Detection of cocaine and its metabolites in breast milk.

A method was developed for measuring cocaine and its metabolites, benzoylecgonine, ecgonine methyl ester, norcocaine, ecgonine ethyl ester, cocaethylene, and m-hydroxybenzoylecgonine, in breast milk by gas chromatography/mass spectrometry. Limits of detection for this method ranged from 2.5 to 10 ng/mL, and limits of quantitation ranged from 5 to 50 ng/mL. For each of the compounds measured by this method, linear response was demonstrated to 750 ng/mL. Breast milk was collected from 11 mothers who admitted to drug use during pregnancy and ten drug-free volunteers serving as control subjects. Cocaine was detected in six of the specimens obtained from drug-exposed subjects, and in none of the drug-free control subjects. In breast milk specimens where cocaine and one or more of its metabolites were detected, the concentration of parent compound was greater than any of the metabolites. The highest cocaine concentration found was over 12 microg/mL. Breast-fed infants of cocaine abusing mothers may be exposed to significant amounts of drug orally.

The effects of pyridostigmine bromide, permethrin, and DEET alone, or in combination, on fixed-ratio and fixed-interval behavior in male and female rats.

Concurrent exposure to pyridostigmine bromide (PB), permethrin (PERM) and/or N,N-diethyl-m-toluamide (DEET) may have contributed to the development of a syndrome that appears to have afflicted military personnel who served during the Gulf War. The present experiment sought to evaluate the behavioral effects of these compounds alone, or in various combinations, in male and female rats. Subjects were exposed to a multiple fixed-ratio (FR) 50, fixed-interval (FI) 2-min schedule of reinforcement. PB dose-dependently decreased FR and FI response rates. FR responding was disrupted by lower doses and there were no differences between the sexes. PERM vehicle administration decreased response rates maintained by both schedules of reinforcement; this was offset by an increase in response rate after the administration of the intermediate dose of PERM. The highest dose of PERM decreased both FR and FI response rates. FR rates in male rats were more disrupted than those in female rats. Only the highest dose of DEET decreased FR and FI response rates in male and female rats. FR rates were more disrupted in female rats than in male rats. Synergistic effects were only observed when FI response rates decreased in male rats upon exposure to half the low dose of PB with half the low dose of PERM or half the low dose of PB with half the low dose of DEET. The results of this experiment thus show that small doses of PB, PERM and DEET disrupt well-established, schedule-controlled behavior in male and female rats in a schedule- and gender-dependent manner; schedule-dependent and gender-dependent synergistic effects were also observed. The mechanism by which the compounds exert these behavioral effects remains to be determined.

The effects of pyridostigmine bromide and permethrin, alone or in combination, on response acquisition in male and female rats.

It has been hypothesized that concurrent exposure to pyridostigmine bromide and permethrin may have contributed to the development of neurocognitive symptoms in Gulf War veterans. The present experiment was designed to investigate the effects of pyridostigmine bromide and permethrin alone, or in combination, on the acquisition of a novel response, one measure of normal cognitive functioning. Male and female Sprague-Dawley rats were treated with pyridostigmine bromide (1.5 mg/kg/day, by gavage in a volume of 5 ml/kg) or its vehicle for 7 consecutive days. They then also received an intraperitoneal injection of permethrin (0, 15, or 60 mg/kg) before they were exposed to an experimental session during which they could earn food by pressing a lever in an operant chamber. Serum permethrin levels increased as a function of its dose, and were higher in rats treated with pyridostigmine bromide. Sex differences were observed as permethrin levels were higher in female rats than in male rats following the highest dose. Pyridostigmine bromide delayed response acquisition in male and female rats, and resulted in higher response rates on the inactive lever in female rats than in male rats. Although permethrin levels were higher in subjects treated with pyridostigmine bromide than in those treated with vehicle, there were no differences in the behavioral effects of permethrin. Whether or not these behavioral effects of pyridostigmine bromide are of central or peripheral origin will need to be determined in future studies, as its effects on motor activity and/or gastro-intestinal motility may have affected response acquisition.

Interactions of pyridostigmine bromide, DEET and permethrin alter locomotor behavior of rats.

Drug interactions have been suggested as a cause of Gulf War Syndrome. Pyridostigmine bromide (PB), a prophylactic treatment against potential nerve gas attack, the insect repellent DEET, and permethrin (PERM) impregnated in soldiers' uniforms may have interacted and caused greater than expected toxicity. We tested those 3 drugs singly and in combinations on male and female Sprague-Dawley rats in open field arenas to find the effects on rate of locomotion and thigmotaxis. Administration rates were 10 mg PB/kg; 50, 200, or 500 mg DEET/kg; 15, 30, or 60 mg PERM/kg; 5 mg PB/kg + 100 mg DEET/kg; 5 mg PB/kg + 15 mg PERM/kg; 100 mg DEET/kg + 15 mg PERM/kg; or vehicle by gavage and i.p. injection. Locomotor behavior was quantified by video-computer analysis for 2 h post-treatment. Female rats were tested in either pro- or metestrus. Drug interactions were determined by the isobolographic method. Blood serum drug concentrations were estimated by high performance liquid chromatography or gas chromatography-mass spectrometry. Single drug effects were very limited within the ranges tested. Double-drug administrations at half the single-drug rates resulted in statistically significant interactions in male rats for both locomotion rate and thigmotaxis. Combination of PB + PERM and DEET + PERM significantly affected speed, whereas only the combination of DEET + PERM significantly affected thigmotaxis. Female rats did not show significant interactions. Our data suggest that serum concentrations of PB and DEET may have been higher in females than males. Administration of PB + DEET may have reduced the serum concentration of DEET, and administration of PB + PERM may have increased the serum concentration of PERM.

Repeated coadministrations of pyridostigmine bromide, DEET, and permethrin alter locomotor behavior of rats.

Interactions of pyridostigmine bromide (PB), permethrin (PERM), and the insect repellent DEET (DEET) have been suggested as possible causes of Gulf War Syndrome (GWS) in humans. Open field locomotor studies have long been used in behavioral toxicology. Using male and female Sprague-Dawley rats, video-computer analyses, and the isobolographic method we have determined the effects on locomotor speed and thigmotaxis following repeated administration of single-, double-, or triple-drug or vehicle controls in an open field. The effects were measured 24 hours after 7 daily drug administrations. Single-drug administrations caused no significant effects. Double-drug administrations resulted in significant effects in the following cases: males given PB + DEET had a significantly slower locomotion rate; males given DEET + PERM had a significantly faster locomotion rate; females given PB + DEET had a significantly slower locomotion rate; and females given PB + PERM spent significantly more time in the center zone (less thigmotaxis). Triple-drug administration caused no significant effect. These results in comparison with behavioral studies in chickens and insects show certain similarities. The implications of the lasting effects on animal models are relevant to GWS in humans.

Pyridostigmine bromide alters locomotion and thigmotaxis of rats: gender effects.

Male rats and female rats in the proestrous and metestrous stages of estrus were tested to determine the effects of pyridostigmine bromide on locomotion rate and thigmotactic response using doses of 3.0, 10.0, and 30.0 mg/kg. Thirty minutes after administration of the pyridostigmine bromide the rats were videorecorded for 2 h in a 1 m2 open-field arena. The rats' activities were analyzed for the drug's effect on speed throughout the 2 h and during six 20-min segments. Also, the times that the rats were observed moving through the central 50% of the arena were determined. Locomotion rates decreased significantly, and thigmotaxses increased significantly in all groups of rats as a dose response to pyridostigmine bromide. Habituation occurred over 2 h for both responses, primarily during the first 40 min. Female rats were more affected than males, but metestrous and proestrous females did not differ significantly in their responses. At the 30 mg/kg the effect was persistent throughout the test period. Proestrous females dosed at 30 mg/kg had much higher pyridostigmine bromide serum levels than metestrous females and males.

The effects of acute and repeated pyridostigmine bromide administration on response acquisition with immediate and delayed reinforcement.

This experiment was designed to assess the effects of acute and repeated administration of pyridostigmine bromide (a carbamate with prophylactic and therapeutic uses) on response acquisition. Experimentally naïve, male Sprague-Dawley rats were exposed to a situation in which lever presses were either immediately followed by food-pellet presentation or after a 16-s resetting delay. Different groups of rats received either one acute administration of pyridostigmine bromide (10 mg/kg, by gavage) or repeated pyridostigmine administration for 7 days (1.5 mg/kg/day, by gavage). Other groups were treated with distilled water for the same period of time. Both acute and repeated pyridostigmine bromide administration decreased serum cholinesterase levels by approximately 50%, but neither treatment affected brain cholinesterase levels in our assay. Acute and repeated drug administration produced the same behavioral effects. Subjects exposed to the 0-s delay conditions obtained many more food pellets than those exposed to the 16-s delay conditions. Administration of pyridostigmine bromide delayed the onset of responding in some, but not all, of the subjects in the treated groups, independent of the delay condition to which they were exposed. Many more responses were observed on an inoperative lever during the 16-s delay conditions than during the 0-s delay conditions, especially during the 16-s delay condition in which subjects had received acute vehicle administration. Whether or not these effects of small doses of pyridostigmine bromide on response acquisition are of central or peripheral origin will need to be determined in future studies, as response acquisition in the present experiment may have been affected by pyridostigmine's effects on gastrointestinal functioning and/or motor activity.

Cocaine suppresses fetal immune system.

The effects of cocaine are well documented in the CNS; however, recent evidence suggests that cocaine may suppress the immune system. Maternal cocaine use essentially exposes the fetus to a continuous exposure of cocaine. The objective of this study was to investigate the immunomodulatory effects of cocaine and its metabolites on maternal and fetal immune systems. Subjects were recruited from an Investigational Review Board approved protocol, and biologic specimens were collected. For each subject peripheral blood mononuclear cells (PBMCs) were isolated by density gradient. Each PBMC sample was stimulated in separate wells with phytohemagglutinin and phrobol 12-myristate 13-acetate. Samples were radiolabeled and stimulation was measured. Cytokine measurements were made on the serum via ELISA assay techniques. In both the phorbol 12-myrisate 13-acetate and the phytohemagglutinin group, the PBMCs isolated from fetal cord blood in the cocaine-using group had significantly (p < 0.05) decreased responses compared with control subjects. IL 1 and IL 2 concentrations were suppressed in the cocaine-exposed fetal serum compared with controls (p < 0.005 and p < 0.05, respectively). We have shown that in utero cocaine exposure results in a nonspecific suppression of fetal T lymphocyte response. The clinical consequences of prenatal cocaine-induced immunosuppression need to be further explored.

Detection of cocaine and its metabolites in amniotic fluid and umbilical cord tissue.

The increased use of cocaine by women of child-bearing age has left many health care scientists searching for improved methods of detecting prenatal cocaine exposure. To that end, a study of the determination of cocaine and its metabolites in amniotic fluid and umbilical cord tissue was undertaken. Amniotic fluid (n = 32) and umbilical cord tissue (n = 70) specimens were collected from pregnant subjects admitted to labor and delivery at Shands Hospital at the University of Florida (Gainesville, FL). Subjects were interviewed regarding drug use during each trimester. Subjects reporting cocaine use were designated as target subjects, and those denying use were control subjects. The specimens were subjected to solid-phase extraction and analyzed for cocaine and its metabolites by gas chromatography-mass spectrometry. Cocaine analytes (predominantly benzoylecgonine) were detected in 28.1 and 18.5% of the amniotic fluid and umbilical cord tissue specimens, respectively. Other cocaine analytes frequently detected included ecgonine methyl ester and m-hydroxy-benzoylecgonine in amniotic fluid specimens and ecgonine methyl ester, norcocaine, and m-hydroxybenzoylecgonine in umbilical cord tissue specimens. This study has shown that cocaine and its metabolites are readily detected in specimens of maternal and fetal origin.

Evaluation of immune parameters and lymphocyte production of prolactin-immunoreactive proteins after chronic administration of cocaine to pregnant rats.

This study examined the effect of chronic cocaine exposure on selected immune parameters in pregnant rats. Cocaine hydrochloride, 60 mg/kg, was administered by i.p. injection as a divided daily dose on gestation days 8 to 19. This cocaine treatment regimen did not result in any change in maternal body weight, spleen and thymus body weight ratios or lymphocyte recovery from these organs. Cocaine treatment had no effect on the plasma levels of prolactin, growth hormone and insulin-like growth factor-1; hormones with immunoregulatory potential. In contrast, the plasma immunoglobulin G concentration in cocaine-treated animals was 48% higher (P < .05) than in control animals. Spleen lymphocytes and thymocytes were isolated and evaluated for their proliferative responses in vitro to a panel of T and B cell mitogens. Lymphocytes from cocaine-treated animals showed no significant differences in proliferative responses to concanavalin A (conA), phytohemagglutinin, pokeweed mitogen, interleukin-2 or lipopolysaccharide. The ability of conA-stimulated spleen lymphocytes to synthesize and secrete prolactin-immunoreactive proteins was further assessed by Western immunoblotting. We found that conA-stimulated spleen lymphocytes from cocaine-treated animals showed significantly decreased levels of intracellular and secreted 44,000-mw prolactin-immunoreactive proteins. In contrast, conA-stimulated spleen lymphocytes from control and cocaine-treated groups secreted equivalent amounts of the cytokine interleukin-2. In conclusion, chronic administration of cocaine to female rats during pregnancy significantly altered serum immunoglobulin G levels and lymphocyte production of prolactin-immunoreactive proteins in the absence of changes in lymphocyte proliferation in response to mitogens.

Analgesic and immunomodulatory effects of codeine and codeine 6-glucuronide.

PURPOSE: The antinociceptive and immunosuppressive effects of codeine and codeine 6-glucuronide were determined in rats after intracerebroventricular administration. METHODS: Codeine 6-glucuronide was synthesized using a modification of the Koenigs-Knorr reaction. A lipophilic intermediate formed during synthesis, methyl [codein-6-yl-2,3,4-tri-O-acetyl-beta-D-glucopyranosid] uronate, was also tested. Morphine was used as a positive control to compare antinociceptive potencies of these compounds. RESULTS: All compounds tested produced significant analgesic responses, as assessed by the tail flick model. Additionally, codeine 6-glucuronide showed significantly less immunosuppressive effects than codeine in vitro. CONCLUSIONS: We conclude that codeine 6-glucuronide and related compounds may have clinical benefit in the treatment of pain in immune compromised patients.

Cimetidine or ranitidine in renal transplant patients receiving cyclosporine.

While H2-receptor antagonists are commonly used in renal transplant patients to prevent peptic ulcer disease, they have been associated with immunostimulation, interference with cyclosporine (CsA) metabolism, and inhibition of tubular secretion of creatinine. In renal transplant patients, cimetidine in high doses has been shown to cause a sustained rise in serum creatinine (SCr) and to reduce creatinine clearance (CrCl) with no change in inulin clearance. In this short-term prospective study, we evaluated the effects of single daily doses of cimetidine or ranitidine on renal function, and CsA serum concentration. Fourteen renal transplant patients with stable renal function were assigned to receive either cimetidine 400 mg daily or ranitidine 150 mg daily for 7 days. In patients who received cimetidine, a slight rise in SCr was observed at days 2 and 5 which was not statistically significant, but no significant change in CsA trough level was noted. No changes in SCr or CsA level were noted in the patients who received ranitidine. No changes in GFR were observed in either cimetidine- or ranitidine-treated patients. We conclude that, in our short-term study, cimetidine or ranitidine in the doses used in this study did not affect the GFR or CsA level, or SCr.

Multiple-dose activated charcoal compared to urinary alkalinization for the enhancement of phenobarbital elimination.

BACKGROUND: Urinary alkalinization and multiple-dose activated charcoal are modalities advocated for the enhancement of phenobarbital elimination in poisoned patients. However, no studies exist comparing the efficacy of these two means of elimination enhancement. We compared their effects on the pharmacokinetic disposition of intravenously administered phenobarbital. METHODS: Ten healthy volunteers participated in each of three randomly ordered study phases. During each phase, 5 mg of intravenous phenobarbital per kilogram of body weight was administered. During phase I, no interventions were made in attempt to enhance phenobarbital elimination. In phase II, participants underwent 24 hours of urinary alkalinization. Throughout phase III, volunteers received six doses of activated charcoal and two doses of sorbitol over 24 hours. RESULTS: The phenobarbital elimination half-life was 148 hours, 47 hours and 19 hours during the control, alkalinization and charcoal phases, respectively. Statistically significant differences in the elimination of phenobarbital were detected when each of the following phases were compared: I vs II, I vs III and II vs III. CONCLUSIONS: Both urinary alkalinization and multiple doses of activated charcoal are effective for the enhancement of phenobarbital elimination but multiple-dose charcoal was superior to urinary alkalinization in our study population.

Hypomagnesemia in renal transplant patients: improvement over time and association with hypertension and cyclosporine levels.

Hypomagenesemia is frequently encountered early after kidney transplantation, especially in patients receiving cyclosporine (CsA). However, there have been no studies addressing the natural history of this disorder in adult transplant recipients. We conducted this investigation to study the change in the prevalence of hypomagnesemia over time in renal transplant patients as well as to determine the factors associated with this change. Three patient groups were studied: 24 CsA-treated patients followed longitudinally at 1, 3 and 6 months post-transplant (Group 1a, 1b, 1c); 33 CsA-treated patients at least 2 years post-transplant (Group 2; mean follow-up 55 +/- 25 months); and 31 non-CsA-treated patients at least 2 years post-transplant (Group 3; mean follow-up 132 +/- 57 months). The following parameters were monitored: serum and urine magnesium levels; serum potassium; creatinine clearance; fractional excretion of magnesium; and trough CsA levels. In group 1 patients, longitudinal follow-up showed a significant linear trend for improvement in the serum magnesium over time (1.6 +/- 0.3, 1.7 +/- 0.2, 1.8 +/- 0.2 mg/dl; p = 0.0015) as well as a decline in the whole blood CsA level (316 +/- 103, 251 +/- 82, 194 +/- 67 ng/ml; p = 0.0015) at 1, 3 and 6 months, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

A preliminary report of diltiazem and ketoconazole. Their cyclosporine-sparing effect and impact on transplant outcome.

A prospective randomized trial was conducted to compare the effect of diltiazem (DILT) with ketoconazole (KETO) on sparing of cyclosporine dose and renal transplant outcome. Renal allograft recipients 18 years old and older were eligible for the study. Triple immunosuppression (TRIPLE) including prednisone, azathioprine, and CsA was administered to all patients. The maintenance CsA dose varied by study group. Patients were randomized to receive one of three treatment strategies: group 1-TRIPLE (CsA 8 mg/kg/day); group 2--TRIPLE (CsA 6 mg/kg/day) + DILT (60 mg b.i.d.); group 3--TRIPLE (CsA 3 mg/kg/day) + KETO (200 mg/day). Modification of the DILT dose was allowed as needed to effect blood pressure control in group 2 patients. Mean 1-month CsA dose reductions were 30% and 60% of controls in group 2 and 3, respectively. A continued effect over time was observed in patients administered KETO but not DILT. At 1 year patients taking KETO required an average of 77% less CsA than the average dose necessary to effect similar parent CsA blood levels when no enzyme inhibitor was used. The use of KETO and DILT for 1 year allowed for 53% and 14% reductions in CsA cost, respectively. These savings include the cost of the KETO or DILT. Serum creatinines, mean arterial pressure (MAP), and incidence of liver function abnormalities were similar throughout treatment groups. The rate of rejection, time to rejection onset, and survival (GS/PS) were not different among the groups. Fungal infections were fewer in patients treated with KETO (12%) than in controls (16%) and patients randomized to DILT (19%). KETO failed to prevent Aspergillus infection in one individual. The investigation failed to identify any harmful result of treating renal allograft recipients with either DILT or KETO for the purpose of reducing CsA expense.