Structure-cytotoxic activity relationship for the toad poison bufadienolides.

The toad poison bufadienolides including natural and derivatized compounds were tested for their cytotoxic effects on primary liver carcinoma cells PLC/PRF/5 and their structure-cytotoxic activity relationships were studied. For this study, a ligand-binding model was developed by using a pharmacophore mapping program, Distance Comparisons (DISCO). The structural features that are common to the 3D structures of active bufadienolides were identified to provide approach to a 3D QSAR method by using Comparative Molecular Field Analysis (CoMFA) study and to correlate the steric and electrostatic fields of the molecules to their activities. A valuable model which enables prediction of their activities was obtained from the CoMFA analysis, which may be employed for the drug designs of new bufadienolide analogues.

Dose escalation safety and tolerance study of the competitive NMDA antagonist selfotel (CGS 19755) in neurosurgery patients.

Selfotel (CGS 19755), a competitive N-methyl-D-aspartate antagonist, is neuroprotective in experimental models of ischemic cerebral injury. We studied the safety and tolerability of a single intravenous dose (0.5 to 2.0 mg/kg) of selfotel in neurosurgery patients. Thirty-two neurosurgical patients undergoing intracranial surgery were given ascending doses of selfotel 2 to 14 h before surgery. Serum selfotel levels were measured over a period of 24 h. Cerebrospinal fluid (CSF) levels were measured 1.5 to 18 h after dosing. Frequent side effects included psychomimetic symptoms such as hallucinations, abnormal dreaming, agitation, and paranoia among 20 (66%) patients. Ataxia was seen among five (16%) and dizziness among eight (25%). Symptoms occurred 38 min to 40 h from administration and persisted 5 min to 4 days. Symptom severity worsened with increasing area under the curve measurements and doses above 1.0 mg/kg. All symptoms were reversible and easily treated with intravenous haloperidol. Modest elevations of hepatic enzymes were observed among four patients. No patient had severe adverse reactions. Maximum selfotel levels attained were 143 mumol (serum) and 4.76 mumol (CSF). Peak serum levels among six patients were within potentially neuroprotective ranges. CSF levels remained detectable up to 18 h after dosing. No obvious relationship was seen between CSF drug levels and symptoms. Selfotel in doses of 0.5 to 2.0 mg/kg can be administered safely to neurosurgical patients. Maximum serum levels attained were within the range shown to be neuroprotective in experimental studies. Side effects even at the highest levels are tolerable and reversible. Selfotel use in patients at risk for cerebral injury should be further explored.

Aminopyrine infusion breath test for the determination of changes in P450 metabolism in vivo.

1. An osmotic mini-pump was used to maintain a constant infusion of radiolabelled [N-dimethyl-14C] aminopyrine into a rat. After implanting the mini-pump, 14CO2 expiration rate was constant within 12 h, and this rate was maintained for 192 h. 2. Treatment with 2-diethylaminoethyl-2,2-diphenylvalerate HCl (SKF 525-A) or cimetidine, inhibitors of P450-dependent metabolism, resulted in both dose- and time-dependent inhibition of the expiration of 14CO2.

A chemiluminescent immunoassay for urinary thromboxane B2.

Because of the vasoactive properties of thromboxane A2 and other related prostaglandins, much research has been conducted on drugs which alter their levels. Urinary levels of thromboxane B2 and 2,3-dinor thromboxane B2 (major urinary metabolite of thromboxane B2) are used as an indication of thromboxane production in-vivo. In order to accurately measure urinary TXB2 levels of subjects on investigative drugs which lower TXA2 and subsequently TXB2, a simple and sensitive analytical tool becomes necessary. We have thus developed a non-radioisotopic (chemiluminescent) assay for urinary TXB2. Sensitivity has been demonstrated to 5 pg/ml. The method correlates well with gas chromatography/mass spectrometry (the accepted reference method) even without column chromatographic purification prior to the conduct of the chemiluminescent assay (r = 0.96). In addition, we have demonstrated feasibility for a chemiluminescent assay to measure urinary 2,3-dinor TXB2.

The caffeine breath test and caffeine urinary metabolite ratios in the Michigan cohort exposed to polybrominated biphenyls: a preliminary study.

A field biochemical epidemiology study was conducted using the Michigan cohort consisting of 51 rural residents exposed to polybrominated biphenyls (PBB). The study had three major objectives: a) to determine the serum half-life of the major PBB congener, hexabromobiphenyl (HBB), in the human, b) to determine if the PBB-exposed subjects had elevated cytochrome P-450I function as determined by the caffeine breath test (CBT) and the caffeine urinary metabolite ratio (CMR), and c) to determine the applicability of the CBT and CMR in field studies. PBB serum levels were detected in 36 of the 51 PBB-exposed subjects. The serum half-life of HBB was determined by comparing the current serum HBB values to the subject's previous serum values obtained 5 to 8 years earlier. The median HBB half-life was 12 years (range 4-97 years). The CBT and CMR were elevated in the subjects exposed to PBBs as compared to the values obtained from urban nonsmokers and were similar to those found in adults who smoke. A gender effect was seen in the PBB-exposed subjects, the median CBT and CMR values of the females being lower than the values of males. There was a correlation between the CBT and the HBB serum values (r2 = 0.2, p = 0.01) but not between CMR and HBB serum values. The CBT and CMR were easily conducted in the field and appear to be useful metabolic probes of cytochrome P-450I activity in human environmental toxicology.

Pharmacokinetic screening of o-naphthoquinone 5-lipoxygenase inhibitors.

The o-naphthoquinone derivative, CGS 8515 (I), is a potent inhibitor (IC50, 0.1 microM) of 5-lipoxygenase, but its therapeutic potential is compromised by a short plasma half-life (22 min) and extremely poor oral bioavailability (less than 2%). Poor biopharmaceutical properties of CGS 8515 were attributed to poor aqueous solubility and rapid in vivo hydrolysis of its methyl ester function to an inactive metabolite (IC50, 100 microM). An active amide analogue (II) was synthesized to prevent rapid hydrolysis. While analogue II appeared to be stable in vivo, its plasma half-life was also short (10 min), possibly because of rapid tissue distribution rather than metabolic elimination. Therefore, three potent analogues with increased aqueous solubilities were synthesized and compared with respect to their pharmacokinetic properties. The analogue with the highest aqueous solubility (V) demonstrated a plasma concentration vs time profile with the largest area under the curve (AUC) and the smallest distribution (alpha) phase of all the analogues studied. The percentage AUC of the terminal phase (beta) for three analogs paralleled their aqueous solubilities. The oral bioavailability of V was improved to 27%, compared to 2% for the parent compound, CGS 8515.

Effects of CGS-15943A on the relaxations produced by adenosine analogs in human blood vessels.

The antagonistic effects of CGS-15943A on the relaxations produced by adenosine and its analogs in human blood vessels were investigated in vitro. Donor hearts were the source of coronary arteries, whereas the internal mammary arteries and saphenous veins were obtained from patients undergoing coronary bypass surgery. Adenosine and its analogs, 5'-N-ethylcarboxamidoadenosine (NECA) and 2-chloroadenosine (CAD), produced concentration-dependent relaxations in KCl-contracted coronary rings. CGS-15943A antagonized, significantly, the relaxations produced by adenosine, NECA and CAD in coronary arteries. Similarly, the adenosine receptor antagonist, 8-phenyltheophylline (8PT, 10-mumol/l), caused a significant attenuation of the relaxing responses to adenosine, NECA and CAD in coronary arteries. In rings obtained from internal mammary arteries and saphenous veins, adenosine, NECA and CAD all produced concentration-dependent relaxations. These relaxations were smaller in magnitude than those obtained in coronary arteries, and were slightly greater in rings contracted with 10 mumol/l prostaglandin F2 alpha (PGF2 alpha) as compared to 35 mmol/l KCl. However, the mammary arteries and saphenous veins relaxed completely in response to 100 mumol/l papaverine. CGS-15943A (10 mumol/l) did not antagonize the relaxing effects of adenosine and its analogs in these vessels. The results show that coronary arteries are more responsive than mammary arteries or saphenous veins to the relaxing effects of adenosine analogs and that these relaxing responses are dependent on the contracting agent. Furthermore, CGS-15943A demonstrated antagonism of the adenosine response in coronary arteries.

Variations in demethylation of N-methylnaltrexone in mice, rats, dogs, and humans.

1. Rats and mice have a greater capacity than dogs or humans to N-demethylate the quaternary ammonium compound, N-methylnaltrexone. 2. In dogs, following the i.v. administration of N-[14C-methyl]methylnaltrexone, 50% of the radioactivity was excreted in the urine and an additional 30% in the faeces within 120 h. 3. In humans following the i.v. administration of 14C-N-methylnaltrexone, 40-60% of the radioactivity was excreted in the urine within the first 24 h. The plasma radioactivity-time curves indicated a biphasic decay and a short distribution phase between 6 and 9 min. with a longer elimination phase between 238 and 1320 min.

Inhibition of cholera-toxin-stimulated intestinal secretion by CGS 9343B in rats: a specific calmodulin inhibitor.

In this study, the effect of CGS 9343B on cholera-toxin-stimulated intestinal secretion in rats was determined using in vivo isolated loops. This recently developed compound is a potent and specific inhibitor of calmodulin, but not of protein kinase C. At a luminal dose of 15 mg/kg, CGS 9343B has little effect on basal intestinal absorption but completely inhibited the secretory effects of cholera toxin. The less specific calmodulin inhibitor, trifluoperazine, has a similar antisecretory effect, but unlike with CGS 9343B, severe toxicity was noted at luminal doses of 7.5 mg/kg. In animals where two intestinal loops were created, one with cholera toxin alone and the other with CGS 9343B and cholera toxin, significant inhibition of secretion was observed in both loops, consistent with a systemic effect of this compound. Finally, both CGS 9343B and trifluoperazine inhibited choleratoxin stimulated increases in mucosal cyclic AMP content, whereas basal levels were unaffected. We conclude that CGS 9343B significantly inhibits choleratoxin-stimulated intestinal secretions, possibly by inhibition of calmodulin-dependent adenylate cyclase activity. Its lack of major toxicity at therapeutic doses makes this compound potentially useful for the treatment of enterotoxigenic diarrheal diseases.

Effects of pregnancy on the cytochrome P-450 system in mice.

The effects of pregnancy on the hepatic cytochrome P-450-dependent mixed-function monooxygenase system (P-450) from day 6 to day 18 of gestation were examined in the C57BL/6J mouse. Pregnancy induced an initial increase and then a decrease in total P-450 content, a decrease in microsomal aminopyrine-N-demethylase activity, and had no effect on microsomal ethylmorphine-N-demethylase activity. Pregnancy also induced in the C57BL/6J and the DBA/2J mice a new major isozyme of P-450 (P-450gest) as determined by high performance liquid chromatography and gel electrophoresis.

The aminopyrine breath test does not correlate with histologic disease severity in patients with cholestasis.

To determine whether the aminopyrine breath test can be used to document the presence of cirrhosis in patients with cholestatic liver disease, 19 patients (13 primary biliary cirrhosis, 4 sclerosing cholangitis and 2 chronic extrahepatic bile duct obstruction) underwent clinical and biochemical evaluations, liver biopsies and an aminopyrine breath test. Results were compared with those in 10 patients with biopsy-proven chronic active hepatitis with bridging and/or cirrhosis and in 22 healthy subjects. The aminopyrine breath test results in the 10 cholestatic patients with cirrhosis were not significantly different from the results in precirrhotic cholestatic patients (mean +/- S.D., 11.2 +/- 5.0 vs. 11.6 +/- 2.8% dose per 2 hr, p greater than 0.05) or healthy subjects (11.5 +/- 2.9% dose per 2 hr). In contrast, the results in the patients with chronic hepatitis were markedly depressed (3.2 +/- 1.9% dose per 2 hr, p less than 0.05). The aminopyrine breath test results did not correlate with results of conventional liver function tests in the cholestatic patients. These results demonstrate that the aminopyrine breath test is not clinically useful in identifying the presence of cirrhosis in patients with cholestatic liver disease, and provide further evidence that decreased microsomal enzyme function is a late feature of cholestatic liver disease.

Effects of dietary protein on theophylline pharmacokinetics and caffeine and aminopyrine breath tests.

The effects of low- and high-protein diets on theophylline kinetics and the time course of changes in 13C-labeled caffeine and aminopyrine CO2 breath tests were examined in six young men. With a low-protein diet, mean theophylline clearance fell 21% (P less than 0.04) and the t1/2 rose from 8.0 to 10.6 hours (P less than 0.02). With a high-protein diet, mean theophylline clearance rose 26% (P less than 0.004) and the t1/2 shortened to 7.4 hours (P less than 0.03). Theophylline volume of distribution and protein binding did not change. Renal clearance of theophylline was lowered during the low-protein diet. Theophylline clearance correlated with caffeine breath test values during the low- (r = 0.73) and high- (r = 0.70) protein diets. Theophylline clearance correlated less well with the aminopyrine breath test values during the low- (r = 0.47) and high- (r = 0.55) protein diets. Thus dietary protein significantly influenced theophylline clearance, but the caffeine and aminopyrine breath tests showed a differential response to this important environmental factor.

The effect of age, gender, and sexual maturation on the caffeine breath test.

This study demonstrated the feasibility of utilizing the (3-13C-methyl) caffeine breath test (CBT) in children and adolescents, and examined the effect of gender, age, and puberty on the CBT. The CBT, expressed as the 2-hour accumulative exhalation of labeled CO2 (2-hour CO2), was compared to the CBT results in the adult. The 2-hour CO2 values were higher in the children than the adult, and the decrease in the CO2 values occurred in males during late puberty and in females during early puberty.

Differential haemin-mediated restoration of cytochrome P-450 N-demethylases after inactivation by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) to phenobarbital-pretreated rats results in the destruction of several phenobarbital-inducible cytochrome P-450 isoenzymes and a correspondingly marked loss of benzphetamine N-demethylase and ethylmorphine N-demethylase activities. Accordingly, the ion-exchange h.p.l.c. or DEAE-cellulose-chromatographic profile of solubilized microsomal preparations from such rats revealed a marked decrease in the cytochrome P-450 content of several eluted fractions compared with that of microsomes from corresponding non-AIA-treated controls. Incubation of liver homogenates from such rats with haemin restores not only cytochrome P-450 content from 35 to 62% of original values, but also benzphetamine N-demethylase and ethylmorphine N-demethylase activities, from 23 to 67%, and from 12 to 36% of original values respectively. Moreover, the chromatographic profiles of microsomes prepared from such homogenates indicated increases of cytochrome P-450 content only in some fractions. Reconstitution of mixed-function oxidase activity of cytochrome P-450 by addition of NADPH: cytochrome P-450 reductase to these fractions indicated that incubation with haemin restored benzphetamine N-demethylase activity predominantly, but ethylmorphine N-demethylase activity only minimally. After injection of [14C]AIA, a significant amount of radiolabel was found covalently bound to protein in chromatographic fraction III, and this binding was unaffected by incubation with haemin. Furthermore, the extent of this binding is apparently equimolar to the amount of cytochrome P-450 refractory to haemin reconstitution in that particular fraction. Whether such refractoriness reflects structural inactivation of the apo-cytochrome remains to be determined. Nevertheless, the evidence presented very strongly argues for AIA-mediated inactivation of multiple phenobarbital-induced isoenzymes, only a few of which are structurally and functionally reparable by haemin.

Comparison of the phenacetin and aminopyrine breath tests: effect of liver disease, inducers and cobaltous chloride.

The phenacetin breath test (PBT) has been proposed as an alternative to the aminopyrine breath test (ABT) for the assessment of hepatic function. To investigate the clinical utility of the PBT, we compared the PBT with the ABT in 9 healthy subjects and 18 patients with biopsy-proven liver disease. We also investigated the effects of cytochrome P-450 inducers in humans and rats, and the effect of cobaltous chloride (CoCl2) in rats on the PBT to elucidate the relationship between the rate of phenacetin deethylation and exhaled labeled CO2 derived from phenacetin. In humans with abnormal ABTs, the PBT correlated with the ABT (r = 0.77), but in healthy humans there was no correlation between the two breath tests. Rifampin pretreatment in healthy humans induced the ABT by 27%, but did not induce the PBT. In rats the PBT was not induced by 3-methylcholanthrene pretreatment at phenacetin doses of 1 mg per kg, but was induced by both 3-methylcholanthrene (178%) and phenobarbital (142%) at 10 mg per kg phenacetin. Pretreatment of rats with CoCl2, which reduces cytochrome P-450 content, decreased the PBT by 40% and the ABT by 84%. The insensitivity of the PBT to induction except at high doses of phenacetin suggests that phenacetin deethylation is not the rate-limiting process modulating exhaled labeled CO2 in healthy subjects, and that the PBT does not generally reflect normal or induced phenacetin dealkylation rates. The PBT, however, did reflect hepatic damage and may even be better than the ABT for grading the severity of hepatic damage.