In vivo drug release behavior in dogs from a new colon-targeted delivery system.

The colon-targeted delivery capsule (CTDC), a new capsule-type dosage form for colonic delivery of drugs, was investigated for the in vivo drug release behavior in dogs. A CTDC formulation with prednisolone as a model drug and theophylline as a marker substance for gastric emptying was prepared for this study. The enteric-coated capsule (ECC) formulation with a similar composition was also prepared as the reference. Both formulations were administered to four beagle dogs, and the drug release behavior thereof was compared. Under fasted condition, ECC released prednisolone and theophylline at the same time within 1 h after the gastric emptying. On the other hand the CTDC released prednisolone at 3.2 h after the gastric emptying. Such release behavior of CTDC was approximately consistent with the results obtained from the in vitro dissolution study, suggesting that the pH-sensing and timed-release functions imparted to the CTDC can work in the gastrointestinal tract of dogs as programmed. Under non-fasted condition, however, the gastric emptying of CTDC was found to be considerably delayed, up to about 14 h, and in this case the in vivo dissolution lag time of prednisolone at the small intestine was shortened to about 1.5 h.

Evaluation of colonic absorbability of drugs in dogs using a novel colon-targeted delivery capsule (CTDC).

A series of dog studies were performed to examine the in vitro/in vivo relationship of drug release behavior of the newly developed colon-targeted delivery capsule (CTDC). The four kinds of CTDCs containing theophylline, each of which has a different in vitro dissolution lag time, were orally administered to four beagle dogs under fasted condition, and the onset times of drug absorption were compared. The CTDC with longer in vitro lag time had a later onset of drug absorption. It was also found that the time difference between the gastric emptying and the onset of drug absorption was almost equal to the in vitro dissolution lag time of the capsule, suggesting a similar performance of CTDC in the gastrointestinal tract. From the comparison to the absorption behavior of the colon arrival marker, i.e. sulfasalazine, it was proved that the CTDC with the lag time of 3 h can deliver the drug directly to the colon. This result implied that the CTDC can be used as a non-invasive means for assessing the regional absorbability of drugs in the gastrointestinal tract. To evaluate the absorbability of drugs in the colon, three model drugs, theophylline (THEO), acetaminophen (ACET), and phenylpropanolamine hydrochloride (PPA) were directly delivered to the colons of beagle dogs using the CTDC with the lag time of about 3 h. The obtained relative bioavailabilities to the solution form were as high as 94.2%, 71.0%, and 91.5% for THEO, ACET and PPA, respectively, suggesting that the colonic absorbability of those drugs is essentially good.

Stereoselective metabolism of bisoprolol enantiomers in dogs and humans.

To clarify the mechanism of the species difference in the metabolism of bisoprolol enantiomers, in vitro metabolic studies were performed using dog liver microsomes and human cytochrome P450 (CYP) isoforms. The O-deisopropylation of bisoprolol enantiomers showed biphasic kinetics in dog liver microsomes. The intrinsic clearance (Vmax/Km) for O-deisopropylation of R(+)-bisoprolol was higher than S(-)-isomer in both high-affinity and low-affinity components. The R/S ratio of the intrinsic clearance in high- and low-affinity components was 1.34 and 1.65, respectively. The inhibition studies in dog liver microsomes using CYP isoform-selective inhibitors indicated that the O-deisopropylation of both bisoprolol enantiomers was mediated via the CYP2D and CYP3A subfamily, and suggested that high-affinity oxidation was dependent on CYP2D. The kinds of CYP subfamilies in dogs, which contribute to the metabolism of bisoprolol enantiomers, were the same as those in humans. The intrinsic clearance for O-deisopropylation of R(+)bisoprolol by human recombinant CYP2D6 was also different from that of S(-)-enantiomers (R/S:1.50). However, unlike the dog microsomes, the intrinsic clearance by the human recombinant CYP3A4 did not show a stereoselective difference. Therefore, the species difference in the R/S ratio of metabolic clearance for the oxidation of bisoprolol enantiomers (dog > human) is mainly due to the species difference in the stereoselectivity of one of the cytochrome P450 subfamilies (CYP3A).

Stereoselective pharmacokinetics of bisoprolol after intravenous and oral administration in beagle dogs.

The stereoselective pharmacokinetics of bisoprolol, a highly beta 1-selective adrenoceptor blocking agent, was studied in dogs. After intravenous and oral administration of the racemate, there was a difference in the plasma concentration between S(-)- and R(+)-bisoprolol. The area under the curve (AUC) of concentration versus time of S(-)-bisoprolol was approximately 1.5 times higher than that of R(+)-bisoprolol and the elimination half-life of S(-)-bisoprolol was approximately 1.4 times longer than that of R(+)-bisoprolol. However, no differences were observed in the volume of distribution, absolute bioavailability, and renal clearance between the two enantiomers. The plasma protein binding of S(-)-bisoprolol was also the same as that of the R(-)-isomer. No chiral inversion or enantiomer-enantiomer interaction was observed, when enantiomers were solely administered via the intravenous route. The comparison of the oxidative metabolic rate of two enantiomers using dog liver microsomes demonstrated that the metabolite was more slowly formed from S(-)- than from R(+)-bisoprolol. Consequently, we concluded that the stereoselective difference in the metabolic clearance between S(-)- and R(+)-bisoprolol caused the difference in the disposition of bisoprolol enantiomers.

Pharmacokinetics and metabolism of bisoprolol enantiomers in humans.

The plasma concentrations and urinary excretions of bisoprolol enantiomers in four Japanese male healthy volunteers after a single oral administration of 20 mg of racemic bisoprolol were evaluated. The AUC(infinity) and elimination half-life of (S)-(-)-bisoprolol were slightly larger than those of (R)-(+)-bisoprolol in all subjects. The metabolic clearance of (R)-(+)-bisoprolol was significantly (P < 0.05) larger than that of (S)-(-)-bisoprolol (S/R ratio: 0.79+/-0.03), although the difference was small. In contrast, no stereoselective in vitro protein binding of bisoprolol in human plasma was found. An in vitro metabolic study using recombinant human cytochrome P450 (CYP) isoforms indicated that oxidation of both bisoprolol enantiomers was catalyzed by the two isoforms, CYP2D6 and CYP3A4. CYP2D6 metabolized bisoprolol stereoselectively (R > S), whereas the metabolism of bisoprolol by CYP3A4 was not stereoselective. The S/R ratio of the mean clearance due to renal tubular secretion was 0.68, indicating a moderate degree of stereoselective renal tubular secretion. These findings taken together suggest that the small differences in the pharmacokinetics between (S)-(-)- and (R)-(+)-bisoprolol are mainly due to the stereoselectivity in the intrinsic metabolic clearance by CYP2D6 and renal tubular secretion.

In situ perfusion system for oral mucosal absorption in dogs.

To evaluate oral mucosal absorption of drugs in dogs, a newly designed in situ perfusion system with a circulating perfusion chamber was developed. The utility of the perfusion system was investigated by using three drugs: salicylic acid (SA), sulfadimethoxine (SM), and diltiazem (DIL). The oral mucosal absorption of the drugs could be adequately described by first-order rate processes. The absorption rate was independent of the amount of un-ionized drug, which varied with the pH of the solution. The absorption of SA was similar for various oral mucosal sites and for repeated experiments using the same site. Pharmacokinetic analysis for the plasma or medium concentration of SA after perfusion showed that SA was absorbed at the rate constant of 0.071 h-1, and that approximately 70% of SA absorbed from oral mucosa was transferred to the circulating blood.

Scintigraphic evaluation of a new capsule-type colon specific drug delivery system in healthy volunteers.

Colonic drug delivery is intended for local or systemic therapies. The lack of predictive in vitro or animal model leads to considerable time delays in colonic product development. The objective of this scintigraphic study was to provide "proof of concept" for a novel capsule-type colonic delivery system (Colon-Targeted Delivery Capsule) in healthy volunteers. The human data validates the design concept behind the release mechanism, in that capsule disintegration, and hence drug release, did not start until 5 h after gastric emptying, irrespective of whether the product was administered to fasted or fed subjects. However, the potential for prolonged gastric residence for large enteric coated products intended for intestinal targeting was also observed; overall, the study provides a focus for subsequent product development and highlights the role of scintigraphy in dynamically visualizing the drug delivery process.

Radioimmunoassay for imidapril, a new angiotensin-converting enzyme inhibitor, and imidaprilat, its active metabolite, in human plasma and urine.

A radioimmunoassay (RIA) was investigated for the determination of imidapril and its active metabolite, imidaprilat, in human plasma and urine. Imidapril is a new angiotensin-converting enzyme inhibitor and an oral prodrug of imidaprilat. Imidapril was determined after conversion to imidaprilat with esterase. Antiserum was raised in rabbits against the p-amino derivative of imidaprilat conjugated to bovine serum albumin. Radioligand was prepared by iodination (125I) of the p-hydroxybenzoylamino derivative of imidaprilat. Cross-reactivities of anti-imidaprilat antiserum for imidapril, its metabolites and several cardiovascular drugs were low. The calibration range was 0.1-100 ng ml-1 using a 100 microliters of human plasma of urine. Intra- and inter-day variations of imidaprilat assay in plasma were 2.0-7.9 and 4.1-6.2%, respectively, and intra- and inter-day variations of imidapril assay in plasma were 5.4-10.7 and 7.9-18.1%, respectively. The variations of the assay in urine were a little smaller than those in plasma. The recovery of imidaprilat and imidapril spiked in plasma or urine samples was approximately 100%. A good correlation between RIA and high-performance liquid chromatograpy was observed for both plasma and urine samples. Furthermore, this method was applied to the determination of imidaprilat and imidapril in human plasma and urine samples, for the evaluation of the pharmacokinetics of imidapril in humans. From the results, it was demonstrated that the developed RIA was useful for the determination of imidaprilat and imidapril in human plasma and urine, and was applicable to pharmacokinetic studies in humans.

High-performance liquid chromatographic analysis of bilirubin and biliverdin from jaundiced broilers.

A sensitive and rapid high-performance liquid chromatographic (HPLC) method was developed and used for the simultaneous determination of bilirubin and biliverdin in pericardial fluid samples collected from broilers at a poultry inspection site. A photodiode array detector distinguishing the bilirubin (UV 450 nm) and biliverdin (365 nm) was used as an analytical detector for HPLC system. An internal-surface reversed-phase silica support column was used, and the mobile phase consisted of acetonitrile: 0.5 M Tris HCl buffer (20:80, pH 7.2). Bilirubin was detected from all of the jaundiced pericardial fluid samples, and a small amount of biliverdin was detected with bilirubin in some samples. These jaundiced broilers had hepatic or bile duct lesions similar to those found in edible animals. From these results, a working definition of jaundiced broilers for poultry inspection sites was suggested: bilirubin is detectable from pericardial fluid and the carcass is in a state of yellow color change.

Malignant aortic body tumor in a Holstein cow.

A malignant aortic body tumor was observed in a 5-year-old female Holstein cow. The neoplastic mass, of 22 x 17 x 15 cm in size, was located at the base of the left atrium, having irregular lobular structures. The tumor cells had slightly eosinophilic cytoplasm, and a round or oval nucleus. Metastasis was only present in the premediastinal lymph node. The tumor cells exhibited intense immunoreactivity for neuron-specific enolase (NSE) and synaptophysin, and were moderately positive for chromogranin A. Electronmicroscopy revealed membrane-limited granules in the cytoplasm. The cultured cells were spindle in shape, and having projectional cytoplasm. They were intensely positive for NSE, synaptophysin, chromogranin A, and neurofilament (200 kD). Consequently, this case was diagnosed as a malignant aortic body tumor from the neuroecrodermal origin.

Rapid analysis of four bilirubins in domestic animal sera using high-performance liquid chromatography.

A rapid method was developed to analyze delta-bilirubin (B delta), diconjugated bilirubin (DCB), monoconjugated bilirubin (MCB), and unconjugated bilirubin (Bu) by direct injection of sera using high-performance liquid chromatography (HPLC) with an internal-surface reversed-phase silica support (ISRP) column. Sharp bilirubin peaks were obtained using a simple mobile phase of acetonitrile: 0.5 M Tris-HCl buffer (20:80, v/v, pH 7.2). A variable-wavelength detector set at 450 nm, 0.01 absorbance unit full scale (AUFS), and a recorder set at 4 mm/min were used for detection. Peaks for B delta, DCB, MCB and Bu appeared at 4.4, 6.4, 9.2 and 14.5 min, respectively, in human serum from subject with obstructive jaundice which was used as a bilirubin standard throughout this experiment. The mean recovery rate after direct addition of Bu in swine serum was 91.9% and that of DCB was 95.9%. When sera from icteric cattle, pigs and horses were analyzed using the direct injection technique, four bilirubin peaks were obtained and there was reliable correlation between the sum of the bilirubin peak heights observed on HPLC and the total bilirubin value measured by a standard reference procedure.

[Pharmacokinetic study on aspoxicillin transfer into pulmonary and tracheal tissues].

Each of 36 patients who underwent tracheotomy for removal of malignant or benign tumors or for treatment of pneumothorax was infused with 2 g of aspoxicillin (ASPC, Doyle injection) intravenously over 1-hour period. ASPC concentrations determined at 1 postoperative time-point in tissues of the lung and trachea and in serum of each patient were analyzed pharmacokinetically to elucidate the transfer of ASPC to the thoracic tissues. The preventive effect of ASPC against postoperative infections was also investigated in 39 tracheotomy patients. 1. The analysis of ASPC concentrations in 36 patients with tracheotomy gave the following results; 1) The peak blood level (about 80 micrograms/ml) was attained at the end of infusion. The serum level then decreased with time to below about 10 micrograms/ml at 6 hours after the start of infusion, with an elimination half-life of about 1.4 hours, which was comparable to that in healthy adults. 2) Peak levels in the lung and tracheal tissues were achieved at about 30 minutes after the start of infusion, at levels of about 30 and 40 micrograms/g, respectively, which decreased to about 5 micrograms/g in both tissues at 6 hours after the start of infusion. 2. Thirty nine patients who were treated with ASPC before operation were examined for the preventive effect of ASPC against postoperative infections for 1 week after operation. No postoperative infection was noted in any patients and ASPC was found to be useful for prevention of postoperative infections. 3. No side effects or abnormal laboratory findings were noted in any patients. Based on the results of the transfer into the tissues of respiratory organs and preventive effect against postoperative infections, we have concluded that ASPC is useful for prevention of infections after thoracic operation.

[Effect of a new selective alpha 2 adrenergic blocker, midaglizole, on isolated airway smooth muscle].

The role of alpha-adrenoceptors in asthma is still unclear. However, several studies have shown bronchodilatation after single doses of different alpha-adrenoceptor antagonists in patient's with asthma. The clinical efficacy of midaglizole, a new selective alpha 2 blocker was demonstrated by a more recent investigation. The present investigation was carried out to examine the effects of midaglizole on isolated airway smooth muscle obtained from humans and guinea pigs. Human bronchial smooth muscle was relaxed in a dose-dependent manner by midaglizole. EC50, molar concentration of midaglizole required to produce 50% reversal of carbachol-induced pre-contraction was (6.0 +/- 0.19) X 10(-5) M. Isoproterenol (5 X 10(-10) M) and midaglizole (3 X 10(-5) M) produced 30.0 +/- 9.5% and 40.8 +/- 7.0% of maximal relaxation, respectively. However, they produced almost 100% of maximal relaxation when used together. Isoproterenol, in combination with midaglizole, was associated with a significant increase of human bronchial relaxation as compared to either of the drugs singly. The same findings were obtained when the drugs were used on the guinea pig trachea. Midaglizole had no effect on the binding of the radiolabeled beta adrenergic antagonist [3H] dihydroalprenolol to particulates prepared from the lung. Propranolol did not inhibit the relaxant effect of midaglizole on airway smooth muscle. These results suggest that midaglizole may be effective for the treatment of asthma.

Effect of deuterium oxide (D2O) on the IgE-mediated Ca2+ influx, arachidonic acid and histamine release in rat basophilic leukemia cells.

Deuterium oxide (D2O), which is known to stimulate microtubule aggregation, enhanced the IgE-mediated 45Ca2+ influx, (14C)-arachidonic acid and histamine release in rat basophilic leukemia cells (RBL-2H3) in the same dose-dependent manner (up to 90% (v/v]. We compared the interaction between D2O and a variety of groups of pharmacological agents. A microtubule depolymerizing agent, demecolcine, which inhibited the IgE-mediated (14C)-arachidonic acid and histamine release without affecting 45Ca2+ influx, was counteracted by 45% D2O. Taxol, a microtubule stabilizing agent, which had an inhibitory effect on the above three steps, was also reversed by 45% D2O. These results would support the previous data on the interaction between D2O and microtubules and would further suggest that the status of microtubule aggregation may be related to the secretory process. Calmodulin inhibitors (W-7, trifluoperazine) blocked the IgE-mediated 45Ca2+ influx, (14C)-arachidonic acid and histamine release in the same dose-dependent manner, but were counteracted by 45% D2O. In contrast, the effects of proteinase inhibitors (TPCK, TLCK), an adenylate cyclase inhibitor (ddAdo), a phosphodiesterase inhibitor (aminophylline), a phospholipid methylation inhibitor (DZA + Hcy) and microfilament blockers (cytochalasin B and D) were not counteracted by 45% D2O. These results would suggest that D2O may be associated with calmodulin directly or indirectly possibly through some relationship between calmodulin and microtubules.

Aldehyde dehydrogenase 2 as a potential protective factor for renal insufficiency in Japanese subjects with heart failure: a pilot study.

The association between the aldehyde dehydrogenase 2 (ALDH2, rs671) genotypes and the estimated glomerular filtration rate (eGFR) was investigated in Japanese hypertensive patients with/without coronary artery disease or with ischemic heart failure (HF), and age/sex-matched normotensive healthy controls. The eGFRs were significantly lower in the HF subjects with the ALDH2 *2/*2 genotype than in those with the other genotypes. Multiple regression analyses adjusted by the potentially confounding factors showed the *2/*2 genotype to be significantly associated with the decreased eGFR, compared to the *1/*1 genotype (ß = 31.99 ml min1 per 1.73 m2, P < 0.01).

Improvement of dissolution rate and oral bioavailability of a sparingly water-soluble drug, (+/-)-5-[[2-(2-naphthalenylmethyl)-5-benzoxazolyl]-methyl]- 2, 4-thiazolidinedione, in co-ground mixture with D-mannitol.

We investigated the usefulness and efficiency of the co-grinding method with D-mannitol to improve the bioavailability of a sparingly water-soluble drug, (+/-)-5-[[2-(2-naphthalenylmethyl)-5-benzoxazolyl]methyl]-2, 4-thiazolidinedione (174), and compared it with those of the single-grinding method. The co-ground mixtures in drug/carrier weight ratios up to 1:5 gave fine particle sizes of less than about 3 microns, which showed a marked increase in the dissolution rate with reduction of particle size, compared with the single-ground powder, even with a similar particle size. The oral bioavailability study of co-ground powders in beagle dogs exhibited a dramatic increase, as did the dissolution rate, according to finer particle size. Finally, complete bioavailability was obtained at the finest particle size of 1.2 microns (drug/carrier ratio of 1:5, w/w) as was a solution of the drug. Bioavailability had a good linear correlation with the dissolution rate. These findings suggested that the co-grinding method with D-mannitol dramatically increased the available surface area, caused by a reduction of particle size, which not only accelerated the dissolution rate but also resulted in greater enhancement of the bioavailability of 174.