Henning Breithaupt

Enzymatic characteristics of crotalus phospholipase A2 and the crotoxin complex

Abstract H. Breithaupt . Enzymatic characteristics of crotalus phospholipase A 2 and the crotoxin complex. Toxicon 14, 221–233, 1976.— Crotalus durissus terrificus phospholipase A, a component of the crotoxin complex, is of the A 2 type. The enzyme hydrolyzes aqueous dispersions of egg yolk lipoproteins with zero order kinetics. Optimum activity occurs at 53–57°C and pH 7·0–8·0. The rate of enzymatic attack on monomeric substrates is negligible. Rapid hydrolysis is observed when the substrate is above the critical micelle concentration. The enzymatic phospholipase activity is 1100 μmoles per min and mg enzyme using aqueous solutions of diheptanoyllecithin, containing 50 mM Ca 2+ , or egg yolk emulsions, containing 400 mM Ca 2+ as substrate. Crotalus phospholipase A 2 requires Ca 2+ for catalytic activity. The activity of the enzyme is also enhanced by Na + , K + , Mg 2+ , and deoxycholate. Zn 2+ , Cu 2+ , Na 2 -EDTA, phosphate, oxalate, sulfide, cyanide, citrate, fatty acids, and lysolecithin inhibit the enzyme. Low concentrations of Ba 2+ increase the enzymatic activity, whereas high concentrations inhibit the enzyme. Crotalus phospholipase A 2 is an extremely stable enzyme, resisting heating (85°C), lyophilization, repeated thawing, urea (6 M), and acidic solvents such as 10% formic acid. The enzyme is inactivated by exposure to pH values higher than 9·0 or by contact with cellophane membranes. Crotapotin, the second component of the crotoxin complex, has no phospholipase activity, but inhibits the crotalus phospholipase A 2 . Three moles of crotapotin decrease the activity of 1 mole of phospholipase A 2 to a residual activity of 10%, independent of the pH range or preincubation time used. The inhibition can be prevented by deoxycholate or by Ca 2+ , but not by high concentrations of NaCl or KCl. In egg yolk suspension, hydrolysis by the phospholipase-crotapotin complex occurs after a lag period. The duration of latency depends on the amounts of crotapotin added to the enzyme.

Clinical results and pharmacokinetics of high-dose cytosine arabinoside (HD ARA-C).

Four patients with acute nonlymphoblastic leukemia and one malignant teratoma refractory to conventional chemotherapy were treated with high doses of cytosine arabinoside (HD ARA‐C). They received up to 12 cycles of 1.8 to 3 g/m2 every 12 hours applied by 2‐hour infusions. A total of 55 HD ARA‐C infusions was performed. All leukemic patients responded. A complete clearance of blasts from the bone marrow was observed in two patients following 8–12 cycles of 3 g/m2. However, relapses occurred after three and seven weeks, in one case with resistance to HD ARA‐C. The patient with malignant teratoma did not respond. No severe toxicity emerged even after repeated applications. Adverse reactions included moderate nausea and vomiting (4 patients), diarrhea (2 patients), hepatic dysfunction (1 patient), bone pain (1 patient), blurred vision (1 patient), conjunctivitis (1 patient), and exanthema with partial epidermiolysis (1 patient). Granulocytopenia occurring between 3–8 days after having started the therapy, subsided within 4–25 days. Plasma levels of ARA‐C and the metabolite uracil arabinoside (ARA‐U) were monitored. At steady state plasma concentrations of ARA‐C were 32–97 μM (8–24 μ/ml). ARA‐C disappeared from the plasma mono‐ or biphasic with a terminal half‐life (t50%) of 7.8–12.6 minutes. The total clearance (Cl) or ARA‐C varied between 1.7 and 2.9 liters/kg · h, and the distribution volume (Vss) between 0.44 and 0.86 liters/kg. Cerebrospinal fluid (CSF) levels of ARA‐C reached 10–15% of steady state concentrations in plasma.

Neurotoxic and myotoxic effects of crotalus phospholipase A and its complex with crotapotin

Summary1.The acidic peptide crotapotin potentiated the toxicity of the basic crotalus phospholipase A in all species tested. The order of sensitivity to the lethal action of the phospholipase was: chick > mouse ≧ rabbit > rat. After a latency period of at least 20 min the animals died of respiratory paralysis. Rabbits survived for more than 10 h, if they were artificially respired. The animals recovered very slowly from respiratory depression.2.In rabbits even high doses of the basic crotalus phospholipase A or its complex with crotapotin1 did not affect the respiratory center nor its reactivity to asphyxia. Conduction of action potentials in the phrenic nerve was not changed.3.Phospholipase-crotapotin complexes decreased the contractile response of isolated phrenic-hemidiaphragms of rats to direct and indirect stimulation in an irreversible manner. A latency period of 20–100 min preceded the paralysis.4.A similar block of neuromuscular transmission developed in vivo. After i. v. injection of PC-complexes the contractile response of isolated rat phrenic diaphragms to nerve stimulation was considerably lower than to direct stimulation.5.Crotalus phospholipase A alone as well as its complex with crotapotin reduced the contractions of the isolated chick biventer cervicis muscle but did not cause a contracture, thus indicating that the phospholipase and PC-complexes act not as depolarizing blockers.6.Blood pressure, heart rate and electrocardiogram were not substantially altered, when phospholipase A or PC-complex were injected slowly i.v. into rabbits or rats. No cardiotoxic effect was observed in the Langendorff preparation of rat hearts perfused with phospholipase A alone (6×10−6 M) or together with crotapotin (10−5 M). Rapid i.v. injection of the venom produced hypotension. The degree of haemolysis correlated with the enzymatic activity, and was low for the highly toxic PC-complexes.

Determination of cytarabine and uracil arabinoside in human plasma and cerebrospinal fluid by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of the antineoplastic agent cytarabine and its main metabolite uracil arabinoside in human plasma and cerebrospinal fluid is described. Complete separation from endogenous constituents was achieved by isocratic reversed-phase chromatography using phosphate buffer (0.05 M, pH 7.0) as the eluent. The limit of detection was 50 ng/ml. Day-to-day coefficients of variation were below 10%. The applicability of this rapid, simple and specific method for pharmacokinetic studies and monitoring of therapy was demonstrated.

Rapid high-pressure liquid chromatographic determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino-N10-methylpteroic acid in biological fluids

Abstract A rapid and sensitive high-pressure liquid chromatographic method for determination of methotrexate and its metabolites 7-hydroxymethotrexate and 2,4-diamino- N 10 -methylpteroic acid has been developed. The assay is based on isocratic reversed-phase chromatography with siliceous microparticulate Spherisorb (5 μm, ODS, 15 × 0.4 cm i.d.) as stationary phase and a ternary solvent mixture of citrate-phosphate (0.05 m , pH 3.2)/methanol/tetrahydrofurane (80:16:4, v/v) as eluant. A precolumn of Perisorb (RP2, 30–40 μm, 3 × 0.4 cm i.d.) reasonably protects the analytical column against deterioration by the components of plasma or other biological fluids. Since the samples of plasma, urine, or cerebrospinal fluid are directly injected into the chromatographic system, the method is very rapid. Within 8 min as little as 50 ng of methotrexate and its metabolites per milliliter (10 −7 m ) can be measured with a precision better than 7%. Structural analogs of methotrexate do not interfere with the determination. There is a good correlation with the results of other methods, e.g., enzyme immunoassay or radioimmunoassay. The applicability for clinical monitoring in patients plasma and urine is demonstrated.

Simultaneous quantification of cefotaxime, desacetylcefotaxime, ofloxacine and ciprofloxacine in ocular aqueous humor and in plasma by high-performance liquid chromatography

Cefotaxime, given intravenously, is currently used as a broad-spectrum antibiotic for prophylaxis of intra- and postoperative infections in ocular lens surgery. A proposed therapeutic and economic alternative is the use of orally active fluoroquinolone ofloxacine as prophylactic agent. A HPLC method was developed for determination of both antibiotics in ocular aqueous humor and plasma in order to optimize dosage for safe surpassing minimal inhibitory concentration in the humor compartment. For plasma determinations a solid-phase extraction procedure was used with ciprofloxacine as internal standard. Detection limits for direct HPLC-analysis of ocular aqueous humor was 0.08 microg/ml for all compounds, whereas in plasma 0.31 microg/ml could be determined after solid-phase extraction.

Determination of mexiletine in biological fluids by high-performance liquid chromatography

A high-performance liquid chromatographic method for the determination of the anti-arrhythmic drug mexiletine in human plasma, urine, and cerebrospinal fluid is described. Following extraction with diethyl ether, mexiletine and the internal standard 4-methylmexiletine were derivatized with 2,4-dinitrofluorobenzene. Analyses were performed using an alternating on-column enrichment technique on small Perisorb RP-2 30-40 micrometers pre-columns with pre-column backflushing for direct injection onto the analytical column of Spherisorb ODS 5 micrometers. Complete separation from endogenous constituents of plasma, urine or cerebrospinal fluid was achieved by isocratic reversed-phase ion-pair chromatography with 1-heptanesulfonic acid (0.005 M; PIC B7)-acetonitrile-tetrahydrofuran (42:48:10, v/v) as eluent. Interferences from other drugs were not observed. The limit of detection was 10 ng/ml (C.V. 6.5%). Day-to-day coefficients of variation were below 9%. The applicability of this rapid method for pharmacokinetic studies and clinical routine is demonstrated.

High-Dose Methotrexate for Osteosarcoma: Toxicity and Clinical Results

9 patients with osteosarcoma were treated with a total of 122 infusions of high-dose methotrexate (MTX; 140-350 mg/kg) followed by leucovorin rescue. Plasma kinetics of MTX and 7-hydroxymethotrexate (7-OH-MTX) has been routinely monitored. Due to inadequate hydration and alkalinization, 1 of the 122 high-dose MTX infusions was followed by delayed disappearance of MTX and 7-OH-MTX from plasma with subsequent development of severe mucositis. Serious hepatotoxicity repeatedly developed in another patient with inconspicuous MTX kinetics. The benefit of monotherapy with high-dose MTX for adjuvant treatment of osteosarcoma remains questionable, since 6 of 8 patients with primary osteosarcoma developed pulmonary metastases within 4-12 months (median 5 months), 2 have been disease-free and alive for 25 and 53 months.

Kinetics of oral and intravenous mexiletine: lack of effect of cimetidine and ranitidine

SummaryThe pharmacokinetics of mexiletine, a Class I antiarrhythmic drug, was investigated in 6 healthy volunteers after single oral doses and 15 min intravenous infusions of 3 mg/kg. Cimetidine and ranitidine are commonly used H2-receptor antagonists, which interact adversely with many drugs, e.g. inhibition of the metabolism of Class I antiarrhythmics such as lidocaine and quinidine by cimetidine. To investigate the effects of the two drugs on the kinetics of mexiletine, cimetidine 800 mg·day−1 or ranitidine 600 mg·day−1 were administered orally for one week.Neither H2-receptor antagonist altered the distribution and elimination of mexiletine, nor did they affect its overall kinetics, or excretion of the metabolites para- and 4-OH-methylmexiletine after oral and intravenous administration of mexiletine.

Quantification of pyrazinamide and its metabolites in plasma by ionic-pair high-performance liquid chromatography. Implications for the separation mechanism

Pyrazinamide, the amide of pyrazinoic acid, is one of the basic therapeutic agents currently used in combination for chemotherapy of tuberculosis. A reversed-phase high-performance liquid chromatography method based on ionic pair chromatography, was developed after solid-phase extraction of the analytes from plasma with prior addition of internal standard. The main metabolites, pyrazinoic acid, 5-hydroxypyrazinoic acid and 5-hydroxypyrazinamide, were included as well as uric acid and other purine derivatives to allow detailed study of the pharmacokinetics of the drug, especially in patients with impaired kidney function. Some interesting features of the chromatographic system giving some insight in the retention mechanism and of the solid-phase extraction are discussed in detail.