Central neuropeptide Y enhances bile secretion through vagal and muscarinic but not nitric oxide pathways in rats.

Neuropeptide Y (NPY) acts in the central nervous system to regulate gastrointestinal functions in rats and dogs. The effects of intracisternal injection of NPY on bile secretion and biliary components were investigated in urethane-anesthetized rats with bile duct cannula. Intracisternal NPY (0.02-0.12 nmol) dose-dependently increased bile secretion by 9.2-19.5%. The secretory response occurred within the first 20-40 min and lasted for the 120-min observation period. Intravenous injection of NPY (0.12 nmol) did not modify bile secretion under identical conditions. Biliary bile acid, phospholipid, and cholesterol secretion were not modified by intracisternal injection of NPY (0.12 nmol), whereas bicarbonate was increased by 19.0 +/- 1.7% from 40 to 120 min after NPY injection. Cervical cord transection at the C6 level, acute bilateral adrenalectomy (-120 min), or injection of NG-nitro-L-arginine methyl ester (10 mg/kg, IV, -15 min), an inhibitor of nitric oxide biosynthesis, did not alter intracisternal NPY (0.12 nmol)-induced stimulation of bile secretion. Atropine (2.0 mg/kg, IP, -30 min) and bilateral cervical vagotomy (-120 min) completely abolished the stimulatory effect of intracisternal NPY (0.12 nmol) on bile secretion. These findings indicate that NPY acts in the brain to stimulate bicarbonate-dependent bile secretion through vagal and muscarinic pathways and suggest that peptides in the central nervous system may be involved in the vagal regulation of bile secretion.

Role of calcitonin gene-related peptide and capsaicin-sensitive afferents in central thyrotropin-releasing hormone-induced hepatic hyperemia.

The involvement of capsaicin-sensitive afferent neurons and calcitonin gene-related peptide (CGRP) in the central thyrotropin-releasing hormone (TRH)-induced hepatic hyperemia was investigated in urethane anesthetized rats. Both systemic capsaicin pretreatment and intravenous administration of CGRP receptor antagonist, human CGRP-(8-37), completely abolished the stimulatory effect of hepatic blood flow induced by intracisternal injection of TRH analog (RX-77368; p-Glu-His-(3,3'-dimethyl)-Pro-NH2, 100 ng), assessed by the hydrogen gas clearance method. These data demonstrate the involvement of capsaicin-sensitive afferent neurons and CGRP in the central TRH-induced stimulation of hepatic blood flow.

Effect of central urocortin on carbon tetrachloride-induced acute liver injury in rats.

The effect of intracisternal injection of urocortin, an endogenous ligand for corticotropin-releasing factor (CRF) 2 receptor, on carbon tetrachloride (CCl4)-induced acute liver injury was investigated in rats. Intracisternal injection of urocortin dose-dependently enhanced elevation of serum alanine aminotransferase and aspartate aminotransferase levels induced by CCl4. Intracisternal urocortin also aggravated CCl4-induced histological changes of the liver. The aggravating effect of central urocortin on CCl4-induced acute liver injury was abolished by chemical sympathectomy, but not by vagotomy. These data demonstrate that urocortin acts in the brain to exacerbate acute liver injury through the sympathetic nervous system and suggest a possible involvement of the CRF2 receptor in the central CRF-induced exacerbation of acute liver injury in rats.

A new index, the core erosion ratio, of compression-coated timed-release tablets predicts the bioavailability of acetaminophen.

Although compression-coated tablets are a commonly used timed-release drug delivery technology, their utility is often limited by poor bioavailability. To try to improve the bioavailability of these tablets, the effect of their core composition of compression-coated tablet on in vivo pharmacokinetics was investigated. First, the extent of mass reduction of cores in different compression-coated tablet core formulations was used to establish a new index, the core erosion ratio. The data show that adding excipients with high water solubility to the core results in a greater core erosion ratio. Next, to elucidate the effect of core erosion ratio on in vivo acetaminophen (AAP) release, three compression-coated tablet formulations with similar in vitro AAP release profiles but different core erosion ratios were administered to four fasted dogs. The time for first appearance (TFA) of AAP in plasma did not differ significantly among formulations, indicating that the in vivo lag time was the same for all formulations. In separate experiments, necroscopy revealed that 3h after oral administration, the tablets were located in the ileum and colon and that all three formulations had identical GI transit times. However, the area under the AAP plasma concentration-time curve was greater in dogs given formulations with larger core erosion ratios. These results suggest that a formulation with a large core erosion ratio can significantly increase in vivo drug release from compression-coated tablets, leading to increased drug absorption from the lower GI tract.

Stabilization of minodronic acid in aqueous solution for parenteral formulation.

The composition, concentration, and buffer pH of potential minodronic acid formulations were evaluated for their drug stability and for their tendency to generate particles after storage for up to 4 weeks at 60 degrees C. The results indicate that citrate and tartrate buffers maintain drug stability and inhibit the formation of particles. The stability of minodronic acid in these solutions increased slightly as the buffer concentration increased, exhibiting less particle formation than in other buffers. Since citrate buffer was considered the most promising stabilizer for minodronic acid, the pH-stability relationship in 100 mM citrate with pH ranging from 3 to 7 was evaluated during storage for 4 weeks at 60 degrees C. The results demonstrate that solution pH of 3-5 result in optimal stability of minodronic acid with no formation of precipitates. A white precipitate was observed in citrate-containing sample solutions with pH of 6 and 7. Analysis of the isolated precipitate provided support for the hypothesis that the precipitate is a complex between minodronic acid and aluminum ions apparently leached from the glass of the ampoules.

Release of recombinant human bone morphogenetic protein 2 from a newly developed carrier.

After implantation of a polymer-coated gelatin sponge (PGS) containing either 0.4 or 1.0 mg of 125I-rhBMP-2 for each 1 cm(3) of PGS into the right ulnar of rabbits, changes in the level of radioactivity at the implant site and in the blood were measured for 21 days after implantation, and the cumulative excretion ratio of radioactivity in the urine and feces was calculated. For both doses, radioactivity at the implant site was eliminated biphasically. The concentration of trichloroacetic acid (TCA)-precipitable radioactivity in the blood reached a maximum 6 h after implantation, at which time it was equivalent to 1.41% of the administered dose (0.4 mg/cm(3)). The remaining radioactivity was eliminated rapidly thereafter, falling below the detection limit within 48 h. The t(1/2alpha) was about 0.1 days, the t(1/2beta) was about 3 days, and the mean resident time (MRT) value was about 4 days. By 17 days after implantation, 88.1% of the administered radioactivity had been excreted in the urine, and 1.7% had been excreted in the feces. TCA precipitation test results indicated that most of the radioactivity excreted in urine was a low-molecular weight decomposition product. At 21 days after implantation, the radioactivity of the PGS implant site had declined to 0.5% of the administered amount. Autoradiographs of the implant site taken 28 days after implantation revealed that, at both doses, the residual radioactivity was confined to the area of the implanted PGS. These results indicate that PGS retains an appropriate amount of recombinant human bone morphogenetic protein 2 (rhBMP-2) at the orthotopically implanted site for at least 21 days enough to induce bone regeneration. Thus, PGS shows great clinical potential as a carrier for rhBMP-2.

A new recombinant human bone morphogenetic protein-2 carrier for bone regeneration.

A gelatin sponge was formed by foaming and heat treating a gelatin solution, followed by coating the solid with poly(D,L-lactic-co-glycolic acid) to reinforce the gelatin framework. This sponge was tested for its suitability as a biodegradable porous, recombinant human bone morphogenetic protein (rhBMP)-2 carrier. Incorporation of rhBMP-2 into the sponge was closely related to its bulk density of gelatin sponge. The calcium content in the sponges, as assessed by an ectopic bone formation assay in rats, increased with the increasing sponge bulk density. Histologic and peripheral quantitative computed tomography analysis of implants in this ectopic assay system revealed cell growth throughout the carrier in 4 weeks after implantation regardless gelatin bulk density. The carrier containing rhBMP-2 maintained its three-dimensional structure after implantation; the carrier resisted collapse caused by soft tissue pressure during rapid bone formation as assessed by soft X-ray photographs. These results indicate that this newly developed sponge has excellent carrier characteristics to introduce rhBMP-2 into areas needed for bone regeneration.

Semisynthetic beta-lactam antibiotics. IV. Synthesis and antibacterial activity of 7 beta-[2-(hetero aromatic methoxyimino)-2-(2-aminothiazol-4-yl)acetamido]cephalosporins.

A series of 7 beta-[2-(hetero aromatic methoxyimino)-2-(2-aminothiazol-4-yl)acetamido]- cephalosporins have been synthesized and bacteriologically evaluated. Several substances in this series showed exceptional in vitro activity, especially those with a five-membered hetero aromatic substituent moiety at the 7-position and a quaternary ammonium group as the 3-function of the cephem nucleus. The most active derivative, 7 beta-[2-(imidazol-4-ylmethoxyimino)-2-(2-aminothiazol-4-yl)a cetamido]-3-(pyridiniomethyl)ceph-3-em-4-carboxylate (13a) was the most evenly balanced with respect to activity against Gram-positive and Gram-negative bacteria. Furthermore, 13 was stable to various types of beta-lactamases and had high affinities for penicillin binding protein-3 and -1Bs of both Escherichia coli and Pseudomonas aeruginosa.

Small intestinal absorption of bropirimine in rats and effect of bile salt on the absorption.

The intestinal absorption characteristics of a poorly water-soluble drug, bropirimine, were investigated by the in-situ small intestinal loop method using male Sprague-Dawley rats. Bropirimine in solution was well absorbed in the overall small intestine, following first-order kinetics. The rate determining step for the disappearance of bropirimine from the small intestinal loop after dosing in the suspension was the dissolution process from suspension. Bropirimine was solubilized by sodium glycocholate. The disappearance of bropirimine from the small intestinal loop was suppressed by sodium glycocholate contained in the solution, because of the loss of thermodynamic activity of bropirimine after its involvement in the micellar complex, not by the direct effect of bile salt on the permeability of intestinal mucosa. The disappearance of bropirimine was also suppressed by sodium glycocholate contained in the suspension. The suppression by sodium glycocholate seemed to be caused by the greater influence of sodium glycocholate on the thermodynamic activity of bropirimine than on the dissolution from suspension.

Bioavailability of bropirimine 250 mg tablet in dogs: effect of food.

The postprandial effect on the bioavailability of bropirimine in dogs after oral administration of bropirimine tablets (Bropirimine 250 mg Tablet) was investigated. At a dose of 500 mg bropirimine (two tablets of bropirimine 250 mg), the maximum plasma concentration under the postprandial condition was about twice that observed under the fasting condition, and the area under the plasma concentration vs time curve under the postprandial condition was also twice that under the fasting condition. The absolute oral bioavailabilities of bropirimine were 41.1% under the fasting condition and 83.5% under the postprandial condition. It is considered that the longer gastric residence time and larger volume of the gastric fluid induced by food-intake caused the increase in dissolution of bropirimine which increased the bioavailability after oral dosing of bropirimine 250-mg tablets.

Rapid improvement of icterus and pruritus by the oral administration of colestimide in two cases of drug-induced hepatitis.

We report two cases of drug-induced hepatitis refractory to therapy of ursodeoxycholic acid and prednisolone, who were relieved of icterus and pruritus immediately by the oral administration of colestimide. Their liver dysfunction was not improved, by withdrawal of causative drugs or by treatment with prednisolone and ursodeoxycholic acid. Colestimide (3.0 g/day), a strong basic anion-exchange resin, was orally taken before breakfast and evening meal, leading to rapid and complete relief of icterus and pruritus. These cases suggested that colestimide would be useful for patients with cholestasis in drug-induced hepatitis, because this agent has few side effects and it is easy to take.

Absorption of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN-1417), an analog of thyrotropin-releasing hormone, in rats and dogs.

Plasma levels of gamma-butyrolactone-gamma-carbonyl-L-histidyl-L-prolinamide citrate (DN-1417), an analog of thyrotropin-releasing hormone, were determined by a radioimmunoassay after oral or intravenous administration in rats and dogs. A pharmacokinetic analysis after intravenous injection revealed biphasic elimination of the plasma concentration following a two compartment open model with half lives in alpha-phase of 2.0 min and beta-phase of 19.2 min in rats, and half lives in alpha-phase of 4.0 min and beta-phase of 33.0 min in dogs. Absolute bioavailabilities when administered orally the solution of DN-1417 after 24 h fasting in rats and dogs were 1 and 10%, respectively. The bioavailability was observed to be unchanged at the dose up to 500 mg/kg in rats and at the dose up to 100 mg/dog in dogs. Thus, the absorption of DN-1417 in rats and dogs was proportional to the dose. On the other hand, the absolute bioavailabilities after meal in dogs were 7.9% at the dose of 20 mg/dog and 7.2% at the dose of 2 mg/dog, whereas in the 24 h fasting condition they were 15.7 and 12.0%, respectively, showing the decrease in absorption with food ingestion. These phenomena are somewhat different from the absorption of thyrotropin-releasing hormone.

Radioimmunoassay of thyrotropin-releasing hormone (TRH) in rat, dog, and human blood.

A radioimmunoassay was developed for the measurement of exogenous thyrotropin-releasing hormone (TRH) in the whole blood of rats, beagle-dogs and humans. Even at low temperature, TRH is degraded so quickly in the whole blood that stabilization of TRH in the blood is necessary. For this purpose, the direct extraction with methanol and concentration before the radioimmunoassay was satisfactorily performed. The method is highly sensitive so that a lower quantifiable concentration of 20 pg/ml was detectable, and good reproducibility and standard errors of less than 10% from triplicate standard curves were obtained. At low concentration of TRH, the effect of food ingestion and volume of whole blood on the sensitivity of the radioimmunoassay was observed and thin layer chromatographic treatment improved it.

Effect of central corticotropin-releasing factor on carbon tetrachloride-induced acute liver injury in rats.

Central neuropeptides play important roles in many instances of physiological and pathophysiological regulation mediated through the autonomic nervous system. In regard to the hepatobiliary system, several neuropeptides act in the brain to regulate bile secretion, hepatic blood flow, and hepatic proliferation. Stressors and sympathetic nerve activation are reported to exacerbate experimental liver injury. Some stressors are known to stimulate corticotropin-releasing factor (CRF) synthesis in the central nervous system and induce activation of sympathetic nerves in animal models. The effect of intracisternal CRF on carbon tetrachloride (CCl4)-induced acute liver injury was examined in rats. Intracisternal injection of CRF dose dependently enhanced elevation of the serum alanine aminotransferase (ALT) level induced by CCl4. Elevations of serum aspartate aminotransferase, alkaline phosphatase, and total bilirubin levels by CCl4 were also enhanced by intracisternal CRF injection. Intracisternal injection of CRF also aggravated CCl4-induced hepatic histological changes. Intracisternal CRF injection alone did not modify the serum ALT level. Intravenous administration of CRF did not influence CCl4-induced acute liver injury. The aggravating effect of central CRF on CCl4-induced acute liver injury was abolished by denervation of hepatic plexus with phenol and by denervation of noradrenergic fibers with 6-hydroxydopamine treatment but not by hepatic branch vagotomy or atropine treatment. These results suggest that CRF acts in the brain to exacerbate acute liver injury through the sympathetic-noradrenergic pathways.

Intestinal absorption mechanisms of thyrotropin-releasing hormone.

Intestinal absorption mechanisms of thyrotropin-releasing hormone (TRH) following the oral administration of TRH-tartrate (TRH-T) were studied in animals. When TRH-T was orally administered to rats or beagle-dogs, absorption of TRH showed apparent saturation and decreased with food ingestion. TRH is very stable against gastrointestinal digestive enzymes, homogenized intestine and epithelial cells. First pass effect in the liver was not observed in beagle-dogs. Absorption site specificity was found in rats, namely TRH can be absorbed from only the upper part of the small intestine. A saturation phenomenon was also observed in in situ and everted sac experiments. TRH absorption was inhibited by the existence of oligopeptides and some beta-lactam antibiotics that had been reported to be absorbed by active transport or carrier-mediated transport systems. The transfer of TRH from mucosal to serosal solutions was inhibited by the replacement of medium Na ions by K ions and by the existence of oligopeptides. The transfer rate from serosal side to mucosal side was much slower than that from mucosal side to serosal side. These results suggested that there should be a certain carrier-mediated transport system in the absorption process of TRH.

Synthesis and antibacterial activities of optically active ofloxacin.

Two optically active (100% enantiomeric excess) isomers of ofloxacin [(+/-)-ofloxacin; DL-8280; (+/-)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7 H-pyrido[1,2,3-de] [1,4] benzoxazine-6-carboxylic acid] were prepared by use of their optically resolved synthetic intermediates. One of the isomers, (-)-ofloxacin, was 8 to 128 times more potent in inhibiting the multiplication of gram-positive and gram-negative bacteria than the other, (+)-ofloxacin, and approximately two times more active than the racemate, (+/-)-ofloxacin.

Blood level and brain distribution of thyrotropin-releasing hormone (TRH) determined by radioimmunoassay after intravenous administration in rats.

The blood level and brain distribution of immunoreactive TRH after intravenous administration of TRH tartrate monohydrate (TRH-T) were investigated by radioimmunoassay in rats. Optimal condition for complete inactivation of TRH-degradative enzymes in brain by a microwave irradiation was found to be a 5kW power for 1.5 seconds. The endogenous TRH was present at the highest concentration in the hypothalamus, followed by the brain stem and thalamus, and was the lowest in the cerebral cortex including hippocampus and cerebellum. The time-course of TRH blood levels following administration of TRH-T at 0.5, 5 and 25 mg/kg fitted to a two compartment open model, and the half-lives in beta-phase increased dose-dependently. The administered TRH was incorporated rapidly from blood into whole brain, and the peak brain level corresponding to 0.108--0.166% of the total dose administered was attained 1 min after the administration. The brain TRH was eliminated with a half-life of about 3 min. The administered TRH was distributed throughout the brain, at the highest concentration in the hypothalamus, followed by the brain stem, cerebellum, thalamus and cerebral cortex. TRH tended to be eliminated most rapidly from the cerebellum.

Absorption of thyrotropin-releasing hormone after oral administration of TRH tartrate monohydrate in the rat, dog and human.

Quantitative blood levels of thyrotropin-releasing hormone (TRH) were determined by a sensitive and specific radioimmunoassay after oral administration or intravenous injected of thyrotropin-releasing hormone tartrate monohydrate (TRH-T) in the rat, dog and human. A pharmacokinetic analysis after intravenous injection of the drug revealed biphasic elimination of the whole blood concentration following a two-compartment open model with a half-life in alpha-phase of 2.6 min and beta-phase of 4.6 min in the rat (dose: 500 micrograms/kg); a half-life in alpha-phase of 3.2 min and beta-phase of 18.1 min in the beagle-dog (dose: 146 micrograms/dog); a half-life in alpha-phase of 4.0 min and beta-phase of 20.4 min in the human (dose: 730 microgram/human). The absolute bioavailability of TRH after oral administration of TRH-T solution in 24 h fasting rats were 1.5, 0.4, and 0.2% at 29.2, 146, and 730 mg/kg dosing levels, respectively (e.q. 20, 100, 500 mg/kg of TRH) compared with i.v. injection (dose: 500 microgram/kg). In beagle-dogs, they were 12.6, 9.8, 5.6, and 3.5% at 2.92, 14.6, 29.2, and 146 mg/dog dosing levels, respectively (e.q., 10, 20, and 100 mg/dog at TRH) compared with i.v. injection (dose: 146 micrograms/dog). Those of after meal in beagle-dogs were 6.0 and 2.3% at 2.92 and 29.2 mg/dog dosing levels (e.q. 2, and 20 mg/dog of TRH). Thus, TRH absorption showed apparent saturation and was decreased by food ingestion. The absolute bioavailability in the humans, who were administered 11.7 mg TRH-T (2.92 mg/tablet X four, e.q. 8 mg of TRH) two hours after meal, was 2.0% on the average, and thyroid stimulating hormone levels were significantly increased by oral administration of TRH-T tablets.

Neuropeptide Y in the dorsal vagal complex stimulates bicarbonate-dependent bile secretion in rats.

BACKGROUND & AIMS: Central administration of neuropeptide Y (NPY) enhances bile secretion through vagal pathways in animal models. NPY nerve fibers and receptors are localized in the dorsal vagal complex (DVC), and retrograde tracing techniques have shown that hepatic vagal nerves are projected mainly from the left DVC. However, nothing is known about the central sites of action for NPY to elicit bile secretion. The medullary sites of the action for NPY were investigated in this study. METHODS: The bile duct was cannulated in urethane-anesthetized and bile acid-compensated rats. After measuring basal secretion, NPY was microinjected into the DVC and bile response was observed for 100 minutes. Either left or right cervical vagotomy or hepatic branch vagotomy was performed 2 hours before the peptide. RESULTS: Microinjection of NPY (7-30 pmol) into the left DVC, but not the right DVC, dose-dependently increased bile acid-independent and bicarbonate-dependent bile secretion. Stimulation of bile secretion by NPY was eliminated by left cervical and hepatic branch vagotomy but not by right cervical vagotomy. CONCLUSIONS: NPY acts in the left DVC to stimulate bile acid-independent and bicarbonate-dependent bile secretion through the left cervical and hepatic vagus; these findings suggest that neuropeptides may act in the specific brain nuclei to regulate hepatic function.