Polymer delivery of the active isomer of misoprostol: a solution to the intestinal side effect problem.

SC-53450 is a new polybutadiene-based polymer system with an acid labile diisopropyl silyl ether linker to which the active isomer of misoprostol (SC-30249) is attached covalently at position C-11. It was studied in rats and dogs to define its profile of gastrointestinal effects relative to misoprostol-hydroxypropyl methylcellulose (HPMC) and the systemic availability of prostaglandin from the polymer. Results of rat studies indicate that SC-53450 has a spectrum of mucosal protective activity similar to misoprostol-HPMC, being protective against indomethacin-induced gastric, cysteamine/indomethacin-induced duodenal and indomethacin-induced lower small bowel damage. SC-53450, in contrast to misoprostol-HPMC, was not diarrheagenic in the rat when administered intragastrically. The observation that SC-53450 is more than 4 times more potent than misoprostol-HPMC suggests the possibility of sustained gastric availability of the prostaglandin SC-30249. SC-53450 exhibited gastric antisecretory activity in histamine-stimulated gastric fistula dogs and protected against acidified aspirin-induced gastric damage in normal fasted beagles. Rat and dog experiments indicate that little, if any, polymer-derived prostaglandin is available systemically, suggesting SC-53450 will have reduced abuse potential in abortion induction. SC-53450 is a potential candidate to replace the present misoprostol formulation in the marketplace for the prevention of nonsteroidal anti-inflammatory drug-induced gastric damage.

Catalytic functionalization of polymers: a novel approach to site specific delivery of misoprostol to the stomach.

The application of functionalized polymers to site-directed delivery of the antiulcer prostaglandin, misoprostol, is described. By use of homogeneous catalysis, the simple polymer, polybutadiene, was modified to incorporate the specialized requirements for controlled delivery of misoprostol to the stomach. An acid labile silyl ether bond to the C-11 hydroxyl of misoprostol was installed as the primary rate determining step for drug release, and a series of analogs, in which the steric hindrance about the silicon atom was varied, was prepared and evaluated for in vitro release rates, efficacy against indomethacin induced gastric damage and diarrheagenic activity. The diisopropylsilyl analog, the slowest releasing system studied, showed efficacy equal to misoprostol against indomethacin-induced gastric damage and no diarrhea at the highest dose tested.

Excess of red cell membrane proteins in hereditary high-phosphatidylcholine hemolytic anemia.

Previous descriptions of hereditary high-phosphatidylcholine hemolytic anemia (HPCHA) have highlighted the association of increased erythrocyte membrane phosphatidylcholine with abnormal membrane cation permeability. We studied the function and composition of erythrocyte membranes from three individuals with HPCHA to characterize further the membrane abnormalities in this disorder. Despite significant macrocytosis, HPCHA red cells were dehydrated and showed an increased surface area to volume ratio compared to normal red cells. The passive efflux of K+ from HPCHA erythrocytes was increased fourfold at 37 degrees C. Total membrane phospholipid was increased 7-42%, largely due to excess phosphatidylcholine, which made up 35.8-37.2% of total phospholipid. Membrane cholesterol:phospholipid ratios were in the normal range. It appears that the excess phosphatidylcholine was not acquired during circulation, since plasma lipids were normal and all subpopulations of density-separated HPCHA erythrocytes were similarly abnormal. The ratio of total protein to phospholipid in white ghosts was increased, indicating that membrane protein was increased to an even greater extent than membrane lipids. No abnormal membrane proteins were identified by Coomassie or periodic acid Schiff (PAS) staining. Quantitation of the major membrane proteins indicated that the total protein excess in HPCHA membranes was due to a proportional increase in all major proteins. We conclude that HPCHA erythrocytes have excess membrane proteins and hypothesize that the changes in lipid composition and cation permeability are secondary to underlying protein abnormalities, which remain to be defined.

Chlorpromazine inhibits vesiculation, alters phosphoinositide turnover and changes deformability of ATP-depleted RBCs.

To delineate further the underlying mechanism by which amphiphilic drugs can modulate vesicle release from human RBCs, we studied the effect of chlorpromazine on erythrocyte vesiculation induced by ATP depletion. This was correlated with turnover of the phosphoinositides as well as RBC deformability during the process since phosphoinositide metabolism may be involved in shape regulation of RBCs. Echinocytic shape transformation and subsequent vesiculation of RBCs, which commonly occur during ATP depletion, were inhibited by chlorpromazine. Furthermore, with a newly developed two-dimensional thin-layer chromatography separation of RBC membrane phospholipids, we showed that chlorpromazine significantly decreased the dephosphorylation of phosphatidylinositol-4,5-bisphosphate (PIP2) in both ATP-depleted RBCs as well as in cells with partly maintained ATP levels. Concomitantly, there was a smaller increase in the relative amount of phosphatidylinositol. In addition, chlorpromazine also inhibited the decreased in RBC deformability as well as the shift of osmotic fragility that occurs during ATP depletion of erythrocytes.

Hereditary poikilocytic anemia associated with the co-inheritance of two alpha spectrin abnormalities.

This report describes a black family in which two distinct structural defects of alpha spectrin were inherited singly and in combination. The propositus, who has a poikilocytic hemolytic anemia that shares many of the features of hereditary pyropoikilocytosis (HPP) or homozygous elliptocytosis, is a compound heterozygote for both the spectrin alpha 1/65 and spectrin alpha 1/50a defects as demonstrated by electrophoretic analysis of spectrin tryptic fragments. The spectrin alpha 1/65 defect alone was found in his mother and sibling, while the spectrin alpha 1/50a defect was present in the father and another sibling. The red cell spectrin content was normal in all family members. The functional consequences of inheritance of these two spectrin defects were compared with those found in an unrelated patient with classic HPP who had the alpha 1/50a spectrin defect and was spectrin deficient as well. Prolonged incubation at 37 degrees C resulted in striking budding, fragmentation, and sphering of classic HPP red cells but only minimal changes in propositus cells. The percentage of spectrin dimers was increased tenfold in classic HPP, sevenfold in the propositus, and threefold in other family members. Mechanical stability of erythrocyte ghosts, measured by ektacytometry, was reduced severely in both classic HPP and in the propositus, but only moderately in other family members. Thus, co-inheritance of two alpha spectrin defects can result in a poikilocytic hemolytic anemia milder than that usually found in HPP. The greater clinical severity of HPP may be a consequence of the presence of spectrin deficiency, a finding absent in the propositus.

Spectrin oxidation correlates with membrane vesiculation in stored RBCs.

An increase in spectrin oxidation in a variety of erythrocytes displaying a tendency to vesiculate has been previously described. To explore this relationship in more detail, we have studied blood stored in citrate-phosphate-dextrose-adenine under blood bank conditions because, in this system, vesiculation occurs slowly. Vesiculation was quantitated by measuring acetylcholinesterase release, and the extent of spectrin oxidation was detected by using thiol-disulfide exchange chromatography. A strong correlation (r = .92) was found between the extent of spectrin oxidation and vesiculation when blood from five donors was analyzed at weekly intervals during storage. This strongly suggests that spectrin oxidation plays a role in the formation of spectrin-free vesicles, thereby limiting the shelf life of stored blood.

Red cell vesiculation--a common membrane physiologic event.

Loss of red blood cell membrane material in the form of microvesicles has been noted in sickle cells, in Ca++-loaded and adenosine triphosphate (ATP)-depleted normal red blood cells; and during storage of normal red blood cells. To further understand the vesiculation process, we have studied vesicles generated by a variety of perturbations of the red blood cell membrane. Vesicles were isolated by centrifugation at 30,000 X g from plasma of heparinized pathologic blood samples (sickle cell anemia, hemoglobin H disease, hereditary spherocytosis, hereditary elliptocytosis, and protein 4.1 deficiency) incubated overnight at 4 degrees C. Vesicle formation also was induced in normal erythrocytes by ATP depletion, by heating to 49 degrees C, by incubation at pH 5.4, and by incubation in 5 mmol/L diamide. Membrane protein composition was characterized on denaturing polyacrylamide gels and by immunoblot. The vesicles all contained band 3, glycophorin A, and band 4.1. Spectrin was depleted in all vesicles. Thiol disulfide exchange chromatography revealed evidence of oxidative cross-linking of spectrin in pathologic and normal red blood cells that had undergone vesiculation. This suggests that the mechanism of vesiculation may be related to cross-linking of membrane proteins. Membrane phospholipid composition of sickle cell and acid-induced vesicles was similar to that of normal red cells as determined by thin-layer chromatography. Possible pathophysiologic effects of vesiculation were assessed by using a modified Russell's viper venom assay. All vesicles examined shortened Russell's viper venom clotting time by 55% to 70% of control values. In addition, ektacytometer studies reveal that cells remaining after acid-induced vesiculation are rigid. These observations indicate that the vesicles may play a role in the hypercoagulation seen in some hemolytic disorders and that the process of vesiculation itself may contribute to increased rigidity of red cells and their subsequent removal from the circulation.

Membrane phospholipid organization and vesiculation of erythrocytes in sickle cell anaemia.

This review has examined the lipid composition of the RBC membrane and the methods used to determine the distribution of the phospholipids within the membrane. The importance of the membrane cytoskeletal proteins, in particular spectrin and protein 4.1, in maintaining this distribution has also been described. Membrane vesiculation and altered membrane phospholipid asymmetry in sickle cell anaemia have been reviewed. The relationships between vesiculation and hypercoagulability and an abnormal reticuloendothelial system in sickle cell disease have been examined. It is apparent that the single amino acid substitution leading to the production of sickle haemoglobin has profound effects on the entire erythrocyte, reaching to the limits of the cell, its plasma membrane.

[Quantitative determination of pemoline in serum and urine after ingestion of therapeutic doses (author's transl)]. / Quantitative Bestimmung von Pemolin in Serum und Harn nach Einnahme therapeutischer Dosen.

Serum and urine 5-phenyl-2-imino-4-oxazolidone (pemoline, Tradon) levels have been determined in normal subjects after therapeutic doses (40 mg). Peak levels in serum were reached within 4-6 h (1.1-1.5 mg/1). The half-life was 16-18 h. After the oral administration of 40 mg of pemoline 35-50% of the dose was excreted in urine within 32 h.