Can oral midazolam predict oral cyclosporine disposition?

Effective cyclosporine therapy is confounded by large interindividual differences in oral bioavailability and a narrow therapeutic window. Because cytochrome P450 (CYP) 3A-mediated first-pass metabolism contributes to this unpredictable bioavailability, an in vivo oral CYP3A phenotyping probe could be a valuable tool in optimizing cyclosporine therapy. Based on similarities in the metabolic kinetics of cyclosporine and midazolam by the liver and intestinal mucosa, we evaluated whether midazolam oral clearance would predict cyclosporine oral clearance when the two drugs were administered to 20 medically stable kidney transplant recipients. Despite earlier findings in liver transplant recipients who displayed a strong correlation between the systemic clearances of midazolam and cyclosporine, there was a weak correlation between their oral clearances in the current group of subjects (r(s)=0.50, P=0.03). Differing extents of intestinal first-pass metabolic extraction between the two drugs, inhibition of midazolam metabolism by cyclosporine at the level of the intestine, and/or P-glycoprotein-mediated intestinal efflux of cyclosporine (but not midazolam) may account for this poor correlation. We conclude that although oral midazolam is unlikely to be clinically useful as a probe for cyclosporine disposition, its utility in the prediction of other orally administered CYP3A substrates cannot be out ruled.

Determination of indinavir, a HIV-1 protease inhibitor, in human plasma using ion-pair reversed-phase high-performance liquid chromatography.

Indinavir is widely prescribed as a component of potent antiretroviral therapy for the treatment of HIV-1 infection. Because virologic failure of therapy can result from subtherapeutic drug levels, monitoring of indinavir levels may be important in clinical management. We have developed a simple, accurate, and precise high-performance liquid chromatographic (HPLC) assay for measurement of indinavir concentration in human plasma. In our method, indinavir was extracted from plasma with dichloromethane at pH 10.4, which resulted in quantitative recovery of indinavir and the internal standard (IS), methyl-indinavir (86% and 80%-97%, respectively). Chromatographic separation was accomplished using a Luna C18 (2) (Phenomenex) analytic column with a mobile phase composed of acetonitrile:phosphate buffer (25 mM) and 0.2% triethylamine pH 7.0 (34.5:65.5, v/v). Ion-paired reagent triethylamine was necessary to ensure an appropriate retention time for indinavir and differentiate it from other protease inhibitors that were coextracted. Quantification was performed at 210 nm. The standard curves were linear (r2>0.999) over the concentration range 25-5,000 ng/mL, when 1-mL aliquots of plasma were extracted. Inter- and intraday coefficients of variation were acceptable. The assay was used to determine trough and peak levels of in plasma from 12 subjects who received indinavir 1200 mg every 12 hours, 1000 mg every 12 hours, or 800 mg every 8 hours. The concentrations of indinavir found in this study (trough 26-768 ng/mL; peak at 1 hr 3,309-17,568 ng/mL) has a wider range than defined previously (trough 50-300 ng/mL; peak 6,000-12,000 ng/mL). This study illustrates three potential uses of indinavir monitoring: to assess individual dosing regimen, to assess patient compliance, and to monitor unusual indinavir levels caused by changed drug clearance.

Use of midazolam as a human cytochrome P450 3A probe: I. In vitro-in vivo correlations in liver transplant patients.

The clearance of midazolam (MDZ) in humans is principally due to metabolic biotransformation catalyzed by CYP3A isoforms. A study was conducted in patients who had undergone liver transplants that provides evidence that MDZ can be used as an in vivo probe of interindividual hepatic CYP3A variability. The clearance of MDZ and cyclosporine after i.v. administration were determined in 10 patients approximately 10 days after transplant surgery. Liver biopsy specimens were obtained within 24 hr of the pharmacokinetic study and CYP3A content and MDZ 1'-hydroxylation activity were measured in 13,000 x g tissue supernatants (S-13). The in vitro rate of 1'-hydroxy-MDZ formation was found to correlate significantly with the total CYP3A content in hepatic S-13 fractions (r = .84, P < .01). The total MDZ clearance measured in vivo was highly correlated with the hepatic CYP3A content measured in vitro (r = .93, P < .001) and with in vivo cyclosporine clearance (r = .81, P < .001). For five of the patients, the intrinsic clearance of midazolam to 1'-hydroxy-MDZ (Vmax/Km) in vitro measured in S-13 preparations was scaled for total liver mass and applied to the well stirred model of hepatic clearance to yield a prediction of MDZ clearance in vivo. The mean MDZ clearance predicted from in vitro 1'-hydroxylation data was identical to the mean clearance observed in vivo (0.60 +/- 0.24 versus 0.59 +/- 0.25 liter/min). Together, the results suggest that variability in hepatic CYP3A expression in liver transplant recipients, and possibly in other populations, can be determined by the measurement of MDZ metabolic clearance.

The magnitude of acute and chronic alcohol abuse in trauma patients.

OBJECTIVE: To assess the incidence of acute alcohol intoxication and the proportion of trauma patients with evidence of chronic alcohol abuse. DESIGN: Prospective cohort study. SETTING: Regional level I trauma center. PARTICIPANTS: Patients aged 18 years and older admitted with blunt or penetrating trauma. MAIN OUTCOME MEASURES: Admission blood alcohol concentrations (BACs), the Short Michigan Alcohol Screening Test (SMAST), and biochemical markers for chronic alcohol abuse. RESULTS: Of the 2657 patients enrolled, 47.0% had a positive BAC and 35.8% were intoxicated (BAC > or = 100 mg/dL) on admission to the emergency department. Intoxicated patients were more likely to be 25 to 34 years old, male, and nonwhite; the highest proportion of intoxicated patients was among victims of stab wounds. Three fourths of acutely intoxicated patients had evidence of chronic alcoholism as indicated by a positive SMAST, and 25% to 35% of acutely intoxicated patients had biochemical evidence of chronic alcohol abuse. CONCLUSIONS: The high prevalence of both acute intoxication and chronic alcoholism in trauma patients indicates the need to diagnose and appropriately treat this pervasive problem in trauma victims.

Substance abuse testing of urine by GC/MS in scanning mode evaluated by proficiency studies, TLC/GC, and EMIT.

Using GC/MS in scanning mode as a screening and definitive identification methodology for substance abuse testing, 4500 urine samples have been analyzed. The accuracy and sensitivity of this method was evaluated by the results of 92 proficiency sample analyses, reanalyses by TLC screening with GC confirmation of 125 samples from forensic sources and reanalysis by EMIT screening for seven groups of drugs confirmed by GC/MS of 590 samples from industrial and treatment sources. The results of these studies are presented.

Laboratory aspects of "Mycobacterium genavense," a proposed species isolated from AIDS patients.

"Mycobacterium genavense" is a proposed new species recently reported to cause disseminated infections in 18 patients with AIDS in Europe. We have recovered "M. genavense" as slowly growing fastidious mycobacteria in blood cultures of seven patients with AIDS. In the original studies of "M. genavense," the fastidious organism grew only in BACTEC 13A vials. The Seattle, Washington, isolates of "M. genavense" also failed to grow when subcultured from 13A vials to routine solid media, but dysgonic colonies were produced on Middlebrook 7H11 agar supplemented with mycobactin J. The mycolic acid pattern of patients' isolates closely resembled that of the type strain of Mycobacterium simiae when analyzed by one- and two-dimensional thin-layer chromatography and by high-performance liquid chromatography. Whole-cell fatty acid analyses by gas-liquid chromatography distinguished the isolates from M. simiae but misidentified them as Mycobacterium fortuitum. Sequence determinations of the hypervariable regions of the 16S rRNA gene indicate that these organisms belong to the recently proposed new species "M. genavense." Growth from Middlebrook 7H11 agar supplemented with mycobactin J consistently yielded positive tests for catalase (semiquantitative and at 68 degrees C), pyrazinamidase, and urease which enable mycobacteriology laboratories to presumptively identify "M. genavense" without nucleic acid analyses. The failure of "M. genavense" to grow on conventional mycobacterial solid media suggests that mycobacterial blood cultures should include a broth medium incubated for at least 8 weeks.

Measurement of free desipramine in serum by ultrafiltration with immunoassay.

We developed an ultrafiltration method for assaying free desipramine (DMI) in serum. An ultrafiltrate of DMI-containing serum was prepared by centrifugation through an Amicon Centrifree micropartition filter. Syva DMI solid-phase extraction (SPE) columns were used to extract the DMI from the serum and ultrafiltrate. The Syva monoclonal EMIT assay was used to quantify the DMI in the extract. In some experiments, the percent free DMI was quantified with radioactivity. Nonspecific losses of DMI in serum to the ultrafilter system were low (recoveries > 91%). Extraction of [3H]DMI from phosphate-buffered saline (to mimic serum ultrafiltrate) with the Syva SPE system was quantitative (recoveries of 98.4% +/- 4.6%). Free DMI concentrations, derived from serum containing 2.5-2500 micrograms/L DMI, were determined by ultrafiltration; results agreed well with values determined by equilibrium dialysis, the average percent of free DMI being 18.4% +/- 0.25% and 15.9% +/- 0.51%, respectively. To increase the sensitivity of the free DMI assay in the therapeutic range (total DMI 125-300 micrograms/L), we increased fourfold the ultrafiltrate volume applied to the SPE column. For free DMI at 11-130 micrograms/L, the within-run and between-run CVs for the ultrafiltration method were < 9% and < 15%, respectively. Binding of DMI to serum proteins decreased over the pH range 6.0-8.0, although temperatures between 20 and 28 degrees C did not affect binding. The ultrafiltration assay is fast, accurate, simple, and adaptable to standard laboratory instrumentation.

Measurement of gestational cocaine exposure: sensitivity of infants' hair, meconium, and urine.

We studied the sensitivity of testing the newborn infant's hair, meconium, and urine in detecting gestational cocaine exposure. The infants were born to 59 women who were interviewed to determine their use of cocaine during pregnancy and whose hair was analyzed for the presence of cocaine. Regression analysis was used to evaluate the relationship between cocaine in newborn hair and in maternal hair. Radioimmunoassay of infants' hair and gas chromatography-mass spectrometry of meconium were more sensitive than immunoassay of urine (p less than 0.02), which failed to identify 60% of cocaine-exposed infants. The quantity of benzoylecgonine in the newborn infant's hair correlated best with the proximal-segment maternal hair, representing the last 12 weeks of antepartum hair growth (R = less than R less than 0.83). Approximately half (52%) of the variation in infants' hair was explained by variation in the proximal maternal hair segment. Correlation (R = 0.77) and explained variation (59%) improved slightly when premature infants (n = 9) were excluded. We conclude that analysis of the newborn infant's hair by radioimmunoassay or of meconium by gas chromatography-mass spectrometry is more sensitive than analysis by immunoassay of urine, and can detect fetal cocaine exposure that occurred during the last two trimesters of pregnancy.

Death following accidental sodium azide ingestion.

Two college students developed symptoms of poisoning following ingestion of a salt solution during a college physiology laboratory exercise. Symptoms included nausea, vomiting, diarrhea, and altered consciousness. The ingested solution was identified as isotonic buffered saline containing sodium azide in a concentration of 1.0 g/L. The solution was commercially prepared for instrumentation use only and was used inadvertently for the exercise instead of freshly preparing sodium chloride in water. One student drank three sips of the solution and survived. The other student drank 700 to 800 mL and over several days became progressively ill, suffering myocardial damage and cardiac dysrhythmias, and, finally, died. Toxicologic studies confirmed the presence of azide in an antemortem urine sample from the deceased. Sodium azide is an uncommon but potent poison which can cause serious illness and death.

Drug use in trauma victims.

We examined the prevalence and characteristics of drug use in a large sample of fatally and nonfatally injured trauma victims. Routinely collected urine specimens from 452 emergency room patients and 160 persons autopsied at the Medical Examiner's Office (MEO) were analyzed for the presence of marijuana, cocaine, opiates and benzodiazepines using EMIT enzyme immunoassays. Blood alcohol levels were also measured. Tests were positive for at least one drug in 40.3% of the ER and 18.7% of the MEO samples. Marijuana was the most commonly detected drug in both groups. Specimens were more likely to be positive in younger persons and in males, and in victims of assaults and traffic accidents. Alcohol was present in the blood in more than one third of ER and MEO samples. Only 39.8% of ER samples and 52.3% of MEO samples were negative for both alcohol and drugs.

Disopyramide and N-monodesalkyl disopyramide in serum and breast milk.

Maternal serum and breast milk were obtained to determine the concentration of disopyramide (DP) and its metabolite N-monodesalkyl disopyramide (NMD) from a woman requiring antidysrhythmic drug therapy. Infant serum and urine were also obtained for drug concentrations. DP 450 mg tid resulted in peak maternal serum concentrations of 4.0 micrograms/mL and 2.2 micrograms/mL for DP and NMD, respectively. Breast milk concentrations averaged 1.06 and 6.24 times the serum levels for DP and NMD, respectively. No DP was measurable in the infant's serum except for cord blood, which contained 0.7 micrograms/mL DP, 26 percent of simultaneous maternal concentration, and 0.9 micrograms/mL NMD, which represented 43 percent of the maternal concentration. Infant urine collected over an eight-hour period contained 3.3 micrograms/mL of DP and 3.7 micrograms/mL of NMD.

Tricyclic antidepressant-specific Fab fragments alter the distribution and elimination of desipramine in the rabbit: a model for overdose treatment.

The effects of tricyclic antidepressant (TCA)-specific Fab fragments (Fab) on total and free desipramine (DMI) levels in serum and urine of DMI-treated rabbits were studied to determine the feasibility of using these Fab for antidotal treatment of TCA overdoses in humans. Serial samples of blood and urine were collected from two 3 kg rabbits after arterial injection of 2 mg 3H-DMI and, about 1.5 hr later, after injection of approximately 1 g Fab (prepared from sheep total IgG). Protein-free filtrates of serum and urine samples were obtained by ultrafiltration; concentrations of apparent total and apparent free DMI (apparent DMI, aDMI = DMI + metabolites) were calculated based on the radioactivity in the sample and ultrafiltrate, respectively. Treatment with Fab induced significant changes in the absolute and relative concentrations of free and total aDMI in both serum and urine. Changes in the serum included increases in the total and free aDMI levels. Changes in the urine included the appearances of a protein-bound aDMI fraction and Fab, and an increase in the percent of unmetabolized DMI. These results demonstrate that TCA-specific Fab influence the distribution and elimination of desipramine in DMI-treated rabbits and suggest that further studies on the use of TCA-specific Fab for antidotal treatment of TCA overdose are warranted.

Determination of free digoxin concentrations in serum for monitoring Fab treatment of digoxin overdose.

A rapid method for assessing the free digoxin concentration in the serum of digoxin-overdosed patients receiving treatment with digoxin-specific Fab fragments has been developed. For this method, a protein-free ultrafiltrate is prepared from the patient's serum, and the digoxin in the ultrafiltrate (free digoxin) is measured by fluorescence polarization immunoassay. Both the inaccuracies associated with measurements of total digoxin by immunoassay in the presence of Fab and the long turnaround time associated with measurements of free digoxin by equilibrium dialysis were avoided. Good correlation was observed between measurements of free digoxin by this ultrafiltration technique and by equilibrium dialysis. The ultrafiltration method was used to evaluate the concentrations of free digoxin in a digoxin-overdosed patient treated with Fab at our hospital. In retrospect, the results suggest that her hospital stay could have been shortened by a timely appreciation of her increased concentration of free digoxin. Using the ultrafiltration method, one can determine free digoxin concentrations quickly, conveniently, and accurately in the clinical laboratory. This procedure therefore should be a valuable aid in monitoring the efficacy and adequacy of Fab treatment.

Drug-specific Fab therapy in drug overdose.

The use of digoxin-specific fragments, antigen-binding (Fab) for antidotal therapy of severe digitalis intoxication is rapidly becoming a treatment of choice. Furthermore, studies with this mode of drug-specific therapy using Fab specific for desipramine and for phencyclidine suggest that this treatment may be applicable to a variety of other drugs or drug classes. As Fab technology has advanced, so have laboratory methods for monitoring the efficacy of treatment.

A case of nonneuroleptic malignant syndrome.

Neuroleptic malignant syndrome is a diagnosis that has been reported with increasing frequency in recent years. We report the case of a patient who was not treated with a neuroleptic but in whom a syndrome identical to neuroleptic malignant syndrome developed. The case highlights the overuse of and the confusion about the diagnosis of the syndrome.

Coating of oral beclomethasone dipropionate capsules with cellulose acetate phthalate enhances delivery of topically active antiinflammatory drug to the terminal ileum.

Selective delivery of orally administered topically active antiinflammatory drugs to the terminal ileum and ascending colon could be potentially useful for patients with inflammatory bowel disease involving these sites. Because topical beclomethasone dipropionate (BDP) enemas have been used successfully in the treatment of distal idiopathic colitis, oral formulations of this drug were studied. Enteric-coated or uncoated capsules containing BDP were administered in a single-dose protocol on separate days to 6 healthy volunteers with postcolectomy ileostomies. Ileostomy effluent was collected for a minimum of 8 h and analyzed by high-performance liquid chromatography for BDP, its pharmacologically active derivative beclomethasone monopropionate (BMP), and inactive beclomethasone alcohol. Cellulose acetate phthalate coating of oral BDP capsules significantly increased the mean percentage recovery of BDP + BMP in ileal effluent (43.0% +/- 24.1%) compared to uncoated BDP capsules (13.5% +/- 8.5%, p less than 0.05, Student's paired t-test). We conclude that oral cellulose acetate phthalate-coated BDP capsules may merit clinical trial in Crohn's ileitis and ileocolitis or in conjunction with BDP enemas for topical treatment of ulcerative colitis involving the whole colon.