Systemic complement activation, lung injury, and products of lipid peroxidation.

Previously we have demonstrated that systemic activation of the complement system after intravenous injection of cobra venom factor (CVF) results in acute lung injury as reflected by increases in the vascular permeability of the lung as well as by morphologic evidence of damage to lung vascular endothelial cells. In using the vascular permeability of the lung as the reference, the current studies show a quantitative correlation between lung injury and the appearance in plasma of lipid peroxidation products (conjugated dienes) as well as increased concentrations of lactic dehydrogenase (LDH) and one of its isoenzymes (LDH-4). After injection of CVF, extracts of lungs also showed elevated levels of conjugated dienes, whereas no elevations were found in extracts of liver, kidney, and spleen. There was no evidence in CVF-injected rats of renal or hepatic injury as reflected by the lack of development of proteinuria and the failure to detect increased serum levels of liver-related enzymes. Other peroxidation products identified in plasma of CVF-injected rats involved hydroperoxides and fluorescent compounds with features of Schiff bases. Not surprisingly, malondialdehyde was not found to be a reliable plasma indicator of lipid peroxidation associated with oxygen radical-mediated lung vascular injury. In using a model of oxygen radical-independent lung injury induced by oleic acid, although large amounts of LDH and LDH-4 were found in the plasma, no increases in plasma levels of conjugated dienes were detected. In CVF-injected animals treated with interventions protective against lung injury (neutrophil depletion, catalase, hydroxyl radical scavengers, or iron chelators), there were striking reductions in the plasma levels of conjugated dienes, hydroperoxides, and fluorochromic products. Morphometric analysis of lung sections revealed that the protective interventions did not interfere with the accumulation of neutrophils in lung interstitial capillaries after systemic activation of complement. In vitro studies with phorbol-stimulated neutrophils failed to demonstrate appearance of conjugated dienes, suggesting that the dienes appearing in plasma of CVF-injected animals are not the result of autotoxic changes in neutrophils. The data presented in this paper suggest that acute lung injury mediated by oxygen radicals derived from phagocytic cells can be monitored by the appearance in plasma of products of lipid peroxidation.

Effects of magnesium sulfate on cardiac conduction and refractoriness in humans.

Magnesium has been used empirically for several decades in the treatment of atrial and ventricular arrhythmias in patients with normal and decreased serum magnesium levels. However, a systematic evaluation of the effects of magnesium on cardiac conduction and refractoriness in humans has not been described. In this study, the electrocardiographic and electrophysiologic effects of magnesium were determined in 10 patients with normal baseline serum magnesium and other electrolyte levels. Six grams of magnesium sulfate was administered intravenously over 6 minutes followed by a continuous infusion of 1 additional gram over 1 hour. Serum magnesium levels rose significantly from a baseline of 2.0 +/- 0.2 to 5.4 +/- 0.4 mg/dl (p less than 0.001). No significant change occurred in heart rate at rest, or in duration of the QRS complex or QT or QTc intervals during sinus rhythm. There were significant increases in sinus node recovery time (1,000 +/- 211 to 1,106 +/- 223 ms, p less than 0.01) and corrected sinus node recovery time (279 +/- 87 to 336 +/- 104 ms, p less than 0.05). Significant increases occurred in atrioventricular (AV) node conduction time during sinus rhythm (82 +/- 22 to 97 +/- 17 ms, p less than 0.02), in the atrial paced cycle length at which AV node Wenckebach block occurred (350 +/- 46 to 419 +/- 65 ms, p less than 0.01) and in the AV node relative refractory period (397 +/- 27 to 422 +/- 18 ms, p less than 0.05), functional refractory period (395 +/- 41 to 415 +/- 33 ms, p less than 0.05) and effective refractory period (306 +/- 67 to 338 +/- 38 ms, p less than 0.05).

Role of myocardial ischemia during programmed stimulation in survivors of cardiac arrest with coronary artery disease.

The role of ischemia in the induction of ventricular tachycardia during programmed stimulation was studied in 19 patients who survived a cardiac arrest and were found to have a significant stenosis in at least one branch of the left coronary artery. The arterial-coronary sinus lactate difference was measured during electrophysiologic testing, before the induction of ventricular tachycardia. Ventricular tachycardia was induced in 15 patients; it was sustained and unimorphic in 6 patients and polymorphic in 9. Myocardial ischemia, as reflected by net myocardial lactate production, was present within 60 seconds before the induction of ventricular tachycardia in 8 of the 15 patients with inducible ventricular tachycardia. In 9 of the 15 patients, programmed stimulation was repeated after a 15 minute rest period, with the same coupling intervals that had induced ventricular tachycardia previously. Net myocardial lactate production was not present in any patient during this repeat attempt. In three patients without evidence of ischemia during the first induction of ventricular tachycardia, the arrhythmia was induced again by the specific coupling intervals that had induced it previously. However, in five of six patients with net myocardial lactate production during the first induction of ventricular tachycardia, the same coupling intervals that had induced the arrhythmia in the presence of ischemia no longer induced it in the absence of ischemia. The results of this study suggest that myocardial ischemia may be a requirement for the induction of ventricular tachycardia in some patients with coronary artery disease who survive a cardiac arrest.

Levels of cyclosporin in epidermis of treated psoriasis patients differentially inhibit growth of keratinocytes cultured in serum free versus serum containing media.

Cyclosporin A, which is a specific immunosuppressive compound, has recently been demonstrated to be of significant benefit in the treatment of psoriasis. Because hyperplasia is a major feature of psoriasis, we have investigated whether this drug acts directly to inhibit keratinocyte growth. We have determined the concentration range of cyclosporin in the epidermis of psoriatic patients undergoing cyclosporin therapy and the effect of this concentration range on the growth of cultured keratinocytes. After 7 days of treatment, psoriatic involved epidermis contained 1.1 +/- 0.3 ng cyclosporin/micrograms DNA. Based on tissue wet weight this is approximately 2.8 micrograms cyclosporin/ml. This value was 10 times that of trough blood samples. On day 3 of treatment, involved epidermis contained 7 times more cyclosporin than scale, while on day 7 this ratio was reduced to 2. This suggests that cyclosporin was initially associated with the lower layers of the epidermis and distributed upward with time. Exposure of adult human keratinocytes, cultured on collagen in the presence of human serum, to cyclosporin (0.1-30 micrograms/ml, 0.4-13-fold higher than lesional cyclosporin) for 2-6 d had no effect on the rate of incorporation of thymidine into DNA. Cyclosporin (1-30 micrograms/ml) also had not effect on the reinitiation of DNA synthesis of quiescent cells subsequent to the reintroduction of serum. In contrast, cyclosporin (1-10 micrograms/ml) inhibited growth of keratinocytes cultured on plastic culture dishes in serum free media (MCDB 153). For a given dose of cyclosporin, cell associated drug was 2-3 times greater in serum free compared to serum containing media. This may contribute in part to the sensitivity of keratinocytes in serum free media to growth inhibition by cyclosporin. These data demonstrate that human epidermis contains a high concentration of cyclosporin after oral administration, and that, under certain conditions, concentrations of cyclosporin within the range found in vivo can inhibit growth of cultured keratinocytes. Because cyclosporin regulates lymphocyte function in vivo and in vitro, the demonstrated antiproliferative effects of cyclosporin on psoriatic keratinocytes in vivo may be due to inhibition of a mononuclear leukocyte-derived keratinocyte growth factor(s) in combination with direct inhibition of keratinocyte growth.

Genetic prenatal aqueductal stenosis with hydrocephalus in rat.

A recessive mutation which arose in Wistar albino rats was variably expressed in the homozygous state as prenatal stenosis of the aqueduct with resultant hydrocephalus. The condition was often compatible with survival to adulthood and with successful reproduction. Mildly sparse hair was the constant gene marker. Eye defects and sometimes foot deformities occurred. The first observable ultrastructural alteration was a disruption of the integrity of the neuroepithelial basal lamina in the cephalic neural tube of affected embryos as early as the 11th fetal day (16-24 somite pairs). The hydrocephalic syndrome closely resembled that produced by giving folic acid analogs to, or producing vitamin B12 deficiency in, pregnant rats in the period including the 11th day. Neither vitamin B12 nor folate, nor certain metabolites closely related to their metabolism, prevented the gene's expression. Homozygote mutants mated with homozygote mutants produced 70% hydrocephalic (dome-shaped heads) offspring, but if the mother was heterozygote, there was a "protective" effect and the number of hydrocephalic young was disproportionately smaller.

Erythromycin breath test predicts oral clearance of cyclosporine in kidney transplant recipients.

It has been shown recently that cyclosporine is largely metabolized by P450IIIA (CYP3A), an enzyme whose catalytic activity varies significantly among patients. To determine whether heterogeneity in P450IIIA activity contributes to interpatient differences in cyclosporine dosing requirements, the oral pharmacokinetics of the drug were determined in 20 stable kidney transplant recipients. P450IIIA activity was then measured in each patient by use of the erythromycin breath test. In the 16 patients who were at steady state, the logarithm of the apparent oral clearance of cyclosporine correlated significantly with the rate of 14CO2 exhaled in breath after intravenous administration of [14C N-methyl]erythromycin (r = 0.55, p = 0.03). No significant correlations existed between apparent oral clearance and age, high-density lipoprotein cholesterol or low-density lipoprotein cholesterol, or hematocrit in these patients. We conclude that heterogeneity in P450IIIA activity significantly contributes to interpatient differences in dosing requirements of cyclosporine in kidney transplant patients.

The erythromycin breath test as a predictor of cyclosporine blood levels.

The daily dose of cyclosporine required to attain a desired blood level can vary greatly among patients. Because elimination of cyclosporine depends on its metabolism in the liver by an enzyme (cytochrome P-450IIIA) that also demethylates erythromycin, we reasoned that the ability of patients to demethylate a test dose of erythromycin might be useful in estimating their appropriate daily doses of cyclosporine. Accordingly, the [14C-N-methyl] erythromycin breath test was administered to 32 patients before they received 3.0, 5.0, or 7.5 mg/kg/day cyclosporine to treat psoriasis. We found that a simple mathematical equation incorporating just the 14CO2 production, the age of the patient, and the daily dose of cyclosporine accounted for almost 80% (R2 = 0.78) of the interpatient variability in cyclosporine blood levels we observed. Our data indicate that P-450IIIA activity largely accounts for the relationship between dose of cyclosporine and blood levels for an individual patient. We conclude that the erythromycin breath test may be a convenient guide for cyclosporine dosing.

Prediction of interpatient and intrapatient variation in OG 37-325 dosing requirements by the erythromycin breath test. A prospective study in renal transplant recipients.

OG 37-325 (nva-cyclosporine, cyclosporine G) is structurally similar to cyclosporine A (CsA). We hypothesized that OG 37-325 may, therefore, be metabolized by P450 3A, an enzyme recently shown to metabolize CsA. To test this hypothesis, we employed the erythromycin breath test (ERMBT) to measure P450 3A activity on multiple occasions in twenty OG 37-325-treated renal transplant recipients. When stable dosing was achieved, there was a measured 6-fold variation in the ratio of 12-hr whole-blood parent compound trough concentration (ng/ml, HPLC) to daily OG 37-325 dose (mg/kg) ([OG 37-325]/dose). In support of our hypothesis, there was an inverse correlation between the ERMBT result and the [OG 37-325]/dose ratio (r = -0.71, P < 0.001, n = 20); that is, patients with higher P450 3A activity generally required higher OG 37-325 doses to attain target blood levels. We also found that intrapatient variation in the [OG 37-325]/dose ratio observed over the course of the study correlated with changes in the ERMBT results (r = -0.67, P = 0.002). Inter- and intrapatient differences in [OG 37-325]/dose ratio were not predicted by patient age, serum cholesterol, blood hematocrit, or traditional liver chemistries. We conclude that P450 3A is generally rate limiting in the elimination of OG 37-325 in adult renal transplant recipients. Therefore, most drug interactions observed with CsA should also be expected with OG 37-325. We also conclude that intrapatient changes in OG 37-325 dosing requirements largely result from changes in P450 3A activity. The ERMBT may therefore provide useful information concerning patient compliance and may also serve as a useful guide to OG 37-325 dosing adjustments.

Artifactual hypercalcemia in multiple myeloma.

A calcium-binding IgG K monoclonal protein in a patient with multiple myeloma and asymptomatic hypercalcemia was recognized, isolated, and characterized. In addition to binding by the whole IgG molecules, calcium was bound by purified Fab fragments and recombined heavy and light chains. In a competitive binding study, the isolated myeloma protein did not bind magnesium. Recognition of calcium-binding myeloma proteins is important in order to avoid therapy for hypercalcemia.

Local cyclosporine immunotherapy of heart transplants in rats enhances survival.

High-dose systemic cyclosporine (CyA) administration frequently results in severe side effects. To evaluate a means of limiting the adverse effects of CyA, we implanted CyA-collagen matrices (0.2 or 1 mg/kg/day released) around the cardiac homografts at the time of rat heterotopic (neck) heart transplantation. Control animals received empty collagen (nondrug) matrix implants. A fourth group received CyA matrix (1 mg/kg/day released), implanted in a distal subdermal leg pouch at the time of heart transplantation. Rejection was determined by the absence of contraction in the transplanted heart. No animal received any other immunosuppression. Parallel groups of animals had whole blood, heart, and kidney CyA levels measured on the sixth posttransplant day. Local immunosuppression with high-dose CyA in a controlled-release matrix resulted in a significant survival advantage (mean survival time, 17.1 days; control, 6.9 days; p less than 0.001). The lower dose of CyA also demonstrated significant survival benefits (10.1 days), with clinically negligible blood CyA levels and very low kidney CyA levels. Both doses of epicardial local release CyA were well absorbed locally, resulting in very high CyA levels in cardiac tissue. Local immunotherapy of transplanted hearts with CyA was shown to be an effective means of preventing rejection. If this technology can be developed, this approach may prove advantageous clinically, both in extending transplantation and in minimizing systemic side effects of immunosuppression.

Ectopic creatine kinase MB production in metastatic cancer.

This case concerns the presence of high serum levels of creatine kinase MB isoenzyme in a patient with metastatic cancer. This patient did not have a myocardial infarction, so the source of the CK-MB was investigated. Because of the observation of macro-creatine kinase in patients with cancer, it was necessary to rule out the presence of this form of the enzyme. Extensive laboratory analysis demonstrated that the isoenzyme was true CK-MB, not an atypical or macro CK. Results of the study showed that ectopic production of the isoenzyme was the apparent source of the high serum activity. Homogenization of the cancer tissue demonstrated the presence of a high percentage of CK-MB activity. The implications of CK-MB production in cancer are discussed, including the use of various technics to rule out interfering activities when situations such as this occur.

The effect of ciprofloxacin on the pharmacokinetic and ECG parameters of quinidine.

Ciprofloxacin decreases the clearance of antipyrine and other drugs which, in part, undergo oxidative metabolism. Based on these findings, the authors hypothesized that ciprofloxacin may decrease the clearance of quinidine, a drug which also undergoes oxidative metabolism. The purpose of this study was to evaluate the effect of ciprofloxacin on the pharmacokinetic and ECG parameters of quinidine in seven healthy men. Oral quinidine sulfate 400 mg was administered alone (Phase A) and after oral ciprofloxacin pretreatment (Phase B) in a randomized crossover fashion with a 2-week washout period between each phase. During Phase B, ciprofloxacin pretreatment (750 mg every 12 hours) was administered for 5 days before and 24 hours after quinidine administration. Quinidine serum samples were obtained over a 24-hour period. QRS and QTc intervals were measured over a 12-hour period. There were no significant differences in clearance (20.3 +/- 3.3 L/hr vs 20.1 +/- 2.3 L/hr, P = .836), half-life (7.9 +/- 1 hr vs 7.8 +/- 0.8 hr, P = 0.8), maximum concentration (1.4 +/- 0.6 mg/L vs 1.5 +/- 0.6 mg/L, P = 0.613), or time to maximum concentration (1.5 +/- 0.2 hr vs 1.5 +/- 0.1 hr, P = 0.571) for quinidine between Phase A and Phase B, respectively. The largest decrease in clearance observed for Phase B compared to Phase A was 10%. There was also no significant difference in the degree of QRS and QTc prolongation between Phase A and Phase B. From these results, it appears that ciprofloxacin in the dose given does not alter the pharmacokinetic or ECG parameters of quinidine. Therefore, no adjustment in the dose of quinidine is needed when coadministered with ciprofloxacin.

Intralesional cyclosporine for psoriasis. Relationship of dose, tissue levels, and efficacy.

UNLABELLED: BACKGROUND AND DESIGN--To avoid systemic side effects, topical and intralesional administration of cyclosporine has been used; however, only intralesional administration has been successful. To understand more about the dosing requirements and resultant tissue levels of intralesional cyclosporine, we injected psoriasis plaques in a double-blind fashion with three different concentrations of cyclosporine (17 mg/mL in seven patients, 10 mg/mL in 13 patients, and 2.5 mg/mL in 11 patients) or matching vehicle three times weekly for 4 weeks. RESULTS--Statistically significant improvement was observed in plaques treated with 17 mg/mL (P = .003) compared with vehicle-treated plaques; the improvements in plaques treated with 10 mg/mL (P = .078) and 2.5 mg/mL (P = .054) achieved marginal statistical significance compared with vehicle treatment. Four weeks after discontinuation of therapy, the change from pretherapy in plaques that had received 17 mg/mL of cyclosporine was statistically significantly better (P less than .0001) than that with vehicle treatment. A similar finding but of marginal statistical significance (P = .059) occurred in the plaques that had received 10 mg/mL of cyclosporine. Throughout the study, untreated psoriasis plaques did not improve. Transient pain was the most common side effect noted with both cyclosporine and vehicle injections. Tissue levels of cyclosporine tended to be highest in plaques receiving the 17-mg/mL concentration; blood levels of cyclosporine were low throughout the study. CONCLUSIONS: --Intralesional cyclosporine requires a sufficient dosage to improve psoriasis, apparently by a local mechanism of action. Improvement may persist for 4 weeks or longer.

Special considerations for geriatric therapeutic drug monitoring.

Numerous physiological changes during the normal aging process can potentially affect how drugs are handled by the body. Gastrointestinal changes include increased gastric pH, decreased intestinal motility, and decreased blood perfusion. Age-related changes in body composition and protein concentrations in plasma contribute to alterations in the distribution of drugs. Hepatic metabolism of drugs may be affected, and renal excretion via glomerular filtration or tubular secretion is diminished. The importance of each of these physiological changes in the elderly, as well as the contribution of multi-drug therapy and other external factors, is discussed.

Determination of felbamate in human serum by high-performance liquid chromatography.

A rapid and sensitive high-performance liquid chromatographic method for the analysis of the antiepileptic felbamate in human serum has been developed. Following protein precipitation with acetonitrile containing the internal standard alphenal, the drugs are extracted from serum using dichloromethane. The extraction efficiency averages 98% for felbamate. Chromatography is performed with use of reversed phase octyl (C8) column, monitored at 210 nm. The isocratic mobile phase is acetonitrile:acetate buffer, pH 5.3, flow rate 2.0 ml/min. Felbamate is resolved from other common antiepileptic drugs that may be co-administered. The assay is linear to 200 micrograms/ml in serum. The effect of mobile phase pH changes on the chromatographic characteristics of the antiepileptic drugs and some potential interferents is discussed.

Bleach and LISS: a potential hazard.

Investigation into the corrosion of a copper alloy inlet port of a vacuum aspirator revealed an exothermic reaction between copper, glycine--the primary constituent of low-ionic-strength-saline (LISS)--and the widely used disinfectant sodium hypochlorite (common bleach). The apparatus had been used for several years to aspirate LISS-wash supernatants during the preparation of LISS-suspended red cells for antibody detection tests. A small blue-rimmed hole had formed through the side of the inlet port of the equipment, which had been rinsed with bleach during periodic cleansing. Our observations indicate a need for caution when using bleach to decontaminate waste or spills containing glycine. Contact of such materials with base metal parts of laboratory apparatus should be avoided, especially when copper is involved.

Comparison of cyclosporin G (Nva2-cyclosporin) concentrations measured in whole blood by monoclonal fluorescence polarization immunoassay, monoclonal radioimmunoassay, and HPLC.

Two commercial monoclonal immunoassays for monitoring cyclosporin A were used to measure whole-blood concentrations of the immunosuppressant cyclosporin G (CsG) in renal transplant patients. We performed a three-way comparison of these two immunoassays and HPLC. Although the two immunoassays agreed favorably, both the monoclonal fluorescence polarization immunoassay and the monoclonal RIA yielded higher CsG results for patients' specimens than did the liquid-chromatographic assay. The experimental data indicate that the observed differences are most likely due to the cross-reactivity of CsG metabolites in the immunoassays.

High-performance liquid chromatographic analysis of cyclosporin G (Nva2-cyclosporine) in human blood.

A sensitive high-performance liquid chromatographic method for the analysis of the immunosuppressant cyclosporin G (OG 37-325, Nva2-cyclosporine, CsG) in whole blood has been developed. Sample preparation, employing cyclosporin A (CsA) as internal standard, involves organic extraction with methyl t-butyl ether under sequential acidic and basic conditions. Chromatography is performed using a 2 mm inside diameter x 25 cm column packed with 5 microns octyl (C8) material. An isocratic mobile phase comprised of acetonitrile:methanol:water, at a flow rate of 0.4 ml/min, is utilized. Separation is monitored at 230 nm. Data are also presented that demonstrate the use of CsG as an alternative internal standard to cyclosporin D for liquid chromatographic determinations of CsA.