L-Asparaginase has been encapsulated in Swiss mouse or human erythrocytes by hypotonic haemolysis followed by isotonic resealing and reannealing. The details of incorporation and properties of carrier erythrocytes are presented. When L-asparaginase loaded into 51Cr-labelled erythrocytes, was infused intravenously, the same half-life was found for asparaginase and 51Cr. In addition, L-asparaginase loaded into erythrocytes was much more effective in eliminating plasma asparagine compared with the same dose of free L-asparaginase injected in solution, during a sustained period (14 days).

Rightward shifts of the O2 dissociation curve (ODC) were experimentally obtained in lysed and resealed erythrocytes following encapsulation of inositol hexaphosphate (IHP). This continuous lysing and resealing procedure led to in vitro P50 (Po2 at 50% hemoglobin saturation) increases up to 80 Torr (pH, 7.40; Pco2, 40 Torr; temp, 37 degrees C) for both human and pig erythrocytes. The Hill number of the transformed blood decreased when IHP was fixed on the hemoglobin, but the sigmoid shape of the ODC was maintained. The O2 hemoglobin binding capacity and the mean corpuscular hemoglobin content were found unchanged by the experimental procedure in human and pig erythrocytes. Isovolumic exchange transfusion of high-P50 erythrocytes in anesthetized and ambient air-ventilated piglets (n = 6) led to substantial in vivo P50 increases (range, 8-19 Torr). The rightward shift of the ODC was concomitant with an increase of the arterial Po2 and of the arteriovenous O2 content difference, 19 and 59% respectively above their control values. The mixed-venous Po2 (PVO2) remained unchanged. The cardiac output was shown to be inversely related to the P50 value. In spite of the O2-transport reduction (37%), O2 consumption was maintained due to enhanced O2 extraction.

This study opens new perspectives for the clinical utilisation of L-asparaginase. This mode of administration of the drug is able to improve pharmacodynamic parameters and enzymic efficacy and to increase the general tolerance of the treatment.

The study of the alpha-N-acetylgalactosaminyltransferase in the sera of 19 individuals belonging to the rare Am blood group makes it possible to confirm the heterogeneity of this phenotype established on genetical and immunological criteria. Two groups of subjects, Am and Ay, can be distinguished. For the individuals of the first group, named Am, 15 samples (7 families) have been studied, the phenotype is inherited as an allele at the ABO locus. 14 of these subjects, have an alpha-N-acetylgalactosaminyltransferase whose kinetic properties were similar to those of A1 subjects. In one family, however, the A transferase detected is of the A1 type. On a quantitative level, the enzyme activities of these sera only reached 30-50 percent of the average value observed for A1 or A2 subjects, respectively. These facts suggest the existence of a genetic inhibitor, possibly linked to the ABO locus, preventing either an A1 or A2 gene from acting at the level of some cellular lines and leading therefore to the recognition of phenotypes named A-m-A1 and A-m-A2. On the contrary, under the experimental conditions used, no alpha-N-acetylgalactosaminyl-transferase activity was detected among the four individuals of the second group, named A-y by Weiner et al. (37), and whose appeareance in siblings results from the action of a recessive modifying y-A gene.

To evaluate the modification of pharmacodynamic parameters induced by the administration of L-asparaginase loaded into red blood cells, 13 patients received a single dose of L-asparaginase internalised into the carrier. The enzyme was loaded using a reversible lysis-resealing process. The dose per patient ranged from 30 to 200 i.u. kg-1. Considerable heterogeneity occurred between patients. the level of L-asparaginase circulating after 24 h represented 47% of the total injected dose as compared to 74.8% for red blood cells (RBCs). However, the half-life of the enzyme remaining in the circulation was very similar to that of the RBC carrier, i.e. 29 days and 27 days, respectively, compared with 8-24 h for the free enzyme. Sustained elimination of plasma L-asparagine occurred, the duration of which was dependent on the injected dose. A single injection of 30.i.u.kg-1 was sufficient to eliminate plasma L-asparagine over 10 days. With 150-200 IU.kg-1 the elimination period was extended to 50 days. These data show that the use of RBCs as carriers of L-asparaginase greatly improves the pharmacodynamic parameters of the drug.

A continuous lysing and resealing procedure with erythrocytes permitted incorporation in these cells of inositol hexaphosphate (InsP6), a strong allosteric effector of Hb. This leads to significant rightward shifts of the HbO2 dissociation curves with in vitro P50 (partial pressure of 02 at 50% Hb saturation), values increasing from 32.2 ± 1.8 torr for control erythrocytes to 86 ± 60 torr (pH 7.40; Pco2 40 torr at 370C; 1 torr = 1.333 x 10W Pa). The shape of the dissociation curve was still sigmoidal, although the Hill coefficient was decreased. The life span of InsP6-loaded erythrocytes equaled that of control erythrocytes. The long-term physiological effects of the InsP6-loaded erythrocytes on piglets were increased 02 release and reduced cardiac output. The reduced 02 affinity of the InsP6-loaded erythrocytes was still effective 20 days after transfusion in awake piglets. The electrolyte concentration appeared stable over the 5-day observation period except for a transient, but significant, hyperkalemia immediately after transfusion. The reductions in the 02 affinity of Hb reported here are large compared with previously reported values. Introduction of InsP6 into viable erythrocytes improves tissue oxygenation when, for any reason, normal blood flow is impaired.A conformational modification of intracellular Hb may considerably change its affinity for 02, which, in turn, may have physiological implications whenever a limitation of blood flow or reduction of Hb concentration impairs systemic oxygenation (1). When 02 uptake by erythrocytes was maintained, an in vivo decrease in 02 affinity was observed to enhance tissue 02 delivery (1). However, the consequence of a substantial increase of the in vivo P50 (02 partial pressure at 50% Hb saturation) is not well documented due to the difficulty in obtaining substantial and long-term low 02 affinity. Few methods have been reported to raise P50 in vivo, and the induced P50 increases were always modest and/or sustained for only a short time. We previously reported a method for increasing P50 in anesthetized piglets using inositol hexaphosphate (InsP6) (2). Because InsP6, the most effective Hb allosteric effector (3), cannot diffuse through the erythrocytic membrane, InsP6 was incorporated into erythrocytes by using a reversed osmotic-lysis process (4-6).The aim of this study was to sustain a marked decrease in Hb affinity for several days in piglets after exchange transfusion with InsP §-enriched homologous erythrocytes. Physiological properties and life span of lysed-resealed InsP6-loaded erythrocytes (InsP6-erythrocytes) were studied before exchange transfusion. The time courses of P50 variation and electrolyte concentrations were measured in piglets after massive exchange transfusion with InsP6-erythrocytes suspended in porcine plasma. MATERIALS AND METHODSHigh-Pso Erythrocyte Preparation. Human or porcine blood, collected on acid-citrate-dextrose solution, was stored at 4°C no longer than 5 days. After centrifugation ofone blood unit (1000 x g for 10 min)...

To evaluate the modification of pharmacodynamic parameters induced by the administration of L-asparaginase loaded into red blood cells, 13 patients received a single dose of L-asparaginase internalised into the carrier. The enzyme was loaded using a reversible lysis-resealing process. The dose per patient ranged from 30 to 200 i.u. kg-1. Considerable heterogeneity occurred between patients. the level of L-asparaginase circulating after 24 h represented 47% of the total injected dose as compared to 74.8% for red blood cells (RBCs). However, the half-life of the enzyme remaining in the circulation was very similar to that of the RBC carrier, i.e. 29 days and 27 days, respectively, compared with 8-24 h for the free enzyme. Sustained elimination of plasma L-asparagine occurred, the duration of which was dependent on the injected dose. A single injection of 30.i.u.kg-1 was sufficient to eliminate plasma L-asparagine over 10 days. With 150-200 IU.kg-1 the elimination period was extended to 50 days. These data show that the use of RBCs as carriers of L-asparaginase greatly improves the pharmacodynamic parameters of the drug.

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