In vivo Evaluation of Self Emulsifying Drug Delivery System for Oral Delivery of Nevirapine.

Nevirapine is a highly lipophilic and water insoluble non-nucleoside reverse transcriptase inhibitor used for the treatment of HIV-1 infection. Lymphoid tissue constitutes the major reservoir of HIV virus and infected cells in HIV-infected patients. Self-emulsifying drug delivery system, using long chain triglycerides, is a popular carrier of drugs due to their ability to transport lipophilic drugs into the lymphatic circulation. However, HIV/AIDS patients experience a variety of functional and anatomical abnormalities in gastrointestinal tract that result in diarrhoea and nutrient malabsorption. Medium chain triglycerides are readily absorbed from the small bowel under conditions in which the absorption of long chain triglycerides is impaired. Therefore, nevirapine self-emulsifying drug delivery system containing medium chain fatty acid, caprylic acid and a solubilizer, Soluphor(®) P (2-pyrrolidone) was developed and found to be superior to the marketed conventional suspension with respect to in vitro diffusion and ex vivo intestinal permeability. This self-emulsifying drug delivery system has now been further investigated for in vivo absorption in an animal model. The contribution of caprylic acid and Soluphor(®) P on in vivo absorption of nevirapine was also studied in the present study. The bioavailability of nevirapine from self-emulsifying drug delivery system, after oral administration, was 2.69 times higher than that of the marketed suspension. The improved bioavailability could be due to absorption of nevirapine via both portal and intestinal lymphatic routes. The study indicates that medium chain or structured triglycerides can be a better option to develop self-emulsifying drug delivery system for lipophilic and extensively metabolised drugs like nevirapine for patients with AIDS-associated malabsorption.

Adrenalectomy potentiates the anti-inflammatory activity of a calcium channel blocker.

Calcium movement is important in the activation of inflammatory cells. Prior studies have shown that calcium channel blockers (CCBs) inhibit carrageenan-induced paw edema in rats by acting at pituitary and hypothalamic levels. Here we evaluated the role of calcium channel blockers and their regulation via the hypothalamus-pituitary-adrenal (HPA) axis in acute and chronic models of rat paw inflammation, using both carrageenan (acute) and formalin (chronic) as inflammation inducers. Adrenalectomized (ADX) and intact Sprague Dawley rats (n = 4 per group) weighing 150-250 each were treated with an intraperitoneal injection of nifedipine (400 microg/kg) or vehicle. Edema was assessed plethysmometrically by evaluating paw volume changes. The results show a significant contradiction between acute and chronic inflammation data. In intact animals, nifedipine demonstrated a significant anti-inflammatory effect in the acute inflammatory model but not in the chronic model. In adrenal-ectomized animals, the anti-inflammatory effect of nifedipine was significantly enhanced both in acute and chronic models. We conclude that adrenalectomy plays a significant role in modulating the inflammatory pathway in the presence of calcium channel blockers.

Anti-ulcer activity of Adhatoda vasica Nees.

Adhatoda vasica Nees (Acantheceae), commonly known as Vasaka, is a well-known plant in indigenous systems of medicine and is used for its beneficial effects, particularly in bronchitis. The present investigation was carried out to study the anti-ulcer activity of Adhatoda vasica leaves using two ulcer models (1) Ethanol-induced, and (2) Pylorus ligation plus aspirin-induced models. Adhatoda vasica leaf powder showeda considerable degree of anti-ulcer activity in experimental rats when compared with a control. The highest degree of activity (80%) was observed in the ethanol-induced ulceration model. Results of the study suggest that in addition to its classically established pharmacological activities, the plant also has immense potential as an anti-ulcer agent of great therapeutic relevance.

Permanent partial phenotypic correction and tolerance in a mouse model of hemophilia B by stem cell gene delivery of human factor IX.

Immune responses against an introduced transgenic protein are a potential risk in many gene replacement strategies to treat genetic disease. We have developed a gene delivery approach for hemophilia B based on lentiviral expression of human factor IX in purified hematopoietic stem cells. In both normal C57Bl/6J and hemophilic 129/Sv recipient mice, we observed the production of therapeutic levels of human factor IX, persisting for at least a year with tolerance to human factor IX antigen. Secondary and tertiary recipients also demonstrate long-term production of therapeutic levels of human factor IX and tolerance, even at very low levels of donor chimerism. Furthermore, in hemophilic mice, partial functional correction of treated mice and phenotypic rescue is achieved. These data show the potential of a stem cell approach to gene delivery to tolerize recipients to a secreted foreign transgenic protein and, with appropriate modification, may be of use in developing treatments for other genetic disorders.

Gene therapy progress and prospects: fetal gene therapy--first proofs of concept--some adverse effects.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease based on the hypothesis that prenatal intervention may avoid the development of severe manifestations of early-onset disease, allow targeting of otherwise inaccessible tissues including expanding stem cell populations, induce tolerance against the therapeutic transgenic protein and thereby provide permanent somatic gene correction. This approach is particularly relevant in relation to prenatal screening programmes for severe genetic diseases as it could offer prevention as a third option to families faced with the prenatal diagnosis of a genetically affected child. Most investigations towards in utero gene therapy have been performed on mice and sheep fetuses as model animals for human disease and for the application of clinically relevant intervention techniques such as vector delivery by minimally invasive ultrasound guidance. Other animals such as dogs may serve as particular disease models and primates have to be considered in immediate preparation for clinical trials. Proof of principle for the hypothesis of fetal gene therapy has been provided during the last 2 years in mouse models for Crigler Najjar Disease, Leber's congenital amaurosis, Pompe's disease and haemophilia B showing long-term postnatal therapeutic effects and tolerance of the transgenic protein after in utero gene delivery. However, recently we have also observed a high incidence of liver tumours after in utero application of an early form of third-generation equine infectious anaemia virus vectors with SIN configuration. These findings highlight the need for more investigations into the safety and the ethical aspects of in utero gene therapy as well as for science-based public information on risks and benefits of this preventive gene therapy approach before application in humans can be contemplated.

Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero.

Cystic fibrosis is a common lethal genetic disease caused by functional absence of the cystic fibrosis transmembrane conductance regulator (CFTR). Although a candidate disease for in utero gene therapy, demonstration of potentially therapeutic levels of transgene expression in the fetal airways after minimally invasive gene delivery is a mandatory prerequisite before application of this approach in humans can be considered. We report here on the delivery of a beta-galactosidase expressing adenovirus directly to the airways of fetal sheep in utero using ultrasound-guided percutaneous injection of the trachea in the fetal chest. Injection of adenoviral particles to the fetal airways was not associated with mortality and resulted in low-level expression in the peripheral airways. However, complexation of the virus with DEAE dextran, which confers a positive charge to the virus, and pretreatment of the airways with Na-caprate, which opens tight junctions, increased transgene expression, and a combination of these two enhancers resulted in widespread and efficient gene transfer of the fetal trachea and bronchial tree. Using a percutaneous ultrasound-guided injection technique, we have clearly demonstrated proof of principle for substantial transgene delivery to the fetal airways providing levels of gene expression that could be relevant for a therapeutic application of CFTR expressing vectors.

The hopes and fears of in utero gene therapy for genetic disease--a review.

Somatic gene delivery in utero is a novel approach to gene therapy for genetic disease. It is based on the concept that application of gene therapy vectors to the fetus in utero may prevent the development of early disease related tissue damage, may allow targeting of otherwise inaccessible organs, tissues and still expanding stem cell populations and may also provide postnatal tolerance against the therapeutic transgenic protein. This review outlines the hypothesis and scientific background of in utero gene therapy and addresses some of the frequently expressed concerns raised by this still experimental, potentially preventive gene therapy approach. We describe and discuss the choice of vectors, of animal models and routes of administration to the fetus. We address potential risk factors of prenatal gene therapy such as vector toxicity, inadvertent germ line modification, developmental aberration and oncogenesis as well as specific risks of this procedure for the fetus and mother and discuss their ethical implications.

Long-term transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice.

Inefficient gene transfer, inaccessibility of stem cell compartments, transient gene expression, and adverse immune and inflammatory reactions to vector and transgenic protein are major barriers to successful in vivo application of gene therapy for most genetic diseases. Prenatal gene therapy with integrating vectors may overcome these problems and prevent early irreparable organ damage. To this end, high-dose attenuated VSV-G pseudotyped equine infectious anaemia virus (EIAV) encoding beta-galactosidase under the CMV promoter was injected into the fetal circulation of immuno-competent MF1 mice. We saw prolonged, extensive gene expression in the liver, heart, brain and muscle, and to a lesser extent in the kidney and lung of postnatal mice. Progressive clustered hepatocyte staining suggests clonal expansion of cells stably transduced. We thus provide proof of principle for efficient gene delivery and persistent transgene expression after prenatal application of the EIAV vector and its potential for permanent correction of genetic diseases.

Involvement of endometrial membrane sulphydryl groups in blastocyst implantation: sulphydryl groups as a potential target for contraceptive research.

The role of membrane sulphydryl groups in blastocyst implantation was studied by masking the membrane sulphydryl groups in the endometrium of Swiss albino mice, Mus musculus, using 10(-5) M cobalt chloride and 0.05 mM as well as 0.005 mM n-ethylmaleimide. Here we show that the blocking of sulphydryl groups with cobalt resulted in a decrease in superoxide radical surge and an increase in superoxide dismutase levels at the time of implantation. We hypothesize that it may be due to either a decrease in membrane fluidity or the unavailability of sulphydryl groups of endometrial membrane, thus preventing blastocyst implantation. These sulphydryl groups can be targeted for future contraceptive research.

Improvement in circulating superoxide dismutase levels: role of nonsteroidal anti-inflammatory drugs in rheumatoid arthritis.

Superoxide anion radical plays a significant role in inflammation, like rheumatoid arthritis. Superoxide dismutase enzyme known to dismutate superoxide anion radical does not play any significant role in this multisystem disease. This paper reports that very low levels of circulating superoxide dismutase levels are observed in patients with rheumatoid arthritis and these levels significantly improve with NSAID therapy. The possible mechanism of the action is discussed.

A regulatory role of sulfhydryl groups in modulation of sperm membrane conformation by heavy metals: sulfhydryl groups as markers for infertility assessment.

Sperm membrane sulfhydryl groups, when masked by heavy metals like cobalt and copper at very low concentration (10(-9)) shows inhibition of lipid peroxidation and superoxide dismutase activity. Inhibition of lipid peroxidation is suggested to lead to reduction in membrane fluidity, a prerequisite for normal sperm function. Augmentation of lipid peroxidation by pentoxifylline in oligospermia provides evidence to this hypothesis that membrane sulfhydryls play a regulatory role in membrane modulation. It is therefore suggested that these sulfhydryl groups can be used as a tool for infertility assessment in unexplained male infertility.

Modulation of sperm membrane conformation by pentoxifylline in oligospermia: a biophysical investigation of sperm membrane in vitro.

Low superoxide anion radical levels, low membrane fluidity and high superoxide dismutase activity are some of the defects existing in oligospermia. The phosphodiesterase inhibitor pentoxifylline has been used as a motility stimulant in the treatment of oligospermia in vitro. The mechanism of its action is unclear. Our results show significant inhibition of superoxide dismutase, increase in lipid peroxidation and membrane fluidity after pentoxifylline treatment. A biophysical modulation of the sperm membrane by an oxyradical mechanism in oligospermia is discussed.

Free radical induced biophysical modification of membrane lipids: a novel mechanism proposed for a haemorheological alteration induced by 4,5' 8-trimethyl psoralen.

Haemostasis is the spontaneous arrest of bleeding from damaged blood vessel. Platelet aggregation is the major event in this process. A number of drugs are known to inhibit this process of platelet aggregation. 4,5' 8-trimethyl psoralen a naturally occurring furocoumarin is used as a photochemotherapeutic agent in PUVA therapy. It shows antithrombotic action by preventing platelet aggregation induced by adenosine diphosphate in vitro. It is proposed that a free radical induces a lipid phase transition of the membrane lipids, the platelet membrane undergoes certain biophysical alteration leading to increased membrane fluidity resulting in a loss of aggregating ability.

Metal binding and resultant loss of phototoxicity of alpha-terthienyl: metal detoxification versus alpha-terthienyl inactivation.

The members of the plant family asteraceae are noted for their cosmopolitan distribution and their versatility is attributed to be largely due to their morphological adaptations. Interestingly members of this family are endowed with rich levels of secondary plant metabolites, many of which are photochemically active (Bakker et al. 1979; Kagan et al. 1989). The secondary plant metabolite alpha-terthienyl derived from the plant family asteraceae is among the new class of light activated insecticide. The photobiocidal effects associated with alpha-terthienyl in presence of sunlight and ultraviolet light (300-400 nm), has stimulated a great deal of interest in its toxic mechanism of action against a number of organisms including phytopathogenic fungi, nematodes and mosquito larvae. Trials under tropical conditions indicate a very high level of activity as a larvicide to mosquito. There is no cross resistance to this compound in malathion resistant mosquito larvae (Arnason et al. 1989). Even though many researchers feel that the phototoxicity of secondary plant metabolites has arisen independently many times in evolution as a defense mechanism, the physiological impact of such biologically active compounds in the plant producing them also should be addressed (Arnason et al. 1987). Moreover, the accumulation of secondary plant metabolites in the roots of many asteraceae members as is the case with alpha-terthienyl in marigold (Tagetes sp.) roots, hints towards their functional divergence. In this study, we demonstrate a potent heavy metal quenching activity of alpha-terthienyl.

Photophysical alterations of erythrocyte membrane by 4,5',8-trimethyl psoralen: membrane response towards psoralen phototherapy.

Despite the wide and versatile use of psoralen phototherapy in various dermal diseases including psoriasis and leukoderma, the proposed mechanism of action of psoralen is disputed. This study launches an original documentation of free-radical resulting in erythrocyte membrane damage studied by spin-labeling using the probes malnet and 5 doxyl stearate as protein and lipid domains, respectively. Both Type I and Type II photodynamics appear plausible and the therapeutic aspects of this innovation are being verified.

Generation of superoxide anion radical by alpha-terthienyl in the anal gills of mosquito larvae Aedes aegypti: a new aspect in alpha-terthienyl phototoxicity.

The present study documents that the secondary plant metabolites, especially alpha-terthienyl, exert phototoxic action through inhibition of certain enzymes and generation of singlet oxygen. Some of the reports have emerged exhibiting involvement of free radical generation in vitro by alpha-terthienyl. We provide evidence for the generation of a free radical viz., superoxide anion radical, by alpha-terthienyl employing spin-trapping techniques, probably due to the extension of the latter reaction. On the basis of this observation the phototoxic action of alpha-terthienyl on Aedes aegypti larvae is explained.

Enhanced axial symmetry at the Fe(3+)-heme center of peroxidase by ascorbate: a basis for the ascorbate-dependent peroxidase action.

In the absence of its substrate hydrogen peroxide, peroxidase exhibits perturbations in its Fe(3+)-heme center, when incubated with ascorbic acid. The electron paramagnetic pattern sprang towards a higher g-value side, denoting a sharpening of the rhombic axial symmetry around the heme-center. The interpretation is that the ascorbate dependent peroxidase action starts with the formation of an Fe(3+)-ascorbate charge transfer complex intermediate.

Superoxide dismutase activity regulation by spermine: a new dimension in spermine biochemistry and sperm development.

Amines are a group of highly important compounds of biological importance; they are known promoters of cell growth, can complex with nucleic acids and can stimulate DNA-primed RNA polymerase activity. Spermine, a polyamine abundantly present in the secretions of the male accessory sex organs, has received no functional attributes till date. This study had been a pioneer attempt to validate the hypothesis of a metalloenzyme activity modulation by spermine and implicit correlations have been drawn.

Superoxide dismutase activation in thyroid and suppression in adrenal. Novel pituitary regulatory routes.

This study reports that administration of TSH in young female mice results in a concomitant augmentation of SOD activity in the thyroid gland. A strong thyroid-adrenal interdependence was also evident in the form of a marked loss of SOD activity in the adrenal gland in response to TSH administration. Very recently SOD/O2.- system had been identified as a potent H2O2 generator which provides substrate for the action of key enzyme in thyroxine and progesterone biosynthesis, viz. the peroxidase. Thus, these results strongly suggest that trophic hormones tonically stimulate hormone biosynthesis by modulating activation/suppression of specific enzymes, which could be the basis of the tuning sequence.

Superoxide dismutase in the anal gills of the mosquito larvae of Aedes aegypti: its inhibition by alpha-terthienyl.

The anal gills of the mosquito larvae of Aedes aegypti were shown to possess superoxide dismutase (EC 1.15.1.1) activity, which increased with the maturation of the larvae from instar 1 to instar 4. This enzyme was highly inhibited upon treatment of the larvae with alpha-terthienyl (2,2':5,2"-terthiophene) and subsequent exposure to long-wave ultraviolet light. Inhibition also occurred with treatment of the crude enzyme extract in a similar fashion. Exposure of the enzyme to the ultraviolet light alone or the treatment of the enzyme with alpha-terthienyl in darkness could not manifest this inhibition. This finding adds a new dimension to the complex mechanism(s) proposed for the photodynamic toxicity of alpha-terthienyl.