Taher Nassar

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.

Novel double coated nanocapsules for intestinal delivery and enhanced oral bioavailability of tacrolimus, a P-gp substrate drug.

This study proposes a new concept of double coated nanocapsules to improve the oral bioavailability of a P-glycoprotein (P-gp) substrate drug, tacrolimus, without modulating the physiological activity of the P-gp pump. Tacrolimus was incorporated in nanocapsules with different ratios of two polymethacrylate polymers followed by microencapsulation of these nanocapsules within hydroxypropylmethylcellulose using a spray drying technique. The influence of different formulations of tacrolimus administered orally to rats and pigs on the drugs absorption was investigated. Histopathological studies were performed on rats to follow the nanocapsule path in enterocytes. The novel formulations that released mostly drug loaded nanocapsules in the intestine were shown to enhance markedly the oral absorption of tacrolimus. The relative oral bioavailability of tacrolimus was 4.9 and 2.45 fold compared to the commercial product in rats and pigs respectively. Although there is no direct evidence that intact nanocapsules internalized in the enterocytes, numerous small oil cores were detected within the enterocytes showing the potential of P-gp substrates incorporated in such nanocarriers to escape the efflux pump.

Neutralizing the neurotoxic effects of exogenous and endogenous tPA

The clinical use of tissue-type plasminogen activator (tPA) in the treatment of stroke is profoundly constrained by its serious side effects. We report that the deleterious effects of tPA on cerebral edema and intracranial bleeding are separable from its fibrinolytic activity and can be neutralized. A hexapeptide (EEIIMD) corresponding to amino acids 350–355 of plasminogen activator inhibitor type 1 (PAI-1) abolished the tPA-induced increase in infarct size and intracranial bleeding in both mechanical and embolic models of stroke in rats, and reduced brain edema and neuronal loss after traumatic brain injury in pigs. These experiments suggest mechanisms to reduce the neurotoxic effects of tPA without compromising its fibrinolytic activity, through the use of selective antagonists and new tPA formulations.

Oral delivery system prolongs blood circulation of docetaxel nanocapsules via lymphatic absorption

Significance Oral drug delivery is the most convenient administration route for patients. Docetaxel, a potent anticancer drug, elicits severe side effects following intravenous administration. Furthermore, oral docetaxel absorption is prevented by biochemical barriers in the intestine. An oral formulation of docetaxel nanocapsules (NCs) embedded in microparticles was developed and elicited higher plasma docetaxel concentrations than intravenous administration of the commercial product. These unexpected results were explained by the penetration of the docetaxel NCs within the enterocytes, circumventing the barriers, where their coating was reinforced prior reaching, intact, the circulation via the lymphatic system. The oral formulation significantly improves docetaxel anticancer efficacy. This delivery concept has potential for clinical translation, allowing docetaxel chemotherapy to be switched from intravenous to oral delivery. An original oral formulation of docetaxel nanocapsules (NCs) embedded in microparticles elicited in rats a higher bioavailability compared with the i.v. administration of the commercial docetaxel solution, Taxotere. In the present study, various animal studies were designed to elucidate the absorption process of docetaxel from such a delivery system. Again, the docetaxel NC formulation elicited a marked enhanced absorption compared with oral Taxotere in minipigs, resulting in relative bioavailability and Cmax values 10- and 8.4-fold higher, respectively, confirming the previous rat study results. It was revealed that orally absorbed NCs altered the elimination and distribution of docetaxel, as shown in the organ biodistribution rat study, due to their reinforced coating, while transiting through the enterocytes by surface adsorption of apoproteins and phospholipids. These findings were demonstrated by the cryogenic-temperature transmission electron microscopy results and confirmed by the use of a chylomicron flow blocker, cycloheximide, that prevented the oral absorption of docetaxel from the NC formulation in an independent pharmacokinetic study. The lipoproteinated NCs reduced the docetaxel release in plasma and its distribution among the organs. The improved anticancer activity compared with i.v. Taxotere, observed in the metastatic lung cancer model in Severe Combined Immune Deficiency-beige (SCID-bg) mice, should be attributed to the extravasation effect, leading to the lipoproteinated NC accumulation in lung tumors, where they exert a significant therapeutic action. To the best of our knowledge, no study has reported that the absorption of NCs was mediated by a lymphatic process and reinforced during their transit.

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.

Antibody Conjugated PLGA Nanoparticles for Targeted Delivery of Paclitaxel Palmitate: Efficacy and Biofate in a Lung Cancer Mouse Model

Aberrant signaling of the epidermal growth factor receptor (EGFR) is common to a variety of human cancers and is also found to be over-expressed in most cases of non-small cell lung cancer. For the development of a molecularly targeted therapy, cetuximab-conjugated nanoparticles (immunonanoparticles, INPs) are designed and loaded with the lipophilic paclitaxel palmitate (pcpl) prodrug. Oleyl cysteineamide (OCA) is synthesized whereby its amphiphilic nature enables interfacial anchoring and thiol surface functionalization of PLGA NPs, facilitating bioconjugation to cetuximab by thioether bonds. It is demonstrated that the in vitro targeting efficiency and improved cellular internalization and cytotoxicity of this targeted delivery system in lung cancer cells over-expressing EGFR. A quantitative measure of the high binding affinity of INPs to EGFR is demonstrated using surface plasmon resonance. In vivo tolerability and enhanced efficacy of cetuximab pcpl INPs in a metastatic lung cancer model are reported. Its therapeutic efficacy in A549-luc-C8 lung tumors is shown using non-invasive bioluminescent imaging. Intravenous administration of cetuximab pcpl INPs to mice results in significantly higher inhibition of tumor growth and increased survival rates as compared to the non-targeted drug solution, drug-loaded nanoparticles or blank INPs. Pharmacokinetics and organ biodistribution of the prodrug and parent drug are evaluated by LC-MS/MS in lung tumor bearing mice. No enhanced total accumulation of nanoparticles or INPs is found at the tumor tissue. However, persistent pcpl levels with sustained conversion and release of paclitaxel are observed for the encapsulated prodrug possibly suggesting the formation of a drug reservoir. The overall results indicate the potential of this promising targeted platform for the improved treatment of lung cancer and other EGFR positive tumors.

A safety and tolerability study of differently-charged nanoparticles for local pulmonary drug delivery

Nanoparticle (NP) based drug delivery systems provide promising opportunities in the treatment of lung diseases. Here we examined the safety and tolerability of pulmonary delivered NPs consisting of PEG-PLA as a function of particle surface charge. The rationale for such a comparison should be attributed to the differential pulmonary toxicity of positively and negatively charged PEG-PLA NP. Thus, the local and systemic effects of pulmonary administered NPs were investigated following 5days of daily endotracheal instillation to BALB/c mice that were euthanized on the eighth or nineteenth day of the experiment. We collected bronchoalveolar lavages and studied hematological as well as histochemistry parameters. Notably, the cationic stearylamine based PEG-PLA NPs elicited increased local and systemic toxic effects both on the eighth and nineteenth day. In contrast, anionic NPs of similar size were much better tolerated with local inflammatory effects observed only on the eighth experimental day after pulmonary instillation. No systemic toxicity effect was observed although a moderate change was noted in the platelet count that was not considered to be of clinical significance. No pathological observations were detected in the internal organs following instillation of anionic NPs. Overall these observations suggest that anionic PEG-PLA NPs are useful pulmonary drug carriers that should be considered as a promising therapeutic drug delivery system.

Blood–brain barrier permeability and tPA-mediated neurotoxicity

Tissue type plasminogen activator (tPA) can induce neuronal apoptosis, disrupt the blood-brain barrier (BBB), and promote dilation of the cerebral vasculature. The timing, sequence and contributions of these and other deleterious effects of tPA and their contribution to post-ischemic brain damage after stroke, have not been fully elucidated. To dissociate the effects of tPA on BBB permeability, cerebral vasodilation and protease-dependent pathways, we developed several tPA mutants and PAI-1 derived peptides constructed by computerized homology modeling of tPA. Our data show that intravenous administration of human tPA to rats increases BBB permeability through a non-catalytic process that is associated with reversible neurotoxicity, brain damage, mortality and contributes significantly to its brief therapeutic window. Furthermore, our data show that inhibiting the effect of tPA on BBB function without affecting its catalytic activity, improves outcome and significantly extends its therapeutic window in mechanical as well as in thromboembolic models of stroke.

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.