatin capsules of which the inner and outer surfaces were coated with 7G ethylcellulose (EC, 30 mu m thickness) and an enteric coating material, hydroxypropyl methylcellulose phthalate (HP-55), respectively; and form 4, gelatin capsules of which the inner surface is coated with 10G EC (60 mu m thickness). The difference between forms 2 and 3 was the amount of citric acid contained in the capsule, namely 100 mg in form 2 and 200 mg in form 3. One hundred milligrams of KNI-272 was placed in each capsule after being dissolved with propylene glycol (PG). These capsules were used to deliver KNI-272 to the stomach for form 1, to the upper part of the small intestine for forms 2 and 3, and to the middle part of the small intestine for form 4. As a reference, 50.0 mg of KNI-272 was administered to the same dogs by intravenous (IV) infusion for 15 min. By measuring the plasma drug levels with the HPLC method, BAs were estimated for each test dosage form. Form 1 showed the highest BA of 26 center dot 2 +/- 7 center dot 0% (mean +/- SE), though the other capsules showed BAs of approximately 10%, namely 6 center dot 6 +/- 0 center dot 4% for form 2, 10 center dot 3 +/- 1 center dot 1% for form 3 and 14 center dot 2 +/- 1 center dot 0% for form 4. Therefore, as the site where KNI-272 is released from the capsule becomes higher, the BA increases. In addition, as the amount of citric acid contained in a capsule increases, the BA value tends to increase. These results suggest that KNI-272 is stable and not extensively hydrolysed in the gut after oral administration, that the dissolution process into GI fluids is important for the BA of KNI-272, and that the most appropriate absorption site of KNI-272 in dogs is the duodenum. The potential of this new tripeptide compound as an orally active anti-AIDS drug has been confirmed.

Absorption of new HIV-1 protease inhibitor, KNI-272, after intraduodenal and intragastric administrations to rats: effect of solvent.

KNI-272 is a tripeptide drug that has a strong pharmacological potential for treating human immunodeficiency virus type 1 (HIV-1). We have already reported the pharmacokinetic characteristics of KNI-272 after intravenous and intraduodenal (ID) administrations to rats. In this study, KNI-272 was administered to rats as a solution and the effect of four kinds of solvent on the bioavailability (BA) of KNI-272 was determined using rats. The mixtures included propylene glycol (PG) and water (70% PG), a solution of PG (100% PG), a solution of Tween 80 (Tween 80), and a mixture of PG and HCO60, a polyoxyethylated, 60 mumol, castor oil derivative (PG:HCO60 = 7:3). After ID administration to rats at a dose of 50.0 mg kg-1, the mean peak plasma concentrations, Cmax, were 2.58 +/- 0.53 (SE) (70% PG), 3.28 +/- 0.51 (100% PG), 3.15 +/- 0.51 (Tween 80), and 4.66 +/- 0.68 micrograms mL-1 (PG:HCO60). The highest BA, 44.6%, was obtained after ID administration of KNI-272 dissolved in PG:HCO60. On the other hand, after intragastric (IG) administration of KNI-272 solution in which the drug was dissolved with PG:HCO60, the Tmax, the Cmax, and the BA were 1.25 +/- 0.60 h, 2.33 +/- 0.65 micrograms mL-1, and 24.2%, respectively. The Cmax and BA values were equal to half of the values obtained after ID administration of KNI-272 dissolved in the same solution. In this study, as the PG concentration in the solution increased and the other additives (Tween 80 and HCO60) were coadministered, the BA of KNI-272 after ID administration increased. These results suggest that, for the development of an oral dosage form of KNI-272, a non-ionic surfactant that dissolves in the duodenum or small intestine and that enhances the absorption of this drug from the gastrointestinal tract into the enterocytes is needed.

Plasma pharmacokinetics and urinary and biliary excretion of a new potent tripeptide HIV-1 protease inhibitor, KNI-272, in rats after intravenous administration.

The pharmacokinetic (PK) characteristics of KNI-272, a potent and selective HIV-1 protease inhibitor, were evaluated in rats after intravenous (IV) administration. The effect of dose on KNI-272 plasma kinetics, and the urinary and biliary elimination kinetics of KNI-272, were examined. After IV administration of 10.0 mg kg-1 KNI-272, the mean terminal elimination half-life, t1/2 lambda zeta, was 3.49 +/- 0.19 (SE) h, the total plasma clearance, CLtot, was 15.1 +/- 1.2 mL min-1 and the distribution volume at steady state, Vd,ss, was 3790 +/- 280 mL kg-1. On the other hand, after 1.0 mg kg-1 IV administration, t1/2 lambda zeta was 3.04 +/- 0.11 h, CLtot was 15.9 +/- 0.2 mL min-1, and Vd,ss was 6950 +/- 600 mL kg-1. The PK parameters of KNI-272 after IV administration showed that the disposition of KNI-272 in the rat plasma is linear within the dose range from 1.0 to 10.0 mg kg-1. Using an equilibrium dialysis method, the plasma binding of KNI-272 was measured in vitro. The free fractions were 17.7 +/- 0.6%, 12.1 +/- 1.5%, and 13.8 +/- 1.4% at the total concentration ranges of 9.898 +/- 0.097 microgram mL-1, 0.888 +/- 0.008 microgram mL-1, and 0.470 +/- 0.55 microgram mL-1, respectively. The percentages of the dose excreted into the urine and bile as the unchanged form were 1.20 +/- 1.06% and 1.61 +/- 0.32% at 1.0 mg kg-1 dose, and 0.164 +/- 0.083% and 1.42 +/- 0.26% at 10.0 mg kg-1 dose, respectively. The renal clearance (CLR) and the biliary clearance (CLB) were calculated to be 0.191 and 0.256 mL min-1 for 1.0 mg kg-1, and 0.0248 and 0.215 mL min-1 for 10.0 mg kg-1, respectively. When comparing these values with the CLtot values, the urinary and biliary excretion of KNI-272 are minor disposition routes.

Comparison of a new orally potent tripeptide HIV-1 protease inhibitor (anti-AIDS drug) based on pharmacokinetic characteristics in rats after intravenous and intraduodenal administrations.

Recently, a series of KNI compounds such as KNI-227 and KNI-272 has been synthesized and shows potent and selective HIV-1 protease inhibitory activity in vitro. In this study, we developed an HPLC assay system for KNI-227 and KNI-272 in rat plasma and examined the pharmacokinetic characteristics in rats after both intravenous (i.v.) and intraduodenal (i.d.) administrations to obtain the disposition characteristics and bioavailabilities of these new anti-AIDS drugs. After i.v. administration of KNI-227, 10.0 mg kg-1, the mean terminal elimination half-life, t1/2 lambda zeta, was 0.808 +/- 0.161(SE) h, the total body clearance, CLtot, was 11.7 +/- 3.3 ml min-1 and the distribution volume at steady state (Vd,ss) was 1410 +/- 460 ml kg-1. On the other hand, after i.v. administration of KNI-272, 10.0 mg kg-1, t1/2 lambda zeta was 2.86 +/- 0.78 h, CLtot was 15.3 +/- 1.4 ml min-1 and Vd,ss was 3440 +/- 670 ml kg-1. In the case of the i.d. administration of drugs, the mean peak plasma concentrations, Cmax, of KNI-227 and KNI-272 were 0.374 +/- 0.110 microgram ml-1 and 0.900 +/- 0.093 micrograms ml-1, respectively. The bioavailabilities (BA) of KNI-227 and KNI-272 to infinity, BA(0-infinity), were 5.90% and 42.3%, respectively. As compared with the lead compound, KNI-174, the BA of KNI-272 was improved about 10 times. Although the anti-AIDS virus activity of these two drugs has not been investigated in vivo, KNI-272 is expected to be a better candidate for oral anti-AIDS therapies.

Distribution characteristics of immunosuppressants FK506 and cyclosporin A in the blood compartment.

Using two representative immunosuppressants, FK506 (FK) and cyclosporin A (CyA), of which the mechanism of pharmacological action is the same although there is a great difference in the pharmacological intensity, the distribution characteristics were studied in both in vivo and in vitro experiments using rat, dog, and human blood. Blood samples were fractionated by means of sedimentation in Ficoll-Paque, and the drug contents in the diluted plasma fraction, erythrocyte fraction, and lymphocyte fraction were measured by an HPLC method. FK distributes to the lymphocyte fraction to a level about three times greater than that of CyA, while CyA distributes to the erythrocyte fraction to a level ten times that of FK. The distribution pattern of these fractions was independent of the drug concentration and species after correcting the drug concentration in each fraction with the blood drug concentration. The uptakes of FK and CyA in the isolated lymphocytes obtained from the rat spleen and human peripheral blood were also studied. The amount of FK taken up by the spleen lymphocytes is five times greater than that of CyA. In the case of the uptake study using human peripheral blood lymphocytes, the concentration of FK in the lymphocyte is 100-fold higher than that of CyA. This difference in the lymphocyte level between the two immunosuppressants is thought to be one of the reasons why FK is more potent than CyA, a difference of about 100-fold in the in vitro pharmacological study and about tenfold in the in vivo organ transplantation experiments.

Pharmacokinetic study of a tripeptide HIV-1 protease inhibitor, KNI-174, in rats after intravenous and intraduodenal administrations.

Recently, as a new type of anti-AIDS drug, an HIV-1 protease inhibitor, KNI-174, has been synthesized; it shows a potent and selective HIV-1 protease inhibitory activity in vitro. In this study, we developed an HPLC assay system for KNI-174 in rat plasma and examined the pharmacokinetics of KNI-174 in rats using this assay method after both intravenous (i.v.) and intraduodenal (i.d.) administrations to obtain the disposition characteristics and bioavailability of this new anti-AIDS drug. This HPLC assay method is specific to KNI-174 and the standard curve was linear from 0.02 to 30 micrograms ml-1 plasma. After i.v. administration, 10.0 mg kg-1, KNI-174 disappeared from the rats' plasma in a three-exponential decay. The mean terminal elimination half-life, t1/2 lambda z, was 3.97 +/- 0.19 (S.E.) h, the total body clearance, CLtot, was 9.53 +/- 1.08 ml min-1 and the distribution volume at steady state, Vd,ss, was 7070 +/- 960 ml kg-1. In the case of the i.d. administration, 10.0 mg kg-1, the mean peak plasma concentration, Cmax, and the peak time, tmax, were 0.196 +/- 0.076 micrograms ml-1 and 0.444 +/- 0.193 h, respectively. The bioavailability of KNI-174 till infinity, BA(0-infinity), was 5.37 per cent. Because the IC50 of KNI-174 against HIV-1 in PHA-PBM was 138 ng ml-1, the time needed for maintaining the concentrations above IC50 after a single i.d. administration of KNI-174 is estimated to be 0.350 +/- 0.184 h.

[Stereoselectivity in N-dealkylation of disopyramide enantiomers in mouse hepatic microsomes].

The oxidative metabolism of disopyramide (DIS) enantiomers to mono-N-dealkyldisopyramide in mouse hepatic microsomes was studied in vitro. When (R-)-DIS was used as a substrate Lineweaver-Bulk plot was a single line with the apparent Km value of 0.23 mM and Vmax value of 1.05 nmol/mg protein/min. When S(+)-DIS was used as a substrate the plot was biphasic, and two apparent Km values (Km1 = 0.04 and Km2 = 0.77, mM) and two Vmax values (Vmax1 = 0.58 and Vmax2 = 1.53, nmol/mg protein/min) were obtained. When racemic DIS was used as a substrate the plot was also biphasic, while the plot observed at the lower concentration region was shifted to the upper region in comparison with that estimated from the sum of the individual N-dealkylase activities of each enantiomer. These results suggest that the N-dealkylation of R(-)-DIS is carried out by very limited cytochrome P-450 isozyme(s), but that of S(+)-DIS is carried out by multiple cytochrome P-450 isozymes; that is, N-dealkylations of R(-)- and S(+)-DIS are involved in the different cytochrome P-450 isozymes each other, particularly at relatively higher concentration of the substrates.