Clinical pharmacology of polyethylene glycol-L-asparaginase

Polyethylene glycol (PEG)-L-asparaginase, at doses ranging from 500 to 8000 units/m2, was infused iv over 60 min in 31 patients of whom 27 were evaluable pharmacokinetically. The plasma disappearance of PEG-L-asparaginase is described by a monophasic curve with a mean half-life of 357 +/- 243 hr which is much longer than that of the unconjugated enzyme (half-life of approximately 20 hr). The rate of total clearance (128 +/- 74 ml/m2 X day) is much slower than that of L-asparaginase (2196 +/- 1098 ml/m2 X day). The volume of distribution is 2093 +/- 643 ml/m2, which is similar to that of L-asparaginase, indicating that PEG-L-asparaginase is mainly localized in the plasma. No enzyme could be measured in urine samples taken from nine patients for a period of up to 4 days. Additionally, no enzyme was measurable in one patient's pleural fluid obtained at the end of infusion and 6 days after infusion of a 1000-unit/m2 dose; the corresponding concentrations in plasma were 0.64 and 0.62 units/ml, respectively. In general, the plasma enzyme concentrations at the end of the 1-hr infusion and at 14 days after drug administration were proportional to the dose given. However, in two patients, a sudden disappearance of enzyme levels occurred which preceded anaphylactic reactions during subsequent treatment. A third patient developed severe bronchospasm 30 min after the first dose, but his enzyme levels were within the normal range.     

Kinetic properties and inhibition of Acinetobacter glutaminase-asparaginase

Kinetic parameters, substrate specificity and exclusivity of ligands at binding sites of L-glutaminase-L-asparaginase purified from Acinetobacter glutaminasificans were studied in order to gain knowledge about the dual activities of this enzyme and its inhibition by structural analogs. Both L-glutamine and L-asparagine, which showed similar Km (4 approximately 7 X 10(-5) M) and Vmax (molecular activity 1.0 min-1) values, were competitive with each other for the substrate binding site. The products, L-glutamic acid and L-aspartic acid, showed competitive inhibition with respect to either L-glutamine or L-asparagine as substrates. Multiple inhibition of the glutaminase activity by L-glutamic acid and L-aspartic acid indicated that these ligands are mutually exclusive at the product-releasing site. The initial rates of both of the enzyme's activities were competitively inhibited by the following inhibitors (in rates of both of the enzyme's activities were competitively inhibited by the following inhibitors (in decreasing order of activity): 6-diazo-5-oxo-L-norleucine (DON), L-methionine sulfoximine, azaserine, and Acivicin. DON and azaserine inhibited both the asparaginase and glutaminase activities in a time-dependent and irreversible manner. The kinetic data suggest an ordered mechanism with glutamine or asparagine as the first substrate and glutamic acid or aspartic acid, respectively, as the last product. These results also suggest that a single mechanism and a single set of binding sites are responsible for catalyzing both of the enzyme's activities. The data also showed that succinylated enzyme, which has a 10-fold increase of plasma half-life in animals and humans and, thus, has benefit as a cancer chemotherapeutic agent, retained its catalytic activity and maintained Km and Vmax values similar to the native enzyme.     

Brain asparaginase, ACE activity and plasma cortisol level in morphine dependent rats: effect of aspartic acid and naloxone

The activities of the brain L-asparaginase and angiotensin converting enzyme (ACE), and the plasma cortisol level were found to be decreased in the rats implanted with morphine (M) containing pellets. Even though 10 mg/kg of naloxone (N) itself showed an inhibitory effect on ACE it abolished the inhibitions seen in the M dependent rats five min following subcutaneous injection. The chronic administration of L-aspartic acid (ASP) during the development of physical dependence or just before the N injection prevented the increase of the plasma cortisol caused by N. It is concluded that in addition to the inhibition of the brain L-asparaginase activity which was previously hypothesized to be the main reason of the development of physical dependence on opiates as a result of the related experimental and clinical data, the inhibition by M of the brain ACE activity may take part in the development of physical dependence. With regard to the plasma cortisol level, the concomitant administration of ASP with M blocks, to a great extent, the development of physical dependence on opiate. The single dose of ASP administration before N injection prevents the effect of N, the manifestation of abstinence syndrome.     

Serious neutropenia in ALL patients treated with L-asparaginase may be avoided by therapeutic monitoring of the enzyme activity in the circulation

The antineoplastic enzyme L-asparaginase is commonly used for the induction of remission in acute lymphoblastic leukemia (ALL). L-Asparagine is an essential amino acid for many lymphoid tumor cells and L-asparaginase catalyzes its conversion to L-aspartic acid and ammonia. The dosage of this highly toxic drug is individualized based on the body surface area of the patient, but monitoring of L-asparaginase activity during the L-asparaginase therapy is not commonly used. We measured L-asparaginase activity in the serum of ten children (aged 3-13 y) with ALL (ALL NOPHO-92 standard or intermediate risk groups) during their L-asparaginase therapy. L-asparaginase was given 30,000 IU/m2 IM during days 37-46 of the induction therapy and no other chemotherapeutic drug except for prednisone was given at the same time. We observed that this dosage schedule resulted in almost 6-fold differences in the serum activity of L-asparaginase between the patients. There was also a relationship between the area under the L-asparaginase activity-time curve (AUC) and even peak L-asparaginase activity in serum during the enzyme therapy and neutropenia after the therapy in the patients: the higher the AUC or peak value was, the more severe was the neutropenia in the patients after treatment. Monitoring L-asparaginase in serum could be useful in optimization of the therapy with this toxic drug.     

Acrylic microspheres in vivo VI: antitumor effect of microparticles with immobilized L-asparaginase against 6C3HED lymphoma

The antitumor effect of immobilized L-asparaginase was tested against lymphoid leukemia in mice with concomitant scanning of the L-asparagine level in serum. L-Asparaginase was immobilized in microspheres of polyacrylamide or polyacryldextran. These particles were used in C3H mice bearing the L-asparagine-dependent lymphoma (6C3HED). The tumor was maintained as an ascites tumor, 1 X 10(6) cells were injected intraperitoneally and on day 4 after inoculation, L-asparaginase was injected intramuscularly or intraperitoneally in microparticles. After injection of 5.0 IU ip of L-asparaginase in microparticles, partial remission was induced, generally, however, the cancer relapsed and killed the mice within 2-3 weeks. To obtain complete regression, it was necessary to inject 20 IU of L-asparaginase in microparticles intraperitoneally. The best therapeutic effect was obtained when the particles were administered intramuscularly. After injection of 5 IU the survival time was prolonged, but complete regression was not achieved. The best effect was obtained when the particles were given intramuscularly in two small doses (2.5 IU) at a 3-day interval. Such treatment induced complete regression; 10 out of 12 treated mice were completely cured and lived for several months. It is concluded that the L-asparagine level in serum has to be depressed to less than 20% of the normal level for at least 6-7 days to obtain complete regression of the tumor.     

Monitoring of Erwinia asparaginase therapy in childhood ALL in the Nordic countries

AIMS: Evaluation of L-asparaginase therapy in the NOPHO-92 ALL-protocol (treatment protocol of acute lymphoblastic leukaemia of the Nordic Society of Paediatric Haematology and Oncology, initiated in 1992) after intravenous and intramuscular administration of Erwinia asparaginase during induction and re-induction therapy. METHODS: Forty children with newly diagnosed acute lymphoblastic leukaemia received Erwinia asparaginase (30 000 IU/m2 i.v. or i.m.) during induction therapy (every day for 10 days), and 19 children received Erwinia asparaginase (30 000 IU/m2 i.v. or i.m.) during re-induction therapy (twice a week for 2 weeks). Within the treatment periods asparaginase trough activity (using a spectrophotometric assay) was determined on specific days. The goal of therapy is complete L-asparagine depletion, which asparaginase activities above 100 IU l(-1) have been shown to ensure. Therefore determination of L-asparagine (using a h.p.l.c. method) was performed only in plasma samples with asparaginase activities below 100 IU l(-1). RESULTS: During induction therapy 92.2% of the trough enzyme activities were above 500 IU l(-1) for the i.v.-treated patients, and 92.4% of the trough enzyme activities were above 500 IU l(-1) for the i.m.-treated patients. During re-induction therapy 64.7% of the trough enzyme activities were below 100 IU l(-1) in the i.v.-treated group, and 73.3% of the trough enzyme activities were below 100 IU l(-1) in the i.m.-treated group. For trough enzyme activities below 100 IU l(-1) L-asparagine depletion was complete in two thirds of the samples. CONCLUSIONS: In the NOPHO-92 ALL-protocol L-asparaginase treatment during induction therapy was unnecessarily intense, but during the re-induction phase it appeared inadequate.     

Pegaspargase: a review of clinical studies

The chemotherapy agent L-asparaginase has been an important part of acute lymphoblastic leukemia therapy for over 30 years. Two of the main disadvantages of the drug are (1) the need for frequent intramuscular injection and (2) a very high rate of allergic reactions. Because of this, L-asparaginase seemed like an ideal target for pegylation and PEG-L-asparaginase was developed in the 1970s and 1980s. The drug has undergone extensive testing and appears to retain its antileukemic effectiveness while allowing less frequent administration than the native compound. While the actual cost to patients for PEG-L-asparaginase is greater than that of multiple injections of other L-asparaginases, the reduced need for physician visits and treatment of complications of therapy may make overall treatment costs considerably less than that of the conventional L-asparaginases. In the review below, we outline the history of therapy with L-asparaginase, the development of PEG-L-asparaginase, and clinical trials in which it has been administered.     

Expanding targets for a metabolic therapy of cancer: L-asparaginase

The antitumour enzyme L-asparaginase (L-asparagine amidohydrolase, EC 3.5.1.1, ASNase), which catalyses the deamidation of L-asparagine (Asn) to L-aspartic acid and ammonia, has been used for many years in the treatment of acute lymphoblastic leukaemia. Also NK tumours, subtypes of myeloid leukaemias and T-cell lymphomas respond to ASNase, and ovarian carcinomas and other solid tumours have been proposed as additional targets for ASNase, with a potential role for its glutaminase activity. The increasing attention devoted to the antitumour activity of ASNase prompted us to analyse recent patents specifically concerning this enzyme. Here, we first give an overview of metabolic pathways affected by Asn and Gln depletion and, hence, potential targets of ASNase. We then discuss recent published patents concerning ASNases. In particular, we pay attention to novel ASNases, such as the recently characterised ASNase produced by Helicobacter pylori, and those presenting amino acid substitutions aimed at improving enzymatic activity of the classical Escherichia coli enzyme. We detail modifications, such as natural glycosylation or synthetic conjugation with other molecules, for therapeutic purposes. Finally, we analyse patents concerning biotechnological protocols and strategies applied to production of ASNase as well as to its administration and delivery in organisms.     

Metabolic fate of carteolol hydrochloride (OPC-1085), a new beta-adrenergic blocking agent. (6) Pharmacokinetics of carteolol in the rat, dog, rabbit and man]

The kinetics (absorption, distribution and excretion) of carteolol were investigated after oral and intravenous administration to man, rats, Beagle dogs and rabbits. The half-life of carteolol in plasma was 1.22 approximately 1.45 hr in rats, 1.73 approximately 2.08 hr in dogs and 1.42 approximately 1.43 hr in rabbits, and was independent of the route of administration. The absorption rate constants, obtained from log(C1-C) approximately time plot, after oral administration were 1.89 hr-1 in rats, 1.04 hr-1 in dogs and 1.54 hr-1 in rabbits. There were no differences between tablet and film coated tablet in the pharmacokinetic parameters of carteolol in man after oral 30 mg (tablet or film coated tablet) administration [half life (t1/2)=4.50 hr (tablet), 4.30 hr (film coated tablet), elimination rate constant (k2) equals 0.154 hr-1 (tablet), 0.161 hr-1 (film coated tablet)]. The elimination rate constant, obtained from Sigma-minus plot after 2, 5 and 10 mg oral administration, was 0.137 approximately 0.160 hr-1.     

PEG-L-天冬酰胺酶的临床研究近况

综述近年来对抗肿瘤药物PEG-L-天冬酰胺酶的临床研究进展,讨论其在急性淋巴细胞白血病治疗中的用药方案和疗效,抗药性以及安全性。PEG-L-天冬酰胺酶由E.coliL-天冬酰胺酶经聚乙二醇共价修饰而得,其免疫原性降低,且药物半衰期延长。     

Effect of adrenaline on the response of erythrocyte 2,3-diphosphoglycerate in rabbits in vivo

• 1.1. A 6 hr time-course response of erythrocyte 2, 3-diphosphoglycerate was studied in rabbits following adrenaline administration.
• 2.2. Eight female New Zealand white rabbits weighing about 3.6 Kg each were injected intra-peritoneally with a total of 0.97 mg/kg of adrenaline (0.56 mg/kg at time 0 min and 0.41 mg/kg at time 0.5 hr), and the venous level of red blood cell (RBC) 2,3-DPG was monitored at 0 hr, 1 hr, 3 hr, and 6 hr, respectively. As controls, the level of 2,3-DPG was also monitored in these rabbits weeks prior to the experiment.
• 3.3. A significant (p<0.05) rise in the mean level of 2.3-DPG (μmol-ml⁻¹ RBC) was reached 3 hr after the initial injection of adrenaline, and the level returned to the preexposure level by the end of 6 hr.
• 4.4. It is speculated that adrenaline may be one of the contributors that increases the level of 2,3-DPG during the resting period following exhaustive exercise because this catecholamine has been reported to increase following this type of hypoxia.

     

Comparison of the suppressive effects of L-aspartic acid and chlorpromazine + diazepam treatments on opiate abstinence syndrome signs in men

According to the hypothesis implying that the main reason of physical dependence on opiate is the inhibition of brain L-asparaginase activity and L-aspartic acid gradually decreases compulsory opiate intake so that physical dependence disappears by itself, 31 opiate addicts were given 8 g L-aspartic acid for 7 days after withdrawal from opiate and appearance of abstinence syndrome signs. The attenuations by L-aspartic acid of the abstinence syndrome signs were statistically compared with those obtained from other 12 opiate addicts received daily 50 mg chlorpromazine + 60 mg diazepam which have long been used to suppress abstinence syndrome because of their multiple receptor blocking and sedative effects. The intensity and duration of 13 signs out of 16 ones were found to be significantly more alleviated and shortened in the addicts treated with L-aspartic acid.     

Formation and disposition of 7-hydroxymethorexate in rabbits

The metabolism of methotrexate in rabbits was investigated following 6 hr of infusion with [3′,5′,7′-³H]methotrexate (50 mg/kg). Methotrexate and its metabolites were analyzed by an enzyme kinetic method, reverse phase high-performance liquid chromatography, and scintillation counting of radioactivity. 7-Hydroxymethotrexate was found to be a major metabolite in plasma, urine, bile and various tissues of rabbits. Cumulative production of 7-hydroxymethotrexate during the first 6 hr was 31.8% of the total dose. The peak 7-hydroxymethotrexate concentration in plasma was reached at the end of the infusion, and this concentration was higher than the methotrexate concentration. The plasma clearance of 7-hydroxymethotrexate was biphasic in nature and slower than that of methotrexate. The highest metotrexate concentration was found in kidney after 6 hr. This concentration was found to be 7.2 times greater than that in plasma. A higher ratio of 7-hydroxymethotrexate/methotrexate was found in liver, small intestine, kidney and testis when compared to plasma. Lung and kidney showed significant conversion of methotrexate to 7-hydroxymethotrexate in vitro, as did the liver.     

Serum methanol concentrations in rats and in men after a single dose of aspartame

Serum methanol concentrations were measured in rats and in humans given oral aspartame. The dose given to rats was the FDA's projected 99th percentile daily intake for humans, assuming aspartame were to replace all sucrose sweeteners in the diet (34 mg/kg). Four male adult volunteers each received 500 mg, equivalent to 6-8.7 mg/kg, which is approximately the FDA's estimate of mean daily human consumption. Both treatments caused a rise in serum methanol. In rats the mean peak value was 3.1 mg/litre 1 hr after administration; serum methanol returned to endogenous values 4 hr after treatment. In the men, the mean rise over endogenous values was 1.06 mg/litre after 45 min. Two hours after treatment, serum methanol had returned to basal levels. The temporary serum methanol increase showed peak values within the range of individual basal levels.     

Acrylic microspheres in vivo V: Immunological properties of immobilized asparaginase in microparticles

L-Asparaginase was immobilized in microparticles of polyacrylamide. Such particles were then injected by intramuscular/subcutaneous, intraperitoneal, or intravenous routes into mice to investigate the immunological consequences of the immobilization. Entrapment of L-asparaginase in microparticles did not prevent the formation of antibodies in intensively treated animals. Intraperitoneal and intravenous injections of particles produced significantly higher antibody levels than soluble L-asparaginase. Antigen administered intramuscularly/subcutaneously in microparticles elicited, however, a weak immune response. Dependent on the route of administration, the particles may thus function as an adjuvant. A modified Arthus reaction in the foot pads of immunized mice indicated that antigenicity decreased when L-asparaginase was immobilized in microparticles. Injection of free L-asparaginase, intramuscularly/subcutaneously (2 x 20 IU) in the preimmunized mice produced no effects on the serum level of L-asparagine, whereas intramuscular/subcutaneous injection of L-asparaginase in microparticles produced a depression of the serum concentration. It is concluded that the intramuscular/subcutaneous injection of L-asparaginase in microparticles is the choice route of administration with respect to duration and the immunological reaction.     

In vivo 31P NMR studies of the hepatic response to L-ethionine in anesthetized rats

Phosphorus-31 surface coil spectroscopy has been used to study the effects of L-ethionine administration on the hepatic metabolism of the anesthetized Sprague-Dawley rat. ATP levels were found to decrease by approximately 30% 3 to 4 hr after administration of 1 mg/g body wt of L-ethionine to the anesthetized rat by gastric gavage. ATP levels returned to control values approximately 8 hr postadministration. The relatively small decrease in ATP level was confirmed by extraction and conventional enzyme assay and is a consequence of the mode of administration of the ethionine. Hepatic inorganic phosphate levels rose concomitantly with the ATP fall. There were no significant changes in either cellular pH or Mg2+ levels as monitored by the 31P shifts of sensitive metabolites. In vivo 31P NMR spectroscopy provides a promising approach to study the effects of hepatotoxicants on cellular ATP, pH, and Mg levels.     

Depletion and replacement of the adrenaline and noradrenaline contents of the rat adrenal gland, following treatment with reserpine

The effect of reserpine on the adrenaline and noradrenaline contents of rat adrenal glands has been studied. Given subcutaneously to male albino Wistar rats in 3 daily doses of 1 mg/kg, it caused, 24 hr after the final dose, a similar percentage loss of adrenaline and noradrenaline of approximately 50%. Subsequently the adrenaline content slowly increased, until at 14 days from the begining of the experiment there was no significant difference between the reserpine-treated and the control glands. In contrast, the recovery of the noradrenaline content was rapid, exceeding the control value at 7 days by approximately 250%. This increased content of noradrenaline declined to the normal level by 21 days. The total amine content returned to normal by 7 days and remained at this level subsequently. The effect of re-depleting the glands of their amine content was investigated. It was found that re-depletion at 7 days caused a preferential release of noradrenaline, followed at 14 days by a peak of noradrenaline at least as high as that obtained following the initial depletion only. Re-depletion at 21 days caused an effect similar to that obtained initially. Denervation of the left adrenal gland did not alter the degree of depletion caused by reserpine, nor did it alter the subsequent replacement of the amines as compared with that in the innervated right gland.     

Enhancing effect of bromovinyldeoxyuridine on antitumor activity of 5'-deoxy-5-fluorouridine against adenocarcinoma 755 in mice. Correlation with pharmacokinetics of plasma 5-fluorouracil levels

5'-Deoxy-5-fluorouridine (DFUR), whether or not combined with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) was pursued in BDF1 mice from both a pharmacokinetic viewpoint, following a single oral dose administration, and an anticancer viewpoint, following 5 daily oral doses in mice inoculated subcutaneously with adenocarcinoma 755 tumor cells. Half-life (t1/2) values for the elimination of DFUR and 5-fluorouracil (5-FU) from plasma following DFUR (100 mg/kg) administration were about 0.80 and 0.39 hr, respectively. Plasma 5-FU AUC (area under the curve) values following oral DFUR (100 mg/kg) was 0.224 micrograms.hr/ml. If DFUR (100 mg/kg) was combined with BVDU (10 mg/kg) the t1/2 and AUC values for 5-FU increased from 0.39 to 1.24 hr, and from 0.224 to 1.699 micrograms.hr/ml, respectively. Thus, BVDU significantly increased the plasma levels of 5-FU. It had no effect on the plasma levels of DFUR. At 100 mg/kg, DFUR did not show a significant antitumor activity. At 500 mg/kg it effected a 90% inhibition in tumor growth. When combined with BVDU (10 mg/kg), DFUR at 100, 200 and 300 mg/kg reduced tumor growth by 96, 100 and 100%, respectively. The antitumor activity achieved by DFUR, in the presence or absence of BVDU, correlated highly significantly with the AUC values for plasma 5-FU.     

Dose-response and time-response biochemical and histological study of potassium dichromate-induced nephrotoxicity in the rat

This study provides quantitative toxicological data on potassium dichromate-induced renal damage and considers the possible difficulties arising from the non-invasive in vivo assessment of renal damage, with particular attention to enzymuria. Renal damage induced in male Wistar rats by single sc injections of potassium dichromate was assessed 52 to 72 hr after doses ranging from 3 to 20 mg potassium dichromate/kg body weight and throughout a 9-day period following a dose of 20 mg potassium dichromate/kg. The earliest and most sensitive non-invasive functional change in the dose-response and time-response studies was an elevation in the rate of urinary excretion of protein. Evidence of tissue damage was observed with elevations in the urinary excretion rates of the brush border enzymes, gamma-glutamyltransferase, alkaline phosphatase and leucine aminopeptidase, the cytosolic enzymes, aspartate aminotransferase and lactate dehydrogenase and the lysosomal enzyme, N-acetyl-beta-D-glucosaminidase. Such changes occurred as early as the abnormal urinary protein excretion, but returned to control or sub-control values sooner. Urinary brush border enzyme excretion returned to control values within 48 hr following potassium dichromate injection, despite histological and histochemical evidence of extensive renal damage and renal dysfunction. Elevations in plasma aspartate aminotransferase and lactate dehydrogenase levels were observed, but histochemical and isoenzyme studies would be needed to determine the source of these increases. The simplest and most persistent indicators of renal damage were the urinary excretion of protein and N-acetyl-beta-D-glucosaminidase.     

Pharmacokinetics of the Aldose Reductase Inhibitor Imirestat Following Topical Ocular Administration

The pharmacokinetics of imirestat following topical ocular administration were evaluated in a series of studies in rabbits and dogs. Following single topical doses to both albino and pigmented rabbits, imirestat was subject to rapid uptake into the cornea followed by an initial rapid decline and then very slow elimination, with a t _1/2 of approximately 130 hr. Drug was rapidly absorbed into aqueous humor, with concentrations declining to nondetectable levels by 12 hr. Imirestat was retained in the lens following topical dosing similar to that in cornea, with an apparent elimination t _1/2 of 140 hr. Vitreous humor concentrations of drug were detectable for up to 72 hr after dosing. There was no apparent difference in the disposition of the drug between albino and pigmented rabbits. Bioavailability following topical dosing increased with dose, although not in a linear fashion. Formulation pH did not have an appreciable effect on ocular bioavailability. There was detectable systemic absorption following topical dosing, with plasma concentrations in rabbit being 50 to 75% of that observed following an equivalent intravenous dose. However, drug levels in the dosed eyes were significantly higher than in contralateral undosed eyes. Multiple dosing of imirestat for 6 weeks resulted in accumulation of drug in rabbit lens. Concentrations were higher in lens cortex than lens nucleus, with the time course for accumulation being different for the two. Our data suggest that imirestat penetrates into ocular tissue following topical dosing and is retained in lens and cornea, potential sites of action for the drug.