Development of Activity Assays for High-Volume Evaluation of Human Immunodeficiency Virus (HIV) Protease Inhibitors in Rat Serum: Results with Ditekiren

We showed previously that a commercially available synthetic tetradecapeptide, Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Leu-Val-Tyr-Ser, produces authentic angiotensin I (Ang I) upon incubation with the HIV-1 protease (S. K. Sharma et al., Anal. Biochem. 198:363, 1991). Therefore, we developed an Ang-I based activity assay for HIV protease inhibitors based on the technology developed earlier (M. J. Ruwart et al., Pharm. Res. 7:407, 1990; S. K. Sharma et al., Anal. Biochem. 186:24, 1990) for tracking renin inhibitors in rat sera. Ditekiren was either extracted from sera with ethyl acetate or assayed after the interfering substances in sera were precipitated with acetonitrile. Purified recombinant HIV-1 protease was added to extracted rat serum and the enzymatic reaction was initiated in the presence of the tetradecapeptide substrate. The inhibition of Ang I production was measured by a commercially available RIA kit. The cleanup methodology also enabled a commercially available Proteinase Scintillation Proximity Assay (SPA, Amersham) to quantify ditekiren in rat serum through the addition of recombinant HIV-1 protease and cleavage of substrate from SPA beads. Results were confirmed by HPLC or by the renin assay for ditekiren, which inhibits both aspartyl proteases. These technologies should prove useful for assessing serum levels of HIV protease inhibitors in rat.     

Cyclosporine metabolism by P450IIIA in rat enterocytes--another determinant of oral bioavailability?

Cyclosporine is converted to its major metabolites (M-17, M-1, and M-21) in human liver by enzymes belonging to the P450IIIA subfamily. These enzymes are also present in rat and human enterocytes; however, the possibility that CsA is metabolized in enterocytes has not been previously investigated. We therefore directly compared metabolism of 3H-CsA in microsomes prepared from liver and jejunal enterocytes. M-17, M-1, and M-21 were the major CsA metabolites produced by enterocyte microsomes. This metabolism appeared to be catalyzed by P450IIIA, because pretreatment of rats with the P450IIIA inducer dexamethasone significantly increased the rate of CsA metabolism in enterocyte microsomes and preincubation of enterocyte microsomes with anti-P450IIIA IgG inhibited the production of CsA metabolites by greater than 95%. To determine if enterocyte P450IIIA metabolizes CsA in vivo, rats were pretreated with the P450IIIA inducer dexamethasone, the P450IIIA inhibitor erythromycin, or vehicle alone. At laparotomy, 2 mg/kg of 3H-CsA was injected into a sealed loop of jejunum, and after collection of the mesenteric venous blood draining this segment for 45 min, the production of M-17 and M-1 was measured. In the control group, a mean of 3.9% of the recovered radioactivity was found as M-1 and M-17. In the rats pretreated with dexamethasone, a mean of 8.4% of the radioactivity was found as M-1 and M-17 (P less than 0.05 relative to control) and this decreased to 2.3% in the group pretreated with erythromycin (P = 0.08 relative to control). We conclude that P450IIIA in jejunal enterocytes readily metabolizes CsA. Furthermore, the metabolism of CsA by enterocytes in vivo is substantial and likely contributes to "first pass metabolism" of orally administered CsA. Our observations provide novel hypotheses to explain some important drug interactions and interpatient differences in CsA dosing requirements.     

In Vitro/in Vivo Models for Peptide Oral Absorption: Comparison of Caco-2 Cell Permeability with Rat Intestinal Absorption of Renin Inhibitory Peptides

Pharmaceutical Research -     

The role of cyclic AMP in canine secretin-stimulated bile flow.

Cyclic nucleotide secretion rates in bile during the physiologic stimulation of ductular bile flow in dogs was evaluated by measurement of bile cyclic AMP concentration during the stimulation of secretin release by infusion of acid into the duodenum of dogs with chronic bile fistulas. The effects of PGA₁ and theophylline on secretin-stimulated bile flow and cyclic AMP secretin rates were also evaluated. Endogenous secretin stimulated bicarbonate-rich bile flow and decreased bile cyclic AMP concentration. PGA₁ significantly decreased secretin-stimulated bile flow but not bile cyclic AMP secretion. Theophylline did not alter secretin-stimulated bile flow or cyclic AMP secretion rates. These data indicate that based on cyclic AMP content in bile, secretin-stimulated ductular bile flow is unrelated to cyclic AMP metabolism.     

Structure-function relationships of peptide fragments of gastrin and cholecystokinin.

This study evaluates the structure-function relationships of the C-terminal peptide fragments of gastrin and cholecystokinin (CCK) in the biliary system and the stomach. Dogs with chronic biliary and gastric fistulas were used. Administration of the common fragments of CCK and gastrin with four and five amino acids and the active fragments of CCK with six through eight amino acids without sulfation of tyrosine in position 7 failed to alter hepatic bile flow from control values while significantly stimulating gastric hydrogen ion output. Administration of the seven and eight amino acid peptide fragments of CCK with sulfation of tyrosine in position 7 significantly increased hepatic bile flow. Administration of the sulfated octapeptide with 4 microgram/kg per h of nonsulfated octapeptide did not result in the inhibition of the choleresis produced by the sulfated peptide. The gastric hydrogen ion response produced by the administration of the nonsulfated and sulfated peptide was equal to that of the nonsulfated peptide alone. These results suggest that in the biliary system the receptor is highly specific as sulfation of the peptide fragment of CCK is essential for combining with the receptor, whereas in the stomach the receptor has little specificity and combines with all of the peptide fragments evaluated.     

The role of accelerated colonic transit in prostaglandin-induced diarrhoea and its inhibition by prostacyclin.

Rats treated with subcutaneous 16,16-dimethyl prostaglandin E2 (16,16-dimethyl PGE2, 100 micrograms kg-1) exhibited diarrhoea even when their ileo-caecal junctions were tied, thereby eliminating contributions from small intestinal transit or fluid accumulation (enteropooling). The origin of the watery stool appeared to be the caecum, since tying the caecal-colonic junction eliminated it. The acceleration of colonic transit is likely to be a primary mechanism of PGE2-induced diarrhoea in the rat, since both normal animals and those with tied ileo-caecal junctions exhibited almost the same incidence of diarrhoea. Subcutaneous prostacyclin (PGI2) (2 mg kg-1 every 60 min) suppressed 16,16-dimethyl PGE2-induced diarrhoea in normal rats and in those with tied ileo-caecal junctions. Colonic transit measured in rats with cannula preimplanted in their proximal colon indicated that 16,16-dimethyl PGE2 enhanced colonic transit and PGI2 suppressed this increase. Thus, PGI2 can inhibit diarrhoea in the rat caused by 16,16-dimethyl PGE2 by suppressing colonic transit exclusive of its effects on small intestinal transit and enteropooling.     

Effect of 16,16-dimethyl prostaglandin E2 on oxygen uptake and microcirculation in the perfused rat liver

Previous work demonstrated that collagen deposition in the liver of rats fed a nutritionally deficient diet for 3 to 4 months was diminished markedly by 16,16-dimethyl prostaglandin E2 treatment. In this study, rats were fed a high-fat diet or a high-fat diet deficient in lipotropes for 2 to 4 weeks prior to liver perfusion. Rates of O2 uptake by the liver were not changed by dietary manipulation. Infusion of 16,16-dimethyl prostaglandin E2 (10 microM), however, decreased O2 uptake by the whole organ by 20 to 40% in both groups. O2 tension was measured at the liver surface with a miniature O2 electrode placed alternatively on periportal and pericentral regions of the liver lobule. Mean O2 tensions in both periportal and pericentral regions were reduced 2- to 3-fold during the infusion of 16,16-dimethyl prostaglandin E2 suggesting an action on the microcirculation. This hypothesis was supported by the observation that fluorescein isothiocyanate-dextran fluorescence detected from the liver surface as well as hepatic vascular volume determined by dye dilution techniques were decreased 30 to 50% by 16,16-dimethyl prostaglandin E2. In addition, 16,16-dimethyl prostaglandin E2 increased portal pressure by about 10 mm Hg in a reversible manner. Thus, it is concluded that pharmacological levels of 16,16-dimethyl prostaglandin E2 affects the microcirculation dramatically in the isolated perfused liver.     

Effect of glucagon on secretin-stimulated bile flow.

The effect of glucagon on secretin-stimulated bile flow was evaluated in dogs with chronic biliary and gastric fistulas. Evaluation of the effects of secretin and glucagon alone on hepatic bile flow indicated that the calculated maximal response (CMR) values of the two agents were similar. Secretin increased the bicarbonate concentration in hepatic bile whereas glucagon did not, suggesting basic differences in mechanism of action. Administration of glucagon to secretin-stimulated bile flow produced an increase in bile flow while decreasing the bicarbonate concentration in secretin-stimulated bile. Since the maximal response for bile flow to glucagon and secretin was significantly greater than the maximal response to either agent alone, glucagon produced potentiation of secretin-stimulated bile. Glucagon increased the CMR value of secretin-stimulated bile from 513 mul/min for secretin alone to 692 mul/min for secretin and glucagon. This was associated with no significant change in the values of the respective D50S. These data suggest that glucagon produced a noncompetitive augmentation of secretin-stimulated bile flow and suggest that the two agents do not utilize the same receptor to stimulate bile flow.     

The effect of vagotomy on the canine enterogastrone mechanism

The effect of truncal vagotomy on the enterogastrone mechanism was evaluated in dogs with Heidenhain pouches and gastric and pancreatic fistulas. During continuous infusion of pentagastrin, 8 microg-Kg.-hr., HCl was infused into the duodenum in doses of 12, 18, and 24 mEq. per hour before and one month following transthoracic vagotomy. The effect of vagotomy on the inhibition of pentagastrin-stimulated Heidenhain pouch hydrogen ion output produced by the endogenous release of duodenal hormones was evaluated. The results indicate that there was no difference in the degree of inhibition produced by 12 and 18 mEq. intraduodenal HCl before and after vagotomy. When 24 mEq. per hour HCl was infused, the percent inhibition was significantly greater after vagotomy compared to values before vagotomy. The D50 of intraduodenal acid for inhibition of Heidenhain pouch hydrogen ion output was not altered by vagotomy, whereas the calculated maximal response (inhibition) was increased after vagotomy. Pancreatic volume was greater after vagotomy when compared to values before vagotomy, when 24 mEq. of HCl per hour was infused into the duodenum. These data suggest that vagotomy does not impair the effectiveness of the enterogastrone mechanism and at high doses of intraduodenal acid may augment it.