Abdullah Haj-Yehia

Effects of the superoxide dismutase-mimetic compound tempol on endothelial dysfunction in streptozotocin-induced diabetic rats.

Evidence exists to support the beneficial effects of superoxide dismutase on endothelial dysfunction induced by hyperglycemia in vitro. In vivo, however, studies of the effects of native superoxide dismutase preparations on the vascular complications accompanying diabetes are limited, and their therapeutic application potential has so far been disappointing. The objective of this study was to evaluate, for the first time in vivo, the effects of long-term administration of tempol, a stable superoxide dismutase-mimic compound, on diabetes-induced endothelial dysfunction in rats. Diabetes was induced by streptozotocin and rats were monitored for 8 weeks with or without treatment with tempol (100 mg/kg, s.c., b.i.d). Diabetic rats showed increased vascular levels of superoxide, which was accompanied by increased levels of the oxidative stress markers malondialdehyde and 8-epi-prostaglandin F(2alpha). In addition, the vasorelaxant as well as the cGMP-producing effects of acetylcholine and glyceryl trinitrate were reduced in diabetic rats. Treatment with tempol abolished not only the differences in the vascular content of superoxide, malondialdehyde and 8-epi-prostaglandin F(2alpha), but also the differences in the relaxation and cGMP responses of aortic rings to both acetylcholine and glyceryl trinitrate between control and diabetic rats. These results support the involvement of reactive oxygen species in mediation of hyperglycemia-induced endothelial dysfunction in vivo, and provide the rationale for potential utilization of stable superoxide dismutase-mimic nitroxides for the prevention of the vascular complications accompanying diabetes.

Microassay of superoxide anion scavenging activity in vitro

We have developed a photometric, platereader-based microassay for superoxide anion scavening activity in vitro. Superoxide anions were generated using a xanthine oxidase/hypoxanthine system and detected by following the reduction of ferricytochrome c at 550 nM. Inhibitory activity was assessed for superoxide dismutase (SOD) and the superoxide anion scavengers tiron and TEMPO together with a number of TEMPO derivatives. The initial rate of change in optical density (OD) at 550 nm, i.e., initial reaction rate, generated by xanthine oxidase (20 mU/ml)/hypoxanthine (100 μM) coupled to ferricytochrome c (100 μM) was effectively abolished by SOD (200 U/ml), tiron (10 mM) and TEMPO (0.3 mM), indicating the involvement of superoxide anions. TEMPO derivatives inhibited the initial reaction rate with the potency order: TEMPO > 4-hydroxy-TEMPO = 4-carboxy-TEMPO. In contrast, 4-hydroxy-TEMPO, which lacks the free radical nitroxide function, was inactive up to 1 mM.

Binding of urokinase to low density lipoprotein-related receptor (LRP) regulates vascular smooth muscle cell contraction.

Urokinase plasminogen activator (uPA) is a multifunctional protein that has been implicated in several physiological and pathological processes involving cell adhesion and migration in addition to fibrinolysis. In a previous study we found that two-chain urokinase plasminogen activator (tcuPA) stimulates phenylephrine-induced vasoconstriction of isolated rat aortic rings. In the present paper we report that uPA−/−mice have a significantly lower mean arterial blood pressure than do wild type mice and that aortic rings from uPA−/− mice show an attenuated contractile response to phenylephrine. In contrast, the blood pressure of urokinase receptor knockout (uPAR−/−) mice and the response of their isolated aortic rings to phenylephrine were normal, indicating that the effect of uPA on vascular contraction is independent of uPAR. Addition of mouse and human uPA almost completely reversed both the impaired vascular contractility and the lower arterial blood pressure in vivo. The in vitro and in vivo effects of infused uPA on aortic contractility and the restoration of normal blood pressure in uPA−/− mice were prevented by antibody to low-density lipoprotein receptor-related protein/α2-macroglobulin receptor (LRP). A modified form of uPA that lacks the kringle failed to restore the blood pressure in uPA−/− mice, notwithstanding having a longer half-life in the circulation. Ligands that regulate the interaction of uPA with LRP, such as PAI-1 or the PAI-1-derived peptide (EEIIMD), abolished the vasoactivity of tcuPA in vitro and in vivo. These studies identify a novel signal transducing cellular receptor pathway involved in the regulation of vascular contractility.

Determination of lipoic acid and dihydrolipoic acid in human plasma and urine by high-performance liquid chromatography with fluorimetric detection.

A highly sensitive method for the determination of alpha-lipoic acid (LA) and dihydrolipoic acid (DHLA) in human plasma and urine has been developed. Samples were acidified and extracted with organic solvent, and the free sulfhydryls of DHLA protected as the dicarboxyethylate by treatment with ethylchloroformate. The free carboxylic function of LA and the SH-protected DHLA were converted into their amide derivatives with the strong fluorophore 2-(4-aminophenyl)-6-methylbenzothiazole in the presence of a coupling agent and a base catalyst. The resulting fluorescent amides of both LA and DHLA were separated on a reversed-phase column (Ultrasphere C8) using simple isocratic elution with acetonitrile-water (80:20) and detected fluorimetrically (excitation 343, emission 423 nm). The method is highly sensitive, reproducible, and is easily applied for the simultaneous determination of LA and DHLA in biological samples.

Urokinase-derived peptides regulate vascular smooth muscle contraction in vitro and in vivo

We examined the effect of urokinase (uPA) and its fragments on vascular smooth muscle cell contraction. Single-chain uPA inhibits phenylepherine (PE) -induced contraction of rat aortic rings, whereas two-chain uPA exerts the opposite effect. Two independent epitopes mediating these opposing activities were identified. A6, a capped peptide corresponding to amino acids 136-143 (KPSSPPEE) of uPA, increased the EC(50) of PE-induced vascular contraction sevenfold by inhibiting the release of calcium from intracellular stores. A6 activity was abolished by deleting the carboxyl-terminal Glu or by mutating the Ser corresponding to position 138 in uPA to Glu. A single-chain uPA variant lacking amino acids 136-143 did not induce vasorelaxation. A second epitope within the kringle of uPA potentiated PE-induced vasoconstriction. This epitope was exposed when single-chain uPA was converted to a two-chain molecule by plasmin. The isolated uPA kringle augmented vasoconstriction, whereas uPA variant lacking the kringle had no procontractile activity. These studies reveal previously undescribed vasoactive domains within urokinase and its naturally derived fragments.

Can we develop improved derivatives of valproic acid

Valproic acid is one of the major antiepileptic drugs. In animal models, valproate showed less anticonvulsant potency than the other three established antiepileptic drugs: phenobarbital, phenytoin and carbamazepine. In addition, two major side-effects, teratogenicity and hepatotoxicity, have been associated with valproate Iherapy. Due to the above and the shortage of new antiepileptic drugs there is a substantial need to develop improved derivatives of valproate. This paper analyses three kinds of valproate derivatives: valpromide, the primary amide of valproate, and its analogues; monoester prodrugs of valproate and an active metabolite of valproate, 2-n-propyl-2-pentenoate. The comparative evaluation was carried out by pharmacokinetic and pharmacodynamic analyses in animals. From the data accumulated so far, we can conclude that 2-n-propyl-2-pentenoatc and/or a valpromide isomer, which does not undergo amide acid biotransformation and preferably is not an epoxide hydrolase inhibitor, may prove to be improved derivatives of the parent compound valproic acid.

Synchronized release of sulpiride and sodium decanoate from HPMC matrices : A rational approach to enhance sulpiride absorption in the rat intestine

AbstractPurpose. (a) To improve the absorption of sulpiride (SP) through the intestinal wall by incorporating it together with sodium decanoate (SD) into erodible matrices, designed to synchronize the release of SP and SD over different periods of time; (b) to test, in vivo the hypothesis that this simultaneous release increases SP absorption from the intestinal lumen. Methods. Matrix tablets, possessing different erosion rates, were prepared by changing the ratios between SD and hydroxypropyl methylcellulose (HPMC). The amounts of HPMC varied from 2.5% to 17% w/w. Double layer tablets, containing similar amounts of SP, SD, and HPMC were used as nonsynchronous controls. The erosion kinetics of the tablets was assessed gravimetrically in vitro in USP basket dissolution apparatus and in vivo in the intestine of the anesthetized rat after intra-intestinal administration. SP absorption was studied after intra-intestinal administration of the different kinds of tablets to anesthetized rats, by monitoring SP blood levels. SP and SD levels in the withdrawn samples from the dissolution systems and blood were analyzed by HPLC. Results. The controlled erosion of the tablets resulted in equal release rates of SP and SD during the initial linear phase of the process. This synchronized release lasted over different time periods depending on the relative amount of HPMC in the formulations (from 1 hour to 4 hours for 2.5 and 17 % w/w of HPMC, respectively). The synchronous matrices increased SP bioavailability after intra-intestinal administration. The increase varied from 1.4 to 2.3-fold for the slow and the fast release formulations, respectively (compared with the nonsynchronous, SD containing control formulations), indicating the ability to control both erosion rate and length of intestinal segment in which absorption is taking place. Conclusions. SP bioavailability after intestinal administration can be improved only if SP is released together with SD along the entire intestinal route. This can be accomplished by the design of synchronous matrices capable of concomitant release of SP and SD despite the differences in their water solubility. The ability to manipulate and control the duration of the synchronous phase of the matrices makes it possible for SP to be absorbed at different parts of the intestine.

2-(4-Carboxyphenyl)-6-N,N-diethylaminobenzofuran: a useful reagent for the sensitive determination of alcohols by high-performance liquid chromatography with fluorimetric detection

A simple and highly sensitive method for the determination of short, medium and long-chain alcohols using high-performance liquid chromatography with fluorimetric detection is described. The alcohols were derivatized to their corresponding esters with (4-carboxyphenyl)-6-N,N-diethylaminobenzofuran. The esterification reaction proceeded rapidly and smoothly in acetonitrile at 60 degrees C with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (a coupling agent) in the presence of 4-dimethylaminopyridine (a base catalyst). The resulting esters of alcohols from methanol to eicosanol (C1-C20-ol) were separated on a reversed-phase column (Ultrasphere C8) with gradient elution (acetonitrile-water) and detected fluorometrically (excitation 387, emission 537 nm). The lower limits of detection (signal-to-noise ratio of 3) for the derivatized alcohols were in the range of 0.2-0.5 pg.

Pharmacokinetic and pharmacodynamic analysis of (E)-2-ene valproyl derivatives of glycine and valproyl derivatives of nipecotic acid

GABA is a major inhibitory neurotransmitter in mammals, whose uptake in glial cells is inhibited by nipecotic acid. In addition to GABA, glycine is an important inhibitory neurotransmitter. Valproic acid (VPA) is one of the four established antiepileptics and (E)-2-ene valproic acid ((E)-2-ene VPA) is its major active metabolite. The described structure-pharmacokinetic-pharmacodynamic relationship (SPPR) study explored the possibility of utilizing valproyl derivatives of glycine and nipecotic acid as new antiepileptics. The pharmacokinetics and pharmacodynamics (anticonvulsant activity and neurotoxicity) of the following conjugation products were investigated: (E)-2-ene valproyl glycinamide (between (E)-2-ene VPA and glycinamide) and valproyl nipecotic acid and valproyl nipecotamide (between VPA and nipecotic acid). Out of the investigated compounds only (E)-2-ene valproyl glycinamide showed a good anticonvulsant profile in both mice and rats due to its better pharmacokinetic and pharmacodynamic profile. (E)-2-ene valproyl glycinamide was more potent than VPA and showed an activity and a safety margin similar to those of its analogous compound valproyl glycinamide. The investigated valproyl derivatives did not operate as chemical drug delivery systems (CDDSs) of glycine or nipecotic acid, but, rather, acted as drugs on their own. (E)-2-ene valproyl glycinamide was partially excreted unchanged in the urine (fe = 7.4%), while its urinary metabolite was (E)-2-ene valproyl glycine. Unlike the new antiepileptic tiagabine, in which nipecotic acid is attached to 4, 4-di-(3-methylthien-2-yl)-3-butenyl and yields an active compound, the conjugation between nipecotic acid or its amide and VPA yielded inactive entities. In contrast to nipecotic acid, the conjugation between VPA or (E)-2-ene VPA and glycinamide gave two active compounds with similar pharmacokinetic and pharmacodynamic profiles.

Development of 3‐nitratomethyl‐proxyl (NMP): A novel, bifunctional superoxide dismutase‐mimic‐nitric oxide‐donor

The vascular endothelium plays a central role in the regulation of physiological functions through the formation, release, and action of various vasoactive factors. Of these, in particular, impairment of activity of locally released nitric oxide (NO) plays a major role in endothelial dysfunction. This dysfunction contributes largely to changes in vascular structure and growth and adhesivity to platelets and leukocytes, resulting in atherosclerosis and thrombosis which ultimately lead to coronary artery disease (CAD). Nitrovasodilators constitute a group of compounds referred to collectively as “NO‐donors.” Of these NO‐donors, the organic nitrate glyceryl trinitrate (GTN) has been the mainstay in treatment of angina pectoris accompanying CAD. Unfortunately, however, early development of tolerance to the vasodilatory effect of the drug, usually accompanied by increased response of blood vessels to endogenous vasoconstrictors (rebound phenomenon), constitutes a major drawback of nitrate therapy. Several mechanisms have been proposed to underlie development of tolerance to organic nitrates and cross‐tolerance to other NO‐donors. Of these, recent reports indicate the primary involvement of superoxide (SO) in mediation of tolerance. Based on these reports and on growing evidence from our laboratories, we herein report the development of a novel organic nitrate; 3‐nitratomethyl‐PROXYL (NMP) that, in addition to being a classical NO‐donor, also possesses a potent antisuperoxide (SOD‐mimic) action. As such, NMP is probably the first compound that can simultaneously and favorably affect both NO and SO. This simultaneous bifunctionality may underlie the potent vasodilatory action of NMP without induction of tolerance. Since the ratio between NO and SO constitutes a major determinant of cellular function, bifunctional agents like NMP may prove useful in the pharmacotherapeutic management of a long series of oxidative stress‐mediated pathologies in which an imbalance between NO and SO exists. Drug Dev. Res. 50:528–536, 2000.