Substituted benzaldehydes designed to increase the oxygen affinity of human haemoglobin and inhibit the sickling of sickle erythrocytes.

Substituted benzaldehydes have been designed to bind preferentially to the oxy conformation of human haemoglobin at a site between the amino terminal residues of the alpha-subunits. Such compounds should stabilize the oxygenated form of haemoglobin and thereby increase its oxygen affinity. The compounds produce the expected effect, left-shifting the oxygen saturation curve of dilute haemoglobin solutions and of whole blood, although the binding pattern to haemoglobin is more complex than envisaged by the design hypothesis. The predicted best compound is also a potent inhibitor, at low oxygen pressure, of the sickling of erythrocytes from patients homozygous for sickle cell disease, and may prove to be a clinically useful anti-sickling agent.     

Developing treatment for sickle cell disease

Sickle cell disease pathophysiology results from sickle haemoglobin polymerisation and its effects on the sickle erythrocyte and the vasculature. Many of the abnormalities of sickle cell disease are secondary to the damage caused by the polymer and the injured red cell. Pharmacological treatment of the disease is focused on the inhibition of sickle haemoglobin polymerisation, prevention or repair of red cell dehydration and interruption of the interaction of sickle cells with the endothelium.     

Developing treatment for sickle cell disease

Sickle cell disease pathophysiology results from sickle haemoglobin polymerisation and its effects on the sickle erythrocyte and the vasculature. Many of the abnormalities of sickle cell disease are secondary to the damage caused by the polymer and the injured red cell. Pharmacological treatment of the disease is focused on the inhibition of sickle haemoglobin polymerisation, prevention or repair of red cell dehydration and interruption of the interaction of sickle cells with the endothelium.     

Autologous immune complex nephritis associated with sickle cell trait: diagnosis of the haemoglobinopathy after renal structural and immunological studies.

A renal tubular epithelial antigen (RTE)--anti-RTE autologous immune complex nephritis associated with sickle cell anaemia (SS) has been reported, but immune complex nephritis has never been described in patients with sickle cell trait (SA). During investigation of a child with "asymptomatic proteinuria" cryoprecipitable complexes of RTE-anti-RTE were detected in the serum and granular deposits of RTE, immunoglobulins, and complement localised on the glomerular basement membranes. Morphological and ultrastructural studies showed increased mesangial matrix, sickled red blood cells in the glomeruli and vessels, and tubular and interstitial abnormalities. These findings prompted haemoglobin electrophoretic studies, which showed previously undiagnosed haemoglobin SA in this patient and her family. These observations suggest that nephritis mediated by similar immunopathogenic mechanisms may be associated with SS and SA haemoglobinopathy. Under some conditions patients with sickle cell trait may experience haemodynamic and oxygenation abnormalities, which may be aetiological factors in the immune complex nephritis associated with SS disease.     

Postextrasystolic potentiation in patients with ischaemic heart disease: influence of inotropic agents.

1. In the present study we have investigated the effects of the relatively low plasma concentrations of gemfibrozil (GFZ) found in clinical practice on the oxygen dissociation curve (ODC) of erythrocytes. 2. ODCs were measured at 30 degrees C and 37 degrees C and at pH 7.4: a) both on HbA solution and erythrocytes incubated in vitro with gemfibrozil and clofibric acid; b) on erythrocytes from healthy volunteers treated with a single oral dose of gemfibrozil. 3. These experiments showed a significant drug-induced shift of the ODC towards lower O2 affinity values without any significant modification of metabolic parameters of erythrocytes such as intracellular pH and intraerythrocytic levels of ATP and DPG. 4. In our experimental conditions gemfibrozil appears to lower both in vitro and in vivo, the partial pressure of oxygen required to give 50% of the haemes saturated with oxygen (P50) of erythrocytes from the control value of 24 +/- 0.5 mm Hg to 29 +/- 0.5 mm Hg (mean +/- s.d.; P < 0.02 by ANOVA). 5. These data clearly indicate that therapeutic doses of gemfibrozil may influence the oxygen transport properties of red cells. This effect could have relevant pharmacological and toxicological implications.     

The pharmacokinetics, tolerability and pharmacodynamics of tucaresol (589C80; 4[2-formyl-3-hydroxyphenoxymethyl] benzoic acid), a potential anti-sickling agent, following oral administration to healthy subjects.

1. Tucaresol (589C80; 4[2-formyl-3-hydroxyphenoxymethyl] benzoic acid) interacts stoichiometrically with haemoglobin to increase oxygen affinity. By decreasing the proportion of insoluble deoxy sickle haemoglobin at capillary oxygen concentrations, tucaresol may be of therapeutic benefit in sickle cell anaemia. 2. In this study, which involved the first administration to man, the pharmacokinetics and pharmacodynamics of tucaresol were studied in healthy male volunteers following oral doses of 200-3600 mg. 3. Peak drug concentrations in plasma and erythrocytes were linearly related to dose; mean (s.d.) values were 95.8 (26.1) and 1035 (67) micrograms ml-1, respectively, at the highest dose. Median tmax in plasma was 6.5 h and in erythrocytes 24.5 h, when approximately 60% of the administered dose was in the target tissue. Plasma drug concentrations fell biexponentially with commencement of the apparent terminal elimination phase at approximately 24 h. The terminal elimination half-life from plasma increased with dose (r = 0.77; P < 0.0001) from 133-190 h at 400 mg to a mean (s.d.) of 289 (30) h at 3600 mg. Erythrocyte drug concentrations declined mono-exponentially with a half-life that was always shorter than the apparent terminal half-life in plasma: overall mean (95% CI) of t1/2 erythrocyte/t1/2 plasma ratio was 0.57 (0.53, 0.61). The erythrocyte AUC/plasma AUC ratio increased with dose (r = 0.67; P < 0.001). 4. The proportion of haemoglobin modified to a form with high oxygen affinity (%MOD) increased in a dose-related manner above doses of 800 mg reaching 19-26% after the 3600 mg dose.(ABSTRACT TRUNCATED AT 250 WORDS)     

The action of propranolol on factors concerned with the delivery of oxygen to the tissues.

1 In a previous study in conscious rats, orally administered propranolol acutely lowered cardiac output by 30.5% and oxygen uptake by 12.2%, while oxygen extraction rose by 31.5%. The present study is concerned with the way in which the rat meets its oxygen requirements against such a fall in perfusion. 2 The effect of known concentrations of propranolol on haemoglobin-oxygen affinity was studied in vitro. The effect of orally administered propranolol (given acutely and chronically) was then determined and this was related to the concentration of the drug in plasma and red cells. Further studies were made on the action of propranolol on the Bohr effect and on arterial oxygen carriage. 3 In vitro, high concentrations of propranolol (1 x 10(-4) M) influenced haemoglobin-oxygen affinity in a biphasic manner and this was associated with changes in haematocrit and red cell and plasma pH. No change occurred in affinity after acute or chronic oral administration of the drug due to insufficient concentration in the blood. No direct action of the Bohr effect was detected. 4 Arterial oxygen content rose acutely due to an increase in haemoglobin concentration. 5 It is concluded that increased oxygen extraction in propranolol-treated rats is not explained by the actions of the drug on haemoglobin-oxygen affinity.     

Pharmacokinetics and pharmacodynamics of tucaresol, an antisickling agent, in healthy volunteers.

1. Tucaresol is an orally administered antisickling agent which increases the oxygen affinity of haemoglobin. 2. The pharmacokinetics, effects on moderate graded exercise and psychometric performance of tucaresol were examined in a double-blind, placebo-controlled, parallel groups study in 12 healthy men. 3. Three doses of tucaresol were given at 48 h intervals intended to modify 15, 25 and 32.5% of a subject's haemoglobin to a high affinity form (%MOD). 4. Mean peak %MOD was 34%. Mean Cmax values in plasma and erythrocytes were 81.4 and 1459 micrograms ml-1, respectively. 5. Heart rate, compared with baseline, increased in the tucaresol group with the greatest changes at the highest %MOD and workload. There were no differences between groups in psychometric test performance. 6. Three volunteers on active drug developed fever, rash and tender cervical lymphadenopathy with onset 7-10 days from the start of dosing, suggesting an immune mechanism. 7. The acute increase in oxygen affinity with tucaresol is physiologically well-tolerated, but the utility of tucaresol in the management of sickle cell disease will depend on the identification of a dosing regimen with a lower incidence of drug allergy.     

Identification of 2-imidazolines as anti-sickling agents

The drugs tolazoline, clonidine, lofexidine, and fenmetozole were found to inhibit the gelation of hemoglobin S in the order of increasing effectiveness. Only the latter, however, reduced the sickling of red cells significantly and normalized the oxygen affinity of SS blood at 5-10 mM concentrations. Since this level of drug is lower than those reported for many other anti-sickling agents to achieve comparable effects, the 2-imidazoline class of compounds may provide important clues for the development of therapy for sickle cell anemia.     

Effects of phenobarbital and diphenylhydantoin on acute vitamin D3 toxicity in the rat.

Methylene chloride is biotransformed in vivo to carbon monoxide (CO), and increased carboxyhemoglobin (COHb) concentrations occur in animals and man following exposure. Binding of CO to hemoglobin causes a shift to the left in the position of the oxyhemoglobin dissociation curve (ODC), theoretically resulting in a diminished ability to release oxygen in tissue. This study was designed to determine the effect of methylene chloride alone and in combination with CO on altering the ODC. Methylene chloride and CO, alone and in combination, were incubated with rat and human blood in vitro and the position of the ODC was measured by the P50 value. The results indicate clearly that methylene chloride alone had no effect on changing the P50 value of the ODC. It was concluded that any shift to the left of the ODC following exposure to methylene chloride is due solely to CO formed from the biotransformation of methylene chloride. Thus, speculation that methylene chloride increases the affinity of Hb for CO is not supported.     

Changes in haemoglobin oxygen affinity and erythrocytic 2,3-BPG in ethanol-treated rats

1. The effects of alcohol on blood oxygen transport properties were studied in rats after a chronic administration of ethanol in drinking water. 2. Ingestion of ethanol provokes an increase in haemoglobin oxygen affinity (at pH 7.4). This is caused by a drop in the MCHC and erythrocyte 2,3-BPG concentration that give rise to a decrease in the 2,3-BPG/Hb ratio. 3. No changes in haemoglobin fractions were observed. 4. The results indicate a depletion in the red blood cell glycolytic pathways. 5. If metabolic acidosis occurs, the expected loss of blood affinity in vivo due to the fall in pH would be compensated by the changes observed in vitro.     

Substituted 5,5'-diphenyl-2-thioxoimidazolidin-4-one as CB1 cannabinoid receptor ligands: synthesis and pharmacological evaluation

A set of 30 substituted 5,5'-diphenyl-2-thioxoimidazolidin-4-one (thiohydantoins) derivatives was synthesized, and their affinity for the human CB(1) cannabinoid receptor has been evaluated. These compounds are derived from the previously described cannabinoid ligands 5,5'-diphenylimidazolidine-2,4-dione (hydantoins). The replacement of the oxygen by a sulfur leads to an increase of the affinity while the function-i.e., inverse agonism-determined by [(35)S]GTPgammaS experiments remains unaffected. Finally, to evaluate the molecular parameters that could influence the affinity of the thiohydantoins, molecular electrostatic potential as well as lipophilicity calculations were undertaken on representative thiohydantoins and hydantoins derivatives. In conclusion, 5,5'-bis-(4-iodophenyl)-3-butyl-2-thioxoimidazolidin-4-one (31) and 3-allyl-5,5'-bis(4-bromophenyl)-2-thioxoimidazolidin-4-one (32) possess the highest affinity for the CB(1) cannabinoid receptor described to date for the hydantoin and thiohydantoins series when compared in a same bioassay.     

Haemoglobin Rahere (beta Lys-Thr): A new high affinity haemoglobin associated with decreased 2, 3-diphosphoglycerate binding and relative polycythaemia.

A new haemoglobin with increased oxygen affinity, beta82 (EF6) lysine leads to threonine (Hb Rahere), was found during the investigation of a patient who was found to have a raised haemoglobin concentration after a routine blood count. The substitution affects one of the 2, 3-diphosphoglycerate binding sites, resulting in an increased affinity for oxygen, but both the haem-haem interaction and the alkaline Bohr effect are normal in the haemolysate. This variant had the same mobility as haemoglobin A on electrophoresis at alkaline pH but was detected by measuring the whole blood oxygen affinity; it could be separated from haemoglobin A, however, by electrophoresis in agar at acid pH. The raised haemoglobin concentration was mainly due to a reduction in plasma volume (a relative polycythaemia) and was associated with a persistently raised white blood count. This case emphasises the need to measure the oxygen affinity of haemoglobin in all patients with absolute or relative polycythaemia when some obvious cause is not evident.     

Modern oxygen carriers: state of art 1990

Three major components of blood with clearly defined functions are: 1) red cells 2) plasma volume 3) coagulation factors At the present time artificial or synthetic coagulation factors are not available. Plasma volume expanders are well known in clinical practice. The main subject of this paper is the complex area of red cell substitutes with particular reference to the oxygen carrying capacity. The history of blood oxygen carrying capacity is briefly presented with particular reference to contributions by G. S. Adair, C. Bohr, M. Perutz and R. R. Benesch. 3 oxygen carrying developments are discussed in detail: 1) perfluorocarbons 2) artificial red cells 3) stroma-free haemoglobin solution Perfluorocarbons were used as carriers of oxygen in experiments and clinical practice. Before their wide clinical application a number of potential problems must be solved. In selected patients (coronary angioplasty, cerebral thrombosis, cardiopulmonary by-pass) perfluorocarbons (Fluosol D-A) could be considered as beneficial. Artificial red cells (neohaemocytes) are available in some centres in which progress of micro-technology appears to lead to encapsulation of purified human haemoglobin. These preparations should receive more attention. Finally stroma-free haemoglobin solution was the subject of our own research for several years. Our preparation met the requirements of the blood substitute, but had the short half-life in the circulation. Recent advances in chemistry in some centres resulted in the modification of hb molecule and linking with P-5-P. The modified poly hb solution is an ideal oxygen delivering fluid.     

Efaproxiral: a novel radiation sensitiser

Efaproxiral (RSR13) is a synthetic allosteric modifier of haemoglobin that reduces its oxygen binding affinity. By facilitating the release of oxygen from haemoglobin, efaproxiral causes an increase in whole blood P 50 (partial pressure of oxygen which results in 50% haemoglobin saturation) and an increase in the PO2 (partial pressure of oxygen) in the tissue. The therapeutic strategy of enhancing oxygen unloading from haemoglobin to tissue emulates and amplifies physiological tissue oxygenation and can enhance the oxygenation of hypoxic tumours. Since hypoxia is known to decrease the effectiveness of radiation therapy, the use of efaproxiral as a radiation sensitiser may be advantageous. Unlike previous radiation sensitisers, efaproxiral does not need to enter the cancer cells to increase radiosensitivity. Phase I-III trial data have defined the safety profile and dosing of the drug, with the potential benefit for extended survival.     

Efaproxiral: a novel radiation sensitiser

Efaproxiral (RSR13) is a synthetic allosteric modifier of haemoglobin that reduces its oxygen binding affinity. By facilitating the release of oxygen from haemoglobin, efaproxiral causes an increase in whole blood P₅₀ (partial pressure of oxygen which results in 50% haemoglobin saturation) and an increase in the Po₂ (partial pressure of oxygen) in the tissue. The therapeutic strategy of enhancing oxygen unloading from haemoglobin to tissue emulates and amplifies physiological tissue oxygenation and can enhance the oxygenation of hypoxic tumours. Since hypoxia is known to decrease the effectiveness of radiation therapy, the use of efaproxiral as a radiation sensitiser may be advantageous. Unlike previous radiation sensitisers, efaproxiral does not need to enter the cancer cells to increase radiosensitivity. Phase I – III trial data have defined the safety profile and dosing of the drug, with the potential benefit for extended survival.     

The effects of sodium nitroprusside and cyanide on haemoglobin function

Incubation of human blood with 0·3 mM sodium nitroprusside (SNP) or 1 mM potassium cyanide (KCN) for 180 min produced a 600-fold increase in red cell cyanide concentration, a 4% decrease in oxygen capacity and a 6% increase in oxygen affinity. These effects were not reproduced in patients receiving SNP by infusion probably because in the clinical situation much smaller amounts of the drug were used and red cell cyanide levels were lower. The in vitro observations could be explained by cyanide either combining directly with the haemoglobin or encouraging its spontaneous oxidation to methaemoglobin during the incubation. Differences in distribution of cyanide between red cell and plasma following incubation with SNP or KCN and the initial lag phase in accumulation of cyanide in the red cell with SNP provide further evidence that nitroprusside breaks down principally within the red cell.     

Desensitized beta-adrenoceptors of C6-glioma cells have distinct binding properties

Summary When C6-rat glioma cells were incubated for 20 min with beta-adrenoceptor agonists, a part of the beta-adrenoceptors was localized in light density vesicles. These receptors have the same affinity for dihydroalprenolol as plasma membrane receptors but have a lowered affinity for agonists as well as for the hydrophilic beta-adrenoceptor antagonist CGP-12177. The affinity of these vesicular receptors for a variety of hydrophobic beta-blockers as well as for the hydrophilic beta-blocker timolol is compared with that of plasma membrane receptors. None of the ligands investigated showed any difference in affinity but CGP-12177. Both, introducing a lipophilic side chain into CGP-12177 or altering the benzimidazol-2-one ring system of CGP-12177 led to an increase in the affinity of the vesicular receptors. Since in the presence of the poreforming agent alamethicin the same affinity was determined for vesicular receptors as for those located on the plasma membrane, it is concluded that the apparent low affinity of the vesicular receptors in the absence of alamethicin is caused by a membrane barrier. The lower affinity of the vesicular receptors for agonists was only slightly increased by alamethicin.Abbreviations P partition coefficient in octanol/water - CGP-12177 4-(3-tertiary-butylamino-2-hydroxypropoxy)-benzimidazol-2-one hydrochloride - CGP-13674 4-{3-[2-(3,4-dimethoxyphenyl)ethylamino]-2-hydroxypropoxy}-benzimidazol-2-one hydrochloride - CGP-12304 8-(tertiarybutylamino-2-hydroxypropoxy)-benzo[3,2-a]-1,4-oxazin-3[4H]-one hydrochloride - CGP 15224, 8-{3-[2-(3,4-di-methoxy-phenyl)ethylamino]-2-hydroxypropoxy}-benzo-[3,2-a]-1,4-oxazin-3[4H]-one hydrochloride - DMEM Dulbecco's modified Eagle's medium     

Sickle cell disease: role of reactive oxygen and nitrogen metabolites

1. Sickle cell disease (SCD) is an inherited disorder of haemoglobin synthesis that is associated with significant morbidity and mortality due to sequelae of episodic vaso-occlusive events: pain crises and multiorgan damage. The microvascular responses to the initiation, progression and resolution of vaso-occlusive events are consistent with an inflammatory phenotype as suggested by activation of multiple cell types, an oxidatively stressed environment and endothelial cell dysfunction. 2. Decreased anti-oxidant defences in SCD patients and mice are accompanied by activation of enzymatic (NADPH oxidase, xanthine oxidase) and non-enzymatic (sickle haemoglobin auto-oxidation) sources of reactive oxygen species. The resultant oxidative stress leads to dysfunction/activation of arteriolar and venular endothelial cells, resulting in impaired vasomotor function and blood cell-endothelial cell adhesion. 3. Changes in substrate and cofactor availability for endothelial cell nitric oxide synthase may underlie reactive oxygen- and nitrogen-induced events that contribute to SCD-induced vasculopathy. 4. The emerging role of reactive oxygen and nitrogen species in the pathogenesis of SCD provides a platform for the development of novel agents to treat this painful and lethal disease.