Preclinical evaluation of radiation and systemic, RGD-targeted, adeno-associated virus phage-TNF gene therapy in a mouse model of spontaneously metastatic melanoma.

The incidence of melanoma in the United States continues to rise, with metastatic lesions notoriously recalcitrant to therapy. There are limited effective treatment options available and a great need for more effective therapies that can be rapidly integrated in the clinic. In this study, we demonstrate that the combination of RGD-targeted adeno-associated virus phage (RGD-AAVP-TNF) with hypofractionated radiation therapy results in synergistic inhibition of primary syngeneic B16 melanoma in a C57 mouse model. Furthermore, this combination appeared to modify the tumor microenvironment, resulting in decreased Tregs in the draining LN and increased tumor-associated macrophages within the primary tumor. Finally, there appeared to be a reduction in metastatic potential and a prolongation of overall survival in the combined treatment group. These results indicate the use of targeted TNF gene therapy vector with radiation treatment could be a valuable treatment option for patients with metastatic melanoma.

Developmental exposure to lead (Pb) alters the expression of the human tau gene and its products in a transgenic animal model.

Tauopathies are a class of neurodegenerative diseases associated with the pathological aggregation of the tau protein in the human brain. The best known of these illnesses is Alzheimer's disease (AD); a disease where the microtubule associated protein tau (MAPT) becomes hyperphosphorylated (lowering its binding affinity to microtubules) and aggregates within neurons in the form of neurofibrillary tangles (NFTs). In this paper we examine whether environmental factors play a significant role in tau pathogenesis. Our studies were conducted in a double mutant mouse model that expressed the human tau gene and lacked the gene for murine tau. The human tau mouse model was tested for the transgene's ability to respond to an environmental toxicant. Pups were developmentally exposed to lead (Pb) from postnatal day (PND) 1-20 with 0.2% Pb acetate. Mice were then sacrificed at PND 20, 30, 40 and 60. Protein and mRNA levels for tau and CDK5 as well as tau phosphorylation at Ser396 were determined. In addition, the potential role of miRNA in tau expression was investigated by measuring levels of miR-34c, a miRNA that targets the mRNA for human tau, at PND20 and 50. The expression of the human tau transgene was altered by developmental exposure to Pb. This exposure also altered the expression of miR-34c. Our findings are the first of their kind to test the responsiveness of the human tau gene to an environmental toxicant and to examine an epigenetic mechanism that may be involved in the regulation of this gene's expression.

Melatonin increases intracellular calcium in the liver, muscle, white adipose tissues and pancreas of diabetic obese rats.

Melatonin, a widespread substance with antioxidant and anti-inflammatory properties, has been found to act as an antidiabetic agent in animal models, regulating the release and action of insulin. However, the molecular bases of this antidiabetic action are unknown, limiting its application in humans. Several studies have recently shown that melatonin can modify calcium (Ca(2+)) in diabetic animals, and Ca(2+) has been reported to be involved in glucose homeostasis. The objective of the present study was to assess whether the antidiabetic effect of chronic melatonin at pharmacological doses is established via Ca(2+) regulation in different tissues in an animal model of obesity-related type 2 diabetes, using Zücker diabetic fatty (ZDF) rats and their lean littermates, Zücker lean (ZL) rats. After the treatments, flame atomic absorption spectrometry was used to determine Ca(2+) levels in the liver, muscle, main types of internal white adipose tissue, subcutaneous lumbar fat, pancreas, brain, and plasma. This study reports for the first time that chronic melatonin administration (10 mg per kg body weight per day for 6 weeks) increases Ca(2+) levels in muscle, liver, different adipose tissues, and pancreas in ZDF rats, although there were no significant changes in their brain or plasma Ca(2+) levels. We propose that this additional peripheral dual action mechanism underlies the improvement in insulin sensitivity and secretion previously documented in samples from the same animals. According to these results, indoleamine may be a potential candidate for the treatment of type 2 diabetes mellitus associated with obesity.

Mini review on blood-brain barrier penetration of pyridinium aldoximes.

This paper reviews the blood-brain barrier (BBB) penetration of newly developed pyridinium aldoximes. Pyridinium aldoximes are highly charged hydrophilic compounds used in the treatment of subjects exposed to organophosphonates because they are effective as acetylcholinesterase reactivators. Pyridinium aldoximes have antidotal effects against poisoning with cholinesterase inhibitors, a frequent problem affecting people working with organophosphate-based insecticides and pesticides. Toxic organophosphonate products such as sarin and tabun can be used by terrorists as chemical warfare agents. This poses a severe challenge to all innocent and peace-loving people worldwide. This review gives a brief summary of BBB transporters and description of the current in vitro and in vivo methods for the characterization of BBB penetration of established and novel pyridinium aldoximes. The authors provide a putative mechanism of penetration, outline some future ways of formulation and discuss the possible advantages and disadvantages of increasing BBB penetration.

Mitigated Tregs and augmented Th17 cells and cytokines are associated with severity of experimental autoimmune neuritis.

Experimental autoimmune neuritis (EAN), an animal model of human Guillain-Barré syndrome, has long been considered as a T helper (Th) 1 cell-mediated autoimmune disorder. However, deficiency of IFN-γ, a signature Th1 cytokine, aggravated EAN, with features of elevated production of IL-17A, despite an alleviated systemic Th1 immune response. We hypothesized that Th17 cells and their cytokines might play a pathogenic role in EAN. To further clarify the roles of these Th and regulatory T cell (Treg) cytokines in the pathogenesis of EAN and their interrelationship, we investigated the expression of Th1/Th2/Th17/Treg cytokines in EAN in this study. We found that the levels of Th17 cells and IL-17A in cauda equina (CE)-infiltrating cells and splenic mononuclear cells (MNCs) as well as in serum paralleled the disease evolution, which increased progressively during the initiation stage and reached higher value at the peak of EAN. The same pattern was also noticed for the expression of IL-22. The diverse expression profiles of FoxP3, IL-17 receptors A and C were seen in CE-infiltrating cells and splenic MNCs in EAN. These findings indicate a major pro-inflammatory role of Th17 cells and IL-17A in the pathogenesis of EAN. Therapeutic interventions may be focused upon inhibiting Th17 cells and their cytokines in the early phase of EAN, so as to delay and suppress clinical signs of the disease, which has relevance for future studies on pathogenesis and treatment of GBS in humans.

Frondoside A enhances the antiproliferative effects of gemcitabine in pancreatic cancer.

Pancreatic cancer has a very poor prognosis. While gemcitabine is the mainstay of therapy and improves quality of life, it has little impact on survival. More effective treatments are desperately needed for this disease. Frondoside A is a triterpenoid glycoside isolated from the Atlantic sea cucumber, Cucumaria frondosa. Frondoside A potently inhibits pancreatic cancer cell growth and induces apoptosis in vitro and in vivo. The aim of the present study was to investigate whether frondoside A could enhance the anti-cancer effects of gemcitabine. Effects of frondoside A and gemcitabine alone and in combination on proliferation were investigated in two human pancreatic cancer cell lines, AsPC-1 and S2013. To investigate possible synergistic effects, combinations of low concentrations of the two drugs were used for a 72 h treatment period in vitro. Growth inhibition was significantly greater with the drug combinations than their additive effects. Combinations of frondoside A and gemcitabine were tested in vivo using the athymic mouse model. Xenografts of AsPC-1 and S2013 cells were allowed to form tumours prior to treatment with the drugs alone or in combination for 30 days. Tumours grew rapidly in placebo-treated animals. Tumour growth was significantly reduced in all treatment groups. At the lowest dose tested, gemcitabine (4 mg/kg/dose), combined with frondoside A (100 µg/kg/day) was significantly more effective than with either drug alone. To conclude: The present data suggest that combinations of frondoside A and gemcitabine may provide clinical benefit for patients with pancreatic cancer.

Metabolism of selegiline [(-)-deprenyl)].

Selegiline (1) [(-)-deprenyl] is used to treat patients with Parkinson's disease. Nevertheless, in much higher doses it has beneficial effects in depression, and dementia of the aged patients. Selegiline (1) undergoes a complex metabolic pathway. Its major metabolites include (-)-desmethyldeprenyl (2), (-)-methamphetamine (3) and (-)-amphetamine (4), deprenyl-N-oxide (5) and formaldehyde (6) as a small metabolic fragment. In addition, more than 40 minor metabolites of selegiline (1) have also been either detected or proposed by investigators and researchers. This review analyses the pharmacological activity, generation pathway and the detection method of the major metabolites of selegiline (1).

A study on ovine pneumonic pasteurellosis: isolation and identification of Pasteurellae and their antibiogram susceptibility pattern in Haramaya District, Eastern Hararghe, Ethiopia.

BACKGROUND: Sheep constitute the second major component of livestock in Ethiopia. However, efficient utilization of this potential resource is hampered by combination of health problems, poor management and feed shortage. Haramaya district is one of the remote settings in Ethiopia where information about the livestock disease is not well documented. Hence this study was conducted to determine the causative agents and their antimicrobial susceptibility pattern of bacterial Pasteurella isolates among pneumonic ovine in Haramaya district, Eastern Hararghe, Ethiopia. RESULTS: Out of 256 samples examined, Pasteurella was isolated in 64 (25%), of which 38 (59.4%) were from lungs and 26 (40.6%) were from nasal cavities. 87.5% of the isolates were Mannheimia haemolytica and 12.5% were Pasteurella multocida. All of the isolates from the lungs were Mannheimia haemolytica whereas 69% of the isolates from nasals cavities were Mannheimia haemolytica. Age and body temperature were significantly associated with Pasteurella isolates from clinic (P < 0.05). Despite diverse in the site of origins, the isolates exhibited uniformity in sensitivity to a majority of the antibacterial agents. The most effective drug was Cholramphenicol (100%) followed by Sulfamethoxazole (89.1%) and Tetracycline (84.4%). Both species were completely resistant to Gentamycin and Vancomycin. CONCLUSION: Mannheimia haemolytica is the most common cause of ovine pneumonic pasteurellosis in the study area. The isolates were susceptible to limited antimicrobial agents. Therefore, the antimicrobial susceptibility test should be conducted before treatment, except for critical cases.

Temporal variations of Hsp60 and HSF-1 in primary rat myocardial cells in vitro under heat stress.

The mechanisms involved in sudden animal death due to acute heart failure during heat stress are not well understood. We examined the relationship between heat stress-induced variations of protective Hsp60 and expression of its regulatory factor, HSF-1, in heat-stressed primary myocardial cells of neonatal rats in vitro through cardiac enzyme detection, immunoblotting, immunocytochemistry, and qPCR. Increases in cardiac damage-related enzyme levels demonstrated injury to myocardial cells after heat exposure at 42°C. Hsp60 expression levels fluctuated during heat stress; they decreased significantly after 20 min, then increased at 120 min and decreased again at 360 min after initiation of heat stress. The highest levels of Hsp60 were observed at 240 min, while the lowest were at 60 min. Damage to myocardial cells was characterized by increases in cardiac enzyme levels and low levels of Hsp60 due to functional disorder of myocardial cells at early stages of heat stress. However, the significant induction of hsp60 mRNA levels from the beginning up to 240 min of heat stress was not consistent with the classic regulatory mechanisms that link transcription and translation, suggesting that Hsp60 expression is delayed due to loss of Hsp60 during the early stages of heat stress. hsf-1 mRNA levels were significantly increased from 10 min of heat stress; however, HSF-1 protein levels did not simultaneously increase, indicating that HSF-1 is not the sole regulator of Hsp60 expression.

Daily subacute paraquat exposure decreases muscle function and substantia nigra dopamine level.

The use of the herbicide paraquat (1,1'-dimethyl-4,4'-bipyridylium dichloride; PQ) which is widely used in agriculture is known to cause dopaminergic neurotoxicity. However, the mechanisms underlying this effect are not fully understood. This present study investigated the behavioral manifestations, motor coordination, and dopaminergic neurodegeneration following exposure to PQ. Male rats were injected with PQ (10 mg/kg i.p.) daily for three weeks. Rotarod systems were used for measuring locomotor activity and motor coordination. The effects of PQ on dorsiflexor, electrophysiologically-induced muscle contraction were studied. Dopamine concentrations in the ventral mesencephalon were measured by high performance liquid chromatography and the number of dopaminergic neurons in substantia nigra pars compacta was estimated by tyrosine hydroxylase immunohistochemistry. PQ induced difficulty in movement and significant reduction in motor activity and twitch tension at the dorsiflexor skeletal muscle. The number of tyrosine hydroxylase positive neurons was significantly less in the substantia nigra pars compacta and nigral dopamine level was significantly reduced in PQ treated animals (20.4+/-3.4 pg/mg) when compared with control animals (55.0+/-2.4 pg/mg wet tissue). Daily treatment of PQ for three weeks induces selective dopaminergic neuronal loss in the substantia nigra and significant behavioral and peripheral motor deficit effects.

Blockade of inhibitors of apoptosis (IAPs) in combination with tumor-targeted delivery of tumor necrosis factor-α leads to synergistic antitumor activity.

In the current study, we examined whether the combination of tumor vasculature-targeted gene therapy with adeno-associated virus bacteriophage-tumor necrosis factor-α (AAVP-TNF-α) and/or the orally administered LCL161, an antagonist of inhibitors of apoptosis proteins (IAPs), enhanced antitumor efficacy without systemic toxicity. M21 human melanoma xenografts were grown subcutaneously in nude mice. Mice were treated according to one of four treatment regimens: AAVP-TNF-α alone (AAVP-TNF-α plus sodium acetate-acetic acid (NaAc) buffer) via tail vein injection; LCL161 alone (phosphate-buffered saline (PBS) plus LCL161) via oral gavage; AAVP-TNF-α plus LCL161; and PBS plus NaAc Buffer as a control group. Tumor volume, survival and toxicity were analyzed. AAVP trafficking and TNF-α production in vivo were detected on days 7 and 21 by real-time PCR, enzyme-linked immunosorbent assay and immunofluorescence. The levels of apoptosis and activation of caspases were assessed on days 7 and 21 by TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling) and immunofluorescence assays. Our results showed that the combination of AAVP-TNF-α and LCL161 significantly inhibited tumor growth and prolonged survival in mice with melanoma xenografts. The combination of AAVP-TNF-α and LCL161 was also significantly more effective than either agent alone, showing a synergistic effect without systemic toxicity.

Active metabolites resulting from decarboxylation, reduction and ester hydrolysis of parent drugs.

Decarboxylation, reduction and hydrolysis can yield active metabolites from the parent drug. Major therapeutic indications and metabolic routes of these drugs are reviewed. Changes in the logP values (determined and calculated) from the parent drug to the active metabolite show certain characteristics in comparison to other phase I metabolic alterations. Metabolic decarboxylation of parent drug is commonly associated with increase in lipophilicity. However, in some cases, decarboxylation may cause a reduction in lipophilicity. Ester hydrolysis generally unmasks either the polar carboxylic or hydroxyl group with the outcome of an increase in hydrophilicity. On the contrary, hydrolysis of phosphate ester means a huge increase in the lipophilic character of the drug, as the highly polar phosphate group is removed.

Aliphatic and aromatic oxidations, epoxidation and S-oxidation of prodrugs that yield active drug metabolites.

About one hundred and fifty of the several thousands of drugs on the market are known to have active metabolites. Medicinal chemistry of the parent drugs as well as that of the metabolites are very important both in medical practice and drug research. The efficacy of a drug will depend on a number of properties including, pharmacokinetic behavior, absorption, tissue distribution, pharmacological potency, toxicity and tissue-specificity. The production and release of active metabolites are important because active drug metabolites may influence the clinical outcome of a drug by increasing the gross level of pharmacologically active compounds (drug + active metabolite) and/or essentially increasing the duration of drug effect, when t(1/2) of active metabolite is much longer than that of the parent drug. Furthermore, certain drug metabolizing enzymes can either be inhibited or induced by other drugs and a variety of food and environmental factors. A careful control of the clinical effects of any drug with active metabolites is important especially in the treatment of the elderly population where the administration of several drugs is not unusual.This review provides a detailed description of the medicinal chemistry of drugs yielding active metabolites after undergoing transformation via aliphatic and aromatic oxidations, epoxidation and S-oxidation. Their respective pharmacologically active metabolites, metabolizing enzymes and changes in lipophilicity are also summarized. The most recent analytical methods used for the reliable quantification of both the parent drugs and their metabolites are also included.

The effect of oxime reactivators on muscarinic receptors: functional and binding examinations.

The antidotal treatment of organophosphorus poisoning is still a problematic issue since no versatile antidote has been developed yet. In our study, we focused on an interesting property, which does not relate to the reactivation of inhibited acetylcholinesterase (AChE) of some oximes, but refers to their anti-muscarinic effects which may contribute considerably to their treatment efficacy. One standard reactivator (HI-6) and two new compounds (K027 and K203) have been investigated for their antimuscarinic properties. Anti-muscarinic effects were studies by means of an in vitro stimulated atrium preparation (functional test), the [(3)H]-QNB binding assay and G-protein coupled receptor assay (GPCR, beta-Arrestin Assay). Based on the functional data HI-6 demonstrates the highest anti-muscarinic effect. However, only when comparing [(3)H]-QNB binding results and GPCR data, K203 shows a very promising compound with regard to anti-muscarinic potency. The therapeutic impact of these findings has been discussed.

Attenuation of stress-induced gastric lesions by lansoprazole, PD-136450 and ranitidine in rats.

Combining restraint with cold temperature (4°C) consistently induces gastric ulceration in rats after 3.5 h. The cold restraint-stress (CRS) method provides a suitable model for acute ulcer investigations. This study compares the antiulcer activities of lansoprazole (a proton pump inhibitor), PD-136450 (CCK(2)/gastrin receptor antagonist) and ranitidine (histamine H(2) receptor antagonist) on CRS-induced gastric ulcers in rats. The results have shown that lansoprazole, which is a potent anti-secretory agent, provides complete protection in this model of ulcer formation. The use of indomethacin pretreatment to inhibit the prostaglandin (PG) synthesis and N(G)-nitro L-arginine methyl ester (L-NAME) pretreatment to inhibit nitric oxide synthase did not alter the lansoprazole-induced inhibition of ulcer index obtained in the untreated Wistar rats indicating that these two systems were not involved in the activation of lansoprazole. PD-136450, an effective anti-secretory agent against gastrin- but not dimaprit-induced stimulation, evoked a dose-dependent inhibition of CRS-induced gastric ulcers. The results show that both PG and nitric oxide pathways can influence the inhibitory effect of PD-136450 against CRS-induced gastric ulcer. The antiulcer activities of both lansoprazole and PD-136450 were compared to that of ranitidine. The results showed that ranitidine was more potent than lansoprazole and PD-136450 in inhibiting CRS-induced gastric ulcers and its effect was shown to be influenced by PG as well as nitric oxide synthase. The results of this study have demonstrated that although lansoprazole, PD-136450 and ranitidine were protective against CRS-induced gastric ulcers, the antiulcer activities of PD-136450 and ranitidine involved both PG and nitric oxide pathways, while lansoprazole acted independently of these two systems during CRS.

High dose epoetin beta in the first weeks following renal transplantation and delayed graft function: Results of the Neo-PDGF Study.

Erythropoietin promotes nephroprotection in animal models of ischemia-reperfusion injury. Neorecormon and Prevention of Delayed Graft Function (Neo-PDGF) is a French open-label multicenter randomized study to evaluate the effect of high doses of epoetin beta (EPO-beta) during the first 2 weeks of renal transplantation on renal function in patients at risk for delayed graft function (DGF). One hundred and four patients were included in the study. Patients randomized in treatment group (A) received four injections of EPO-beta (30.000 UI each), given before surgery and at 12 h, 7 days and 14 days posttransplantation. Patients randomized in control group (B) did not receive EPO-beta. Immunosuppression included induction with basiliximab and maintenance therapy with steroids, mycophenolate mofetil and tacrolimus. At 1 month posttransplant, the estimated glomerular filtration rate (MDRD formula) was 42.5 +/- 19.0 mL/min in the EPO-beta group and 44.0 +/- 16.3 mL/min in the control group (p = ns). The frequency of DGF was similar in both groups (32% vs. 38.8%; p = ns). No difference in the incidence of serious adverse events was observed. (ClinicalTrials.gov number, NCT00815867.).

Chromatographic separation of antiviral/anticancer nucleoside reverse transcriptase inhibitor drugs.

This paper discusses the current methods used for quantitative determination of analogues of nucleotide reverse transcriptase inhibitors (NtRTIs) in body fluids, cells, and tissues. Nucleoside reverse transcriptase inhibitors (NRTIs) prodrugs given to AIDS/herpes/cancer patients conjugate with phosphates at the site of their action. Separation of phosphorylated NRTIs is generally performed by reversed-phase chromatography. After separation, plasma NRTIs can be detected using a variety of methods, including immunoassay through monitoring of UV absorbance, fluorescence, and mass spectrometry. The most recent development in the field of detection of plasma NtRTIs shows a tendency toward the use double- or triple-focusing mass spectrometry, the most specific and sensitive monitoring technique.

Medicinal chemistry of antiviral/anticancer prodrugs subjected to phosphate conjugation.

Certain xenobiotics are given in the "prodrug" form. Either the human body, or one compartment of the body, or the targeted virus itself metabolizes the prodrug into its active form. The bioprecursor form of drugs is used for a wide variety of reasons, namely: to make drug penetration into the target organ (mainly to the brain through the blood-brain-barrier) possible, eliminate unpleasant taste, alter (either increasing or decreasing) the half life of the active component or supply more than one active components to the body.

Parasitological and clinical responses of lambs experimentally infected with Haemonchus contortus (L3) with and without ivermectin treatment

Haemonchus contortus is one of the most important parasites that infects sheep and exerts its pathogenic effects by sucking blood, causing disturbances of organ-functions and thus inducing alterations in various normal physiological parameters. Changes in live body weight, faecal egg count, kinetics of circulating eosinophils and PCV value were studied at weekly interval for a period of 84 days in 18 lambs of local breed after infection with a single dose of 5000 H. contortus (L(3)). In the two groups of lambs infected with nematodes both non-treated and treated with ivermectin (HcNT and HcIT), similar egg excretion patterns was observed starting from third week after infection, with a regular increase in FEC. Examination of whole abomasum of each animal revealed no developmental stage of nematode from treated and control lambs on day 84. The total mean number of H. contortus worms recovered at necropsy from abomasa of untreated infected lambs (group HcNT) was 2576.2 (+/-221.0). The significant loss of body weight, development of heavy worm burden and severe anaemia as indicated by reduced PCV in untreated infected lambs indicated high susceptibility of the lambs to H. contortus. On the other hand, complete absence of the parasite, improved PCV value and body weight after treatment of infected animals (HcIT) proved 100% efficacy of ivermectin against H. contortus.

Parasitological and clinical responses of lambs experimentally infected with Haemonchus contortus (L3) with and without ivermectin treatment.

Haemonchus contortus is one of the most important parasites that infects sheep and exerts its pathogenic effects by sucking blood, causing disturbances of organ-functions and thus inducing alterations in various normal physiological parameters. Changes in live body weight, faecal egg count, kinetics of circulating eosinophils and PCV value were studied at weekly interval for a period of 84 days in 18 lambs of local breed after infection with a single dose of 5000 H. contortus (L(3)). In the two groups of lambs infected with nematodes both non-treated and treated with ivermectin (HcNT and HcIT), similar egg excretion patterns was observed starting from third week after infection, with a regular increase in FEC. Examination of whole abomasum of each animal revealed no developmental stage of nematode from treated and control lambs on day 84. The total mean number of H. contortus worms recovered at necropsy from abomasa of untreated infected lambs (group HcNT) was 2576.2 (+/-221.0). The significant loss of body weight, development of heavy worm burden and severe anaemia as indicated by reduced PCV in untreated infected lambs indicated high susceptibility of the lambs to H. contortus. On the other hand, complete absence of the parasite, improved PCV value and body weight after treatment of infected animals (HcIT) proved 100% efficacy of ivermectin against H. contortus.