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A De Flora

Publications and source records attributed to A De Flora.

At least 73 records · Page 4Linked to original sources
PubMed1992

Interaction of carboplatin with carrier human erythrocytes.

The antineoplastic drug Carboplatin (CBDCA) was encapsulated in human erythrocytes by means of transient hypotonic hemolysis, followed by isotonic resealing. Up to 5 mg/ml of packed cells could be entrapped, with about 70% cell recovery. In vitro incubation of the CBDCA-loaded erythrocytes in autologous plasma caused a very slow release of the drug from the cells (12% approximately in 3 h). The encapsulation conditions, performed at a low hematocrit, in order to obtain high amounts of the drug inside the carriers, impaired the metabolic properties of the loaded erythrocytes significantly. In particular, an almost complete disappearance of GSH was observed. Analysis of the intraerythrocytic metabolism of CBDCA showed that, in spite of its relatively high stability in aqueous solutions, in hemolysates and in the loaded erythrocytes a significant percentage of CBDCA is rapidly converted to other species that still retain an antiproliferative activity in vitro. This fast conversion could be extensively inhibited by previous conversion of oxyhemoglobin to methemoglobin or carbomonoxyhemoglobin, suggesting an important role of heme iron in this process. Encapsulation of CBDCA in selectively targeted human erythrocytes may represent a therapeutic strategy for increasing the drug concentration in specific organs, notably liver.

Carboplatin↗
PubMed1991

Heterogeneity of guanine nucleotide binding proteins in human red blood cell membranes.

Membranes from human erythrocytes bind radioactive GTP and GTP analogs according to apparently homogeneous patterns. In spite of this uniform type of association, multiple guanine nucleotide binding proteins have been identified both by SDS-PAGE analysis of native and of variously ADP-ribosylated membrane preparations and by FPLC chromatography of solubilised erythrocyte membranes preliminarily incubated with [alpha-32P] GTP in the presence of 5 mM MgCl2. From eight to nine peak fractions of pronase-digestible GTP-binding activity were separated on a MA7Q anion exchange column, this pattern being highly reproducible with different membrane preparations. Prior incubation of membranes with [alpha-32P] GTP in the presence of excess unlabeled GDP resulted in displacement of bound labeled nucleotide from all FPLC fractions. The patterns of GTP binding were also markedly modified by preliminary treatment of membranes with N-ethylmaleimide. Detectable GTPase activity was present in each of the FPLC peak fractions. This wide heterogeneity of guanine nucleotide binding proteins raises so far unanswered questions as to their physiological significance in the mature erythrocyte.

Blood Protein Electrophoresis↗
PubMed1991

Pharmacokinetic properties of doxorubicin encapsulated in glutaraldehyde-treated canine erythrocytes.

Canine erythrocytes were loaded with the antineoplastic drug doxorubicin and then treated with 0.16% glutaraldehyde. This procedure has been previously shown to slow down the efflux of doxorubicin from erythrocytes and to result in the selective targeting of the carrier erythrocytes to liver. Three dogs were treated each with 2 different schedules of IV bolus administration of doxorubicin (0.4 mg/kg of body weight): free drug and doxorubicin encapsulated in glutaraldehyde-treated erythrocytes. The 2 treatments yielded consistent differences in the plasma pharmacokinetic properties of doxorubicin and of its only metabolite, doxorubicinol. A triphasic exponential decay of doxorubicin plasma concentrations was observed on injection of the free drug. Conversely, in the case of erythrocyte-encapsulated doxorubicin, 4 phases of plasma concentrations of doxorubicinol were found. The plasma concentrations of doxorubicinol, after a steady increase during the first hour, followed patterns of decay comparable to those of the parent drug. On the basis of the kinetic variables calculated with the 2 administration schedules, area under curve concentrations of plasma doxorubicin were 136 micrograms.h/L (free infusion) and 734 micrograms.h/L (erythrocyte-encapsulated drug). Significant alterations of hematologic and hematochemical factors were not observed in the 3 dogs during and after the 2 treatments. On the basis of our findings, doxorubicin-loaded and glutaraldehyde-treated erythrocytes may potentially be used in the treatment of systemic and hepatic tumors in dogs.

Animals↗
PubMed1990

Multiple small molecular weight guanine nucleotide-binding proteins in human erythrocyte membranes.

Native membranes from human erythrocytes contain the following G proteins which are ADP-ribosylated by a number of bacterial toxins: Gi alpha and Go alpha (pertussis toxin), Gs alpha (cholera toxin), and three proteins of 27, 26 and 22 kDa (exoenzyme C3 from Clostridium botulinum). Three additional C3 substrates (18.5, 16.5 and 14.5 kDa) appeared in conditions of unrestrained proteolysis during hemolysis. SDS-PAGE separation of erythrocyte membrane proteins followed by electroblotting and incubation of nitrocellulose sheets with radiolabeled GTP revealed consistently four GTP-binding proteins with Mr values of 27, 26, 22 and 21 kDa. Although a 22 kDa protein was immunochemically identified as ras p21, the C3 substrate of 22 kDa is a different protein probably identifiable with a rho gene product. Accordingly, at least five distinct small molecular weight guanine nucleotide-binding proteins, whose functions are so far undetermined, are present in native human erythrocyte membranes.

ADP Ribose Transferases↗
PubMed1990

Construction and characterization of adriamycin-loaded canine red blood cells as a potential slow delivery system.

Adriamycin was internalized in canine red blood cells (RBC) by two procedures involving (a) simple diffusion of the drug into cells and (b) hypotonic dialysis followed by isotonic resealing. The two procedures yielded comparable amounts of encapsulated adriamycin, around 35 micrograms/10(9) RBC. Exposure of adriamycin-loaded RBC to 0.16% glutaraldehyde consistently slowed down the rate of efflux of the drug as compared with non-glutaraldehyde-treated cells: after 1 h of incubation at 37 degrees C, greater than 80% of adriamycin was still present inside the glutaraldehyde-treated RBC, while at 24 h it was 66%, compared to 10% and 1%, respectively, in the adriamycin-loaded, non-glutaraldehyde-treated cells. Canine RBC showed a higher rate of transformation of adriamycin than the human cells, the only intracellular metabolite being adriamycinol, which is apparently formed by the NADPH-dependent enzyme aldehyde reductase. Production of adriamycinol was remarkably lower in the glutaraldehyde-treated RBC, as a result of progressive and extensive inactivation of hexose monophosphate shunt activity responsible for NADPH formation. These results, coupled with the known selective targeting of glutaraldehyde-treated RBC to liver, hold promise as to in vivo applications of this drug delivery system in antineoplastic therapy.

Animals↗
PubMed1989

Modulation of erythrocyte Ca2+-ATPase by selective calpain cleavage of the calmodulin-binding domain.

The activity of the membrane-bound and the purified erythrocyte Ca2+-ATPase in the absence of calmodulin was stimulated by calpain digestion but could be further increased to maximal levels by calmodulin (CaM). Thus, CaM sensitivity was retained by the digested ATPase, at least at short times of incubation. In membranes digested at higher temperatures and in the purified ATPase digested at higher calpain/ATPase ratios, the ATPase became fully activated. The membrane-bound and the purified 138-kDa ATPase were converted by calpain to a fragment of approximately 124 kDa which still bound CaM and could be isolated on CaM columns when proteolysis occurred slowly but not when it occurred rapidly. Carboxypeptidase digestion of the purified enzyme and of its fragment of about 124 kDa has shown that calpain attacked the CaM-binding domain near the C terminus of the ATPase. This has also been supported by digestion of the purified enzyme and of its fragment of about 124 kDa. A first cut occurred in the middle of the domain producing a fragment of about 14 kDa and a (CaM-binding) fragment of about 124 kDa. A second cut closer to the N terminus of the domain also produced a fragment of about 124 kDa and accounted for the loss of CaM binding at prolonged times of incubation of the ATPase with calpain.

Amino Acid Sequence↗
PubMed1989

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes.

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.

Binding, Competitive↗
PubMed1989

Encapsulation of doxorubicin in liver-targeted erythrocytes increases the therapeutic index of the drug in a murine metastatic model.

Doxorubicin-loaded, glutaraldehyde-treated murine erythrocytes, once reinjected into circulation, are rapidly taken up by liver and lungs and behave as an organ-targeted, slow delivery system for the encapsulated drug. The antitumor activity of encapsulated doxorubicin (former generic name, adriamycin) was compared with that of the free drug in a murine hepatic and pulmonary tumor model. This was obtained by intrasplenic injection of L1210 lymphoma cells followed by splenectomy. Different schedules of treatment of tumor-bearing mice with erythrocyte-encapsulated or free doxorubicin were investigated. The optimal schedule of treatment for free doxorubicin proved to be i.v. bolus administration on the day of splenectomy. Under these conditions, the dose producing 50% inhibition of metastatic growth in the liver, as measured by inhibition of 5-[125I]iodo-2'-deoxyuridine uptake 9 days after tumor induction, was 6.3 mg/kg for free doxorubicin and 0.48 mg/kg for the encapsulated drug. In these conditions pulmonary tumor development was even more efficiently prevented by encapsulated doxorubicin as compared with the free drug. The values of the therapeutic index (TI), defined as the ratio between the maximal tolerated dose (LD10) and the minimal effective dose (ED90, producing 90% inhibition of liver metastatic growth), were 4.2 and 1.8 for encapsulated and free doxorubicin, respectively.

Alanine Transaminase↗
PubMed1988

Improved metabolic properties of hexokinase-overloaded human erythrocytes.

Human erythrocytes were loaded with homogeneous hexokinase purified from human placenta (an enzyme species apparently identical to the erythrocyte enzyme), using a procedure of encapsulation based on hypotonic hemolysis, isotonic resealing and reannealing. The hexokinase-overloaded erythrocytes contained 4.77 +/- 0.75 IU of hexokinase activity per ml of packed erythrocytes, a value 15-times higher than that of corresponding unloaded or native red cells. The hexokinase-loaded erythrocytes were found to metabolize twice the amount of glucose consumed by the unloaded cells through a nearly doubled glycolytic activity, while the activity of the hexose monophosphate shunt pathway was unmodified. Estimates of glycolytic intermediates showed increased levels of most metabolites with respect to the unloaded erythrocytes, while the intracellular concentrations of adenine nucleotides and 2,3-bisphosphoglycerate were unaffected by entrapment of hexokinase. The new steady-state condition characterized by improved glycolytic function was demonstrated to be directly related to enhanced levels of hexokinase activity and not to the use of a rejuvenation solution during the procedure of entrapment. These results are consistent with suggestions by several investigators that glucose metabolism in human erythrocytes is regulated by hexokinase, and they open new perspectives for manipulating erythrocytes with the ultimate aim of improving their survival under different storage conditions.

Adenine Nucleotides↗
PubMed1988

Conversion of encapsulated 5-fluoro-2'-deoxyuridine 5'-monophosphate to the antineoplastic drug 5-fluoro-2'-deoxyuridine in human erythrocytes.

The fluoropyrimidine deoxyribonucleotide 5-fluoro-2'-deoxyuridine 5'-monophosphate (FdUMP) was encapsulated in human erythrocytes by a procedure based on hypotonic hemolysis and isotonic resealing. Encapsulated FdUMP (up to 9 mumol/ml of packed erythrocytes) did not affect erythrocyte metabolism or morphology. Hemolysates were found to catalyze efficient dephosphorylation of FdUMP to yield nearly stoichiometric amounts of the corresponding deoxyribonucleoside 5-fluoro-2'-deoxyuridine (FdUrd), an antineoplastic drug showing selective cytotoxicity toward liver metastases from colorectal carcinomas. The dephosphorylation reaction had an apparent Km of 7.7 +/- 1.2 mM FdUMP at pH 7.4 and was remarkably slower at pH 8.2. ATP, GTP, and UTP inhibited both the disappearance of FdUMP and the formation of FdUrd in hemolysates. The enzyme responsible for the FdUMP-to-FdUrd conversion was identified with the deoxyribonucleotide-specific isozyme of erythrocyte pyrimidine 5'-nucleotidase (EC 3.1.3.5). Intracellular formation and subsequent release of FdUrd were observed in intact erythrocytes loaded with FdUMP. Inhibition of FdUrd release from these erythrocytes was obtained by raising the pH intracellularly and, alternatively, by coencapsulation of ATP. Autologous FdUMP-loaded erythrocytes might be used as endogenous bioreactors designed for time-programmed and liver-targeted delivery of FdUrd.

Antineoplastic Agents↗
PubMed1988

In vivo liver and lung targeting of adriamycin encapsulated in glutaraldehyde-treated murine erythrocytes.

Treatment of adriamycin-loaded erythrocytes from B6D2F1 mice with 0.1% glutaraldehyde produced the following effects: a considerable decrease in the in vitro leakage of the unmodified drug and a selective liver (and, to a lesser extent, lung) uptake of the encapsulated drug (70% of the injected dose) compared to drug leakage from, and tissue distribution of, carrier erythrocytes not treated with glutaraldehyde. The liver vascular bed was not saturated by five daily intravenous injections of 20 microliters of glutaraldehyde-treated erythrocytes, which allows a total dosage of 200 micrograms of the drug (half the LD50 value) to be administered. No appreciable liver damage results from extensive and prolonged uptake of glutaraldehyde-treated carrier erythrocytes. Entrapment of adriamycin within erythrocytes along with glutaraldehyde treatment of the carrier cells seems to be a promising therapeutic strategy against liver (and lung) tumors.

Animals↗
PubMed1987

Favism: impairment of proteolytic systems in red blood cells.

Red blood cells (RBC) from favic patients are characterized by (a) severe oxidative damage (contributed by autoxidation of divicine and isouramil, two pyrimidine aglycones present in fava beans) and (b) greatly increased calcium levels. In vitro, both autoxidation of divicine and calcium loading produced marked alterations of proteolytic systems in intact RBC. Specifically, autoxidizing divicine inactivated procalpain, the proenzyme species of calcium-activated cytosolic neutral proteinase, or calpain. Inactivation was much greater with glucose-6-phosphate dehydrogenase (G6PD)-deficient RBC than with normal RBC. On the other hand, loading of normal and G6PD-deficient RBC with calcium resulted in conversion of procalpain to calpain and eventual autoproteolytic inactivation of calpain itself, and extensive release of acid endopeptidase activity from the membranes into the cytosol. Damaged RBC from favic patients had significantly lowered procalpain activity and an abnormal subcellular distribution of acid proteinase activity that was found mostly in the cytosol. When purified calpain was incubated with membranes from acetylphenylhydrazine (APH)-treated RBC, significant proteolysis was observed affecting mostly band 3 and hemoglobin chains, ie, the two proteins involved in the onset of aggregation of Heinz bodies. Moreover, exposure of intact RBC to 20 mmol/L APH induced depletion of procalpain activity for which the time course was inversely related to formation of Heinz bodies. These findings support the role of procalpain in protecting G6PD-deficient RBC from oxidant-induced Heinz body formation and imply that exhaustion of the procalpain-calpain system is an important step in the mechanisms of RBC damage and destruction in favism.

Calcium↗
PubMed1987

Alterations of red blood cell proteolysis in favism.

Damaged RBC drawn from favic patients during acute hemolysis showed marked alterations in their two major proteolytic systems. Cytosolic procalpain (i.e., the proenzyme species of Ca2+-activated neutral proteinase, or calpain) had considerably lower activity than in matched RBC from asymptomatic G6PD-deficient subjects. The total RBC activity of the three acid endopeptidases that are normally membrane-bound was not reduced in favism, but its subcellular distribution was mostly cytosolic, suggesting quantitative release from membranes. Changes in procalpain activity are the result of both autoxidation of divicine and of the intracellular elevation of Ca2+ that is found in favism. Changes in acid endopeptidase activity are the consequence of perturbed Ca2+ homeostasis. Overall, the picture shows a marked impairment of the RBC proteolytic machinery that in turn may worsen cellular damage.

Calcium↗
PubMed1987

Hepatic or splenic targeting of carrier erythrocytes: a murine model.

Carrier mouse erythrocytes, i.e., red cells, subjected to a dialysis technique involving transient hypotonic hemolysis and isotonic resealing were treated in vitro in three different ways: (a) energy depletion by exposure for 90 min at 42 degrees C; (b) desialylation by incubation with neuroaminidase; and (c) oxidative stress by incubation with H2O2 and NaN3. Procedure (c) afforded maximal damage, as shown by analysis of biochemical properties of the treated erythrocytes. Reinfusion in mice of the variously manipulated erythrocytes following their 51Cr labeling showed extensive fragilization as indicated by rapid clearance of radioactivity from the circulation. Moreover, both the energy-depleted and the neuraminidase-treated erythrocytes showed a preferential liver uptake, reaching 50 and 75%, respectively, within 2 h. On the other hand, exposure of erythrocytes to the oxidant stress triggered a largely splenic removal, accounting for almost 40% of the reinjected cells within 4 h. Transmission electron microscopy of liver from mice receiving energy-depleted erythrocytes demonstrated remarkable erythrocyte congestion within the sinusoids, followed by hyperactivity of Kupffer cells and by subsequent thickening of the perisinusoidal Disse space. Concomitantly, levels of serum transaminase activities were moderately increased. Each of the three procedures of manipulation of carrier erythrocytes may prove applicable under conditions where selective targeting of erythrocyte-encapsulated chemicals and drugs to either the liver or the spleen has to be achieved.

Animals↗
PubMed1986

Oxidative inactivation of the calcium-stimulated neutral proteinase from human red blood cells by divicine and intracellular protection by reduced glutathione.

Calpain, the micromolar Ca2+-requiring form of Ca2+-stimulated neutral proteinase purified from human red cells, is remarkably inactivated during autoxidation of divicine (2,6-diamino-4,5-dihydroxypyrimidine), an aglycone implicated in the pathogenesis of favism. Inactivation of purified calpain is produced, in decreasing order of efficiency, by transient, probably semiquinonic species arising from autoxidation of divicine, by the H2O2 that is formed upon autoxidation itself, and by quinonic divicine, respectively. Purified procalpain, the millimolar Ca2+-requiring form that can be converted to the fully active calpain form by a variety of mechanisms, is less susceptible than calpain itself to inactivation by the same by-products of divicine autoxidation. When intact red cells are exposed to autoxidizing divicine, procalpain undergoes a significant loss of activity. At 1 mM divicine, intracellular inactivation is observed with procalpain only, while the activity of a number of red cell enzymes is unaffected. Inactivation of procalpain is consistently greater in red cells from glucose-6-phosphate dehydrogenase-deficient subjects than in normal cells. Restoration of normal levels of glucose-6-phosphate dehydrogenase activity by means of entrapment of homogeneous human glucose-6-phosphate dehydrogenase in the deficient red cells results in normal stability of intracellular reduced glutathione; decreased susceptibility of procalpain to inactivation by autoxidizing divicine. These findings suggest that in the glucose-6-phosphate dehydrogenase-deficient red cells the procalpain-calpain system is a major target of divicine cytotoxicity.

Calpain↗
PubMed1986

Calcium-induced alterations in the levels and subcellular distribution of proteolytic enzymes in human red blood cells.

Human red cells were treated with 100 microM Ca2+ and ionophore A 23187. This treatment induces remarkable changes in the activities of the two major proteolytic systems of red cells, i.e. Ca2+-dependent neutral proteinase and acid endopeptidases. Ca2+-dependent neutral proteinase undergoes intracellularly preliminary activation of the inactive proenzyme species, followed by eventual inactivation through self-proteolysis. Transient activation is shown by selective degradation of cytoskeletal proteins known to be targets of this enzyme system. Concomitantly, acid endopeptidase activity is substantially released from the membrane into the cytosol. Preliminary inactivation of the Ca2+-dependent neutral proteinase by exposure of Glucose 6-phosphate dehydrogenase-deficient red cells to auto-oxidizing divicine prevents alterations induced by Ca2+ loading on cytoskeletal membrane proteins, while leaving solubilization of acid endopeptidase activity unaffected. The two events, although dependent on Ca2+ loading, are therefore unrelated to each other.

Calcimycin↗
PubMed1986

Contributions of superoxide, hydrogen peroxide, and transition metal ions to auto-oxidation of the favism-inducing pyrimidine aglycone, divicine, and its reactions with haemoglobin.

The influence of O2-, H2O2 and metal ions on the auto-oxidation of divicine, a pyrimidine aglycone, was studied. In air at pH 7.4, the hydroquinonic form oxidized within a few minutes. Superoxide dismutase (SOD) markedly decreased the initial rate, giving a lag phase followed by rapid oxidation. Although catalase or diethylenetriamine-penta-acetic acid (DTPA) alone had little effect, each in the presence of SOD further slowed the initial rate and increased the lag. H2O2 decreased the lag time, as did Cu2+, Fe2+ or haemoglobin. GSH substantially increased the lag phase, but it eventually reacted with the divicine to form a 305 nm-absorbing adduct. These results indicate that an O2(-)-dependent mechanism of divicine auto-oxidation normally predominates. Auto-oxidation can also occur by a mechanism involving H2O2 and transition metal ions or haemoglobin, and if both these reactions are prevented by SOD and DTPA or catalase, a third mechanism, requiring build-up of an autocatalytic intermediate, becomes operative. Oxyhaemoglobin did not react directly with divicine, but reacted with the H2O2 produced by divicine auto-oxidation to give mainly an oxidized derivative presumed to be ferrylhaemoglobin. Divicine was shown to reduce ferylhaemoglobin to methaemoglobin, and this reaction was probably responsible for the acceleratory effect of haemoglobin on divicine oxidation. These results indicate that O2 rather than oxyhaemoglobin is likely to initiate divicine oxidation in the erythrocyte. Haemolytic crises, which are thought to result from this oxidation, occur only sporadically in glucose-6-phosphate dehydrogenase deficient individuals following ingestion of fava beans. A characteristic of the crises is acute depletion of erythrocyte GSH, and the vulnerability of these cells could relate to the ability of GSH, in combination with SOD, to protect against the autocatalytic mechanism of divicine auto-oxidation. Our demonstration of a variety of auto-oxidation pathways also suggests possible areas of individual variation.

Catalase↗