The mechanism of action of the hypocholesterolemic drug p-(1-adamantyloxy)-aniline.

The effect of p-(1-adamantyloxy)-aniline (AOA) on the biliary secretion of cholesterol, bile salts, and phospholipids was studied in hypercholesterolemic rats. A second study was conducted using hypercholesterolemic rats with 14C-labeled cholesterol pools for the purpose of determining the effects of AOA on cholesterol metabolism. Treatment with AOA (100 mg/kg, orally) daily for 1 week resulted in the increased secretion of biliary cholesterol, but did not affect the secretion of bile salts or phospholipids. This treatment also resulted in an increase in the fecal excretion of 14C-labeled neutral and acidic sterols, and in reductions of both the total radioactivity in the liver and liver cholesterol. The data presented support the conclusion that the hypocholesterolemic action of AOA is due to the increased secretion of cholesterol into the bile and to the increased fecal excretion of cholesterol and bile salts.

Evaluation of a vitamin-cloaking strategy for oligopeptide therapeutics: biotinylated HIV-1 protease inhibitors.

The outstanding limitations to the oligopeptide as a therapeutic agent are poor oral availability and rapid biliary clearance. To address these concerns a series of eight peptidic HIV-1 protease inhibitors containing the structural segment of the vitamin biotin have been prepared. These have been evaluated with regard to the hypothesis that this vitamin would cloak the peptidic character of these oligopeptides, and thus impart to these inhibitors the potential for absorption and distribution via biotin transporters and receptors. By iterative optimization about a -Cha psi[CH-(OH)CH(OH)]Val- core inhibitory insert, three particularly potent inhibitors (K(i) < or = 10 nM) of the HIV-1 protease were obtained. Although excellent cell culture antiviral activity is observed for other peptidic protease inhibitors of comparable affinity, none in this series exhibited satisfactory antiviral activity. This failure is attributed to the incompatibility of the hydrophilic and hydrogen-bonding biotin segment, with the facile membrane permeability and intracellular access presumably required for antiviral activity. The ability of the biotin to cloak the peptide, and thus render the overall appearance of the conjugate as that of a vitamin, was evaluated. Four of this series were evaluated for recognition by the Caco-2 cell intestinal biotin transporter. None inhibited competitively biotin uptake, indicating a lack of recognition. A vitamin may bind to a specific protein carrier, and thus attain an improved serum profile (by resistance to biliary clearance) and advantageous delivery to cells. Therefore, the serum concentrations of three were evaluated following an iv bolus in a rat model for serum clearance. One of the three protease inhibitors (L-idonamide, 6-cyclohexyl-2,5,6-trideoxy-2-(1-methylethyl)-5-[[3-methyl-1-oxo-2-[[5- (hexahydro-2-oxo-1H-thieno[3,4-d]imidazol-4-yl)-1- oxopentyl]amino]butyl]amino]-N-[2-methyl-1-[[(2- pyridinylmethyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta (1R*,2R*,3R*),6 alpha]]-) sustained a more than 5-fold increase in serum concentration at all time points relative to the benchmark structure. The remaining two had serum concentrations at least equal to the benchmark, suggestive of improved resistance to clearance. One (L-idonamide,6-cyclohexyl-2,5,6-trideoxy-5-[[2-[[5-(hexahydro-2-ox o-1H- thieno-[3,4-d]imidazol-4-yl)pentyl]thio]benzoyl]amino]-2-(1- methylethyl)-N-[2-methyl-1-[[(2-pyridinyl- methyl)amino]carbonyl]butyl]-, [3aS-[3a alpha, 4 beta(1R*,2R*),6a alpha]]-) was prepared as a complex with the biotin-binding protein avidin. Avidin may resemble an endogenous serum biotin carrier protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Structure-based design of nonpeptidic HIV protease inhibitors: the sulfonamide-substituted cyclooctylpyramones.

Recently, cyclooctylpyranone derivatives with m-carboxamide substituents (e.g. 2c) were identified as potent, nonpeptidic HIV protease inhibitors, but these compounds lacked significant antiviral activity in cell culture. Substitution of a sulfonamide group at the meta position, however, produces compounds with excellent HIV protease binding affinity and antiviral activity. Guided by an iterative structure-based drug design process, we have prepared and evaluated a number of these derivatives, which are readily available via a seven-step synthesis. A few of the most potent compounds were further evaluated for such characteristics as pharmacokinetics and toxicity in rats and dogs. From this work, the p-cyanophenyl sulfonamide derivative 35k emerged as a promising inhibitor, was selected for further development, and entered phase I clinical trials.

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.

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.

Prazosin inhibits small intestinal transit in the rat.

Gastric emptying, small intestinal transit, and colonic transit were measured in fasted rats preimplanted with either duodenal or colonic cannulae. At the doses stated, prazosin (given subcutaneously) had no effect on gastric emptying or colonic transit, whereas small intestinal transit was significantly delayed.

Evaluation of lincomycin as a cholesterol gallstone dissolution rate accelerator.

These studies were undertaken to test the hypothesis that interfacial resistance may be an important rate-limiting factor in cholesterol gallstone dissolution. The addition of lincomycin hydrochloride to the gallbladder bile of dogs in an in vitro bath system resulted in an acceleration in the rate of dissolution of a compressed cholesterol monohydrate pellet incubating in the bile. However, the constant infusion of lincomycin for 13 d directly into the gallbladders of conscious, unrestrained dogs, which resulted in biliary lincomycin concentrations comparable to that of the in vitro tests, did not alter the dissolution rate of a compressed cholesterol monohydrate pellet which had been surgically placed into the gallbladder. We therefore conclude that the interfacial resistance between the cholesterol monohydrate pellet and the bile may be reduced by the addition of lincomycin to the gallbladder bile which, in the in vitro environment, results in an acceleration in the rate of dissolution of compressed cholesterol pellets. However, the ineffectiveness of lincomycin in accelerating the dissolution of cholesterol pellets in vivo suggests that interfacial resistance is not the only rate-limiting factor in gallstone dissolution. Other factors, such as mixing, may also be critical.

Prostacyclin inhibits gastric emptying and small-intestinal transit in rats and dogs.

Prostacyclin (PGI2) antagonizes 16,16-dimethyl prostaglandin E2-induced diarrhea in rats, presumably by inhibiting the fluid accumulation of "enteropooling" in the small intestine. The effect of PGI2 on gastric emptying, small intestinal transit, and colonic transit was examined in rats and dogs to determine if interference with propulsion might also contribute to the antidiarrheal properties of this compound. Rats implanted with chronic duodenal cannulas were given subcutaneous PGI2 (0.1-1000 microgram/kg) followed 10 min later by intragastric 51Cr and a visually detectable duodenal transit marker. Forty-five minutes later, the animals were killed. Small-intestinal transit was expressed as the percentage of small intestinal length traveled by the visually detected marker. Gastric emptying was expressed as the percentage of the total 51Cr found in the small intestine. Subcutaneous PGI2 inhibited gastric emptying maximally at 10 micrograms/kg. Small-intestinal transit was significantly decreased at 50 micrograms/kg and almost completely suppressed at 1.0 mg/kg. Subcutaneous naloxone (0.5 mg/kg) given 10 min before and 20 min after subcutaneous PGI2 administration did not block PGI2's effects. Intravenous or oral PGI2 in doses as high as 0.2 or 10 mg/kg, respectively, had none of these effects. However, a high-dose intravenous bolus (1.0 mg/kg) or infusion (1.0 mg/kg X 45 min) both inhibited gastric emptying. Small intestinal transit was only decreased by PGI2 infusion, suggesting that this parameter was more sensitive to a sustained blood level than gastric emptying. Hourly injections of subcutaneous PGI2 (0.5 mg/kg) had no effect on rat colonic transit measured over a 3-h period after deposition of the transit marker through a colonic cannula in a manner similar to that described for small-intestinal transit above. Small-intestinal transit was also measured in dogs given a barium suspension through a chronic duodenal cannula. The animals simultaneously received subcutaneous PGI2 (10 micrograms/kg) and were given an additional treatment and an abdominal x-ray every 30 min thereafter. In vehicle-treated dogs, barium reached the cecal area in an average of 2.8 h after instillation. In PGI2-treated dogs, barium never reached the cecum in the 5-h examination period. Thus, PGI2 inhibits gastric emptying in rat and small-intestinal transit in rat and dog but has no effect on rat colonic transit. These properties could contribute to PGI2's antidiarrheal activity.

The effects of PGF2 alpha, PGE2 and 16, 16 dimethyl PGE2 on gastric emptying and small intestinal transit in rat.

Prostaglandins are well known for their ability to stimulate contraction in gastrointestinal smooth muscle, yet very little information is available on how their activity affects propulsion in vivo. Thus, studies were undertaken to determine the effect of various prostaglandins on gastric emptying (GE) and small intestinal transit (SIT) in unanesthetized fasted rats. Rats were treated with intravenous, subcutaneous, or oral PGF2 alpha, PGE2, or 16, 16 dimethyl PGE2 at various doses, followed 1 (intravenous), 20 (subcutaneous) or 10 (oral) mins later by intragastric 51Cr oxide in black ink. Forty-five mins later, rats were sacrificed by CO2 asphyxiation, the pylorus clamped, and the gut excised. SIT was expressed as the percent of intestinal length traveled by the most distal portion of ink. GE was expressed as the percent of the 51Cr emptied into the intestines. If GE was affected by prostaglandin treatment, the experiments were repeated with rats pre-implanted with duodenal cannula. This preparation allowed the visual transit marker to be deposited directly into the duodenum, thus avoiding acceleration or delay of SIT caused by fluctuations in GE. The results of these studies show that: (1) intravenous 16, 16 dimethyl PGE2 (5-50 micrograms/kg), but not PGF2 alpha or PGE2, accelerates GE and delays SIT; (2) oral prostaglandin administration increases SIT; (3) oral 16, 16 dimethyl PGE2 delays GE; (4) subcutaneous 16, 16 dimethyl PGE2 accelerates, has no effect upon, or delays GE depending upon dose, but accelerates SIT at all doses tested; and (5) subcutaneous PGE2 accelerates SIT while PGF2 alpha does not. Thus, the effect of prostaglandins on GE and SIT depends upon the dosage and route of administration as well as type of prostaglandin used.

Prostaglandin stimulation of gastrointestinal transit in post-operative ileus rats.

The prostaglandins PGF2 alpha, PGE2 and 16,16-dimethyl PGE2, when administered intravenously, orally, subcutaneously or intraduodenally to laparotomized rats, decreased gastric emptying, small intestinal transit and colonic transit as compared to unoperated controls. All three prostaglandins increased colonic transit above that found with unoperated controls. This activity was independent of small intestinal fluid accumulation (i.e., enteropooling) since ligating the ileal-cecal junction had no effect on colonic transit. Small intestinal transit was increased, but not normalized, by PGE2 and 16,16-dimethyl PGE2. 16,16-Dimethyl PGE2 completely restored gastric emptying when given intravenously to laparotomized rats of doses greater than 5.0 microgram/kg. This effect on gastric emptying lasted approximately 4 hrs. Thus, 16,16-dimethyl PGE2, when given intravenously, normalized gastric emptying, significantly increased small intestinal transit, and made the colon hypermotile. Prostaglandins may be beneficial in the treatment of postoperative ileus and other conditions of sluggish gastrointestinal propulsion.

Clonidine delays small intestinal transit in the rat.

Subcutaneous clonidine (0.01--1.0 mg/kg) was found to delay small intestinal transit but not gastric emptying in the unanesthetized rat, with a maximal effect seen at 0.1 mg/kg. Gastric emptying was expressed as the percentage of intragastrically administered 51Cr emptied into the small intestine after 45 min. Small intestinal transit was the percentage of the small intestinal length traveled 45 min after oral or duodenal administration of black ink. The depression of small intestinal transit by clonidine to 20 to 30% of control values was blocked by phentolamine and yohimbine, but not by prazosin or phenoxybenzamine, suggesting a presynaptic (alpha-2) agonist action of clonidine. Pretreatment of rats with 6-hydroxydopamine, propranolol, atropine, methysergide, naloxone, mepyramine or metiamide failed to alter the effects of clonidine. These results suggest that an alpha adrenergic receptor, possibly presynaptic, regulates small bowel propulsion in rat without involvement of acetylcholine, norepinephrine, endorphins, histamine or serotonin.

Ionic composition of small intestinal secretion induced by PGE2.

Small intestinal fluid secretion induced by oral prostaglandin E2 in fasted rats was analyzed for various ionic components. Rat intestinal fluid had elevated calcium and potassium as well as decreased sodium and chloride concentrations relative to plasma electrolytes. Either low dose prostaglandin (0.15 mg/kg) or 1 ml of intragastric mannitol (5%) induced accumulation in the small intestine of fluid that had elevated chloride and depressed calcium and sodium concentrations compared to vehicle-treated controls. Higher doses of prostaglandin (1.0 mg/kg) led to secretions with increased sodium and chloride concentrations with respect to mannitol-induced fluid. Electrolyte concentrations in fluid induced by low dose prostaglandin appear to be similar to those in fluid caused by osmotically-induced changes. Higher doses of prostaglandin E2 induce additional electrolyte alterations which may result from modified gut transport of water and ions.

16,16-dimethyl-PGE2 protection against alpha-naphthylisothiocyanate-induced experimental cholangitis in the rat.

Male rats were treated with subcutaneous vehicle or 16,16-dimethyl-PGE2 (dmPGE2, 100 micrograms per kg), 24, 18 and 0.5 hr prior to and 6, 24 and 30 hr after challenge with oral alpha-naphthylisothiocyanate (ANIT, 30 mg per kg). Forty-eight hours after challenge, rats were sacrificed by decapitation; serum and liver samples were taken for biochemical and histological analysis, respectively. Rats treated with vehicle (2% ethanol in saline) and ANIT exhibited elevations in alkaline phosphatase, SGPT and bilirubin as well as cholangitis and mild parenchymal necrosis. Rats treated with dmPGE2 and ANIT had normal serum biochemical findings, no necrosis and only mild proliferation of bile duct epithelium. Thus, dmPGE2 may be able to protect the rat liver against the deleterious effects of orally administered ANIT.

16,16-Dimethyl PGE2 and fatty acids protect hepatocytes against CCl4-induced damage.

16,16-Dimethyl PGE2 (dmPGE2) has previously been shown to protect the in vivo rat liver against CCl4-induced damage. These studies were undertaken to determine if this protection could be demonstrated in vitro where factors of absorption, secretion, and blood flow are not present. Primary hepatocyte cultures were established by perfusing rat liver with collagenase. Hepatocytes were plated at a density of 2 X 10(4) cells/cm, allowed 90 min to attach, then stabilized in L15 medium for 18 h. Hepatocytes were then challenged with CCl4 with concomitant exposure to 10(-9) to 10(-5) M dmPGE2, stearic acid, oleic acid, or ethanol vehicle (0.00001 to 0.1%). After 1 h, challenge was aspirated and cells were stained with 0.04% trypan blue to determine viability. Hepatocytes in the vehicle groups took up more trypan when exposed to CCl4 than those treated with dmPGE2, stearic acid, or oleic acid at concentrations of 10(-9) to 10(-7) M. At 0.1% ethanol vehicle protected as well as all other treatments. Protection against CCl4 by dmPGE2, stearic, and oleic acids as well as high concentrations of ethanol may occur by altering the metabolism of CCl4.

16,16-Dimethyl prostaglandin E2 delays collagen formation in nutritional injury in rat liver.

Chronic nutritional injury was induced in rats by a high-fat, lipotrope-deficient diet. The hepatoprotective effect of 16,16-dimethyl prostaglandin E2 on the deposition of collagen and fat was assessed by histological evaluation and measurement of hydroxyproline. Dose-response studies established that optimal protection was achieved by the twice daily administration of 16,16-dimethyl prostaglandin E2 at 100 micrograms per kg (subcutaneous) or 250 micrograms per kg (oral). 16,16-Dimethyl prostaglandin E2 and a crystalline analog [(p-acetamidobenzamido)phenyl ester of 16,16-dimethyl prostaglandin E2 significantly delayed both the deposition of collagen and the increase in hepatic hydroxyproline content. There was an excellent correlation between the histological assessment of collagen and the biochemical measurement of hydroxyproline. These data provide a rationale for the evaluation of prostaglandins in the treatment of human liver disease.

Hepatic protection by 16, 16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1-induced injury in the rat.

Studies were conducted to assess the possible protective action of 16,16-dimethyl prostaglandin E2 (DMPG) against acute aflatoxin B1 (AFB1) induced hepatic injury in the rat. Evaluation of liver damage by histopathologic techniques and clinical chemistry indicated that hepatic necrosis was ameliorated by treatment with DMPG even though binding of radiolabeled (3H)-AFB1 to hepatic DNA was unaffected by this prostaglandin. However, DMPG did not protect rats against AFB1-induced mortality. These data suggest that hepatic protection by DMPG was due to mechanisms other than an interference with the activation or hepatic binding of AFB1.

Changes in thymidine uptake in the gastrointestinal tract of the rat following treatment with 16,16-dimethyl prostaglandin E2.

Thymidine uptake in the organs of the gastrointestinal tract of the rat was studied to determine if cell synthesis was involved in the increases in weight of the stomach, small intestine and colon which result from treatment with 16,16-dimethyl prostaglandin E2 (16,16-dimethyl PGE2). Animals were treated for 2 days with 16,16-dimethyl PGE2. They were injected with the 3H-thymidine, sacrificed and the organs of interest were removed. The total amount of tritium in the stomach, duodenum, jejunum, ileum, and colon was determined. Thymidine uptake was significantly increased in the duodenum (1.50 times), jejunum (1.53 times), and colon (1.40 times) but not in the stomach and ileum. The increases were dose related in the duodenum and jejunum. The colon showed a similar dose response pattern but the changes with dose did not reach significance. These results confirm and extend a previous report that 16,16-dimethyl PGE2 increased thymidine uptake in the duodenum but not the stomach. This is different from gastrin which has been shown by others to increase thymidine uptake in the stomach, duodenum, ileum and colon.