Controlled Release Gel Encompassing Curcumin Microspheres and Diclofenac Diethylamine for Feat Against Arthritis Inflammation.

BACKGROUND: Diclofenac and curcumin is anticipated to have synergistic action. Hence, topical route of administration can be used in minimizing the issues with oral administration of both drugs. OBJECTIVE: This research aims at formulation of controlled release dosage form containing curcumin microspheres and diclofenac diethylamine and then incorporating it into gel formulation for treatment of inflammation associated with rheumatoid arthritis. METHODS: Curcumin microspheres were prepared, optimized and assayed. Gel containing microspheres was formulated and evaluated for physicochemical parameters like spreadability and viscosity. In vitro and ex vivo diffusion studies were carried out followed by evaluation of efficacy. Efficacy of the developed formulation was evaluated for anti-inflammatory activity. RESULTS: Particle size, Zeta potential, pH, spreadability and viscosity of optimized Batch F1 was found to be in range 0.5 µm - 5 µm,-27.9 mV, 6.2, 105 g cm/s and 7500 cps respectively. In vitro diffusion of developed gel of diclofenac diethylamine and curcumin was found to be 92.16 ± 0.0040 % in 3 h and 92.54 ± 0.0036 % in 12 h as compared to 79.57 ± 0.004 % diffusion in 2 h for marketed gel, thus showing controlled delivery of curcumin. CONCLUSION: Decreased inflammation in formulation treated group by 72.53% and 50.75% in marketed treated group was seen. Thus the formulation developed showed prolonged activity as well as better anti-inflammatory activity.

Ion-pair formation combined with a penetration enhancer as a dual strategy to improve the transdermal delivery of meloxicam.

The aim of the study was to develop a novel drug-in-adhesive patch for transdermal delivery of meloxicam (MLX). The formulation involved a strategy to combine a chemical enhancer with an ion-pair agent. Diethylamine (DETA) was selected as the counter ion to form the ion-pair agent MLX-DETA. MLX-DETA was characterized by nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). The ion-pair lifetime (T life) of MLX-DETA was 164.1 µs. The water solubility of MLX-DETA was increased nearly 9.3-fold, compared with that of MLX. Oleic acid (OA) was selected as the chemical enhancer, and the optimized formulation consisted of 5% (w/w) MLX-DETA, 5% (w/w) oleic acid, and DURO-TAK® 87-4098 adhesive as the pressure-sensitive adhesive matrix. The permeation study in vitro showed that both the counter ion and chemical enhancer were effective in improving the skin permeation of MLX. Tissue distribution studies demonstrated that higher accumulation of MLX following application of the MLX-DETA patch to the skin could be obtained in rats compared with the MLX-patch group. In conclusion, to increase the skin absorption and obtain a sustained release for the transdermal delivery of MLX, preparation of a drug-in-adhesive patch by combining an ion pair (MLX-DETA) with a permeation enhancer (OA) is a suitable strategy.

Acetylated cashew gum-based nanoparticles for transdermal delivery of diclofenac diethyl amine.

Nanoprecipitation and dialysis methods were employed to obtain nanoparticles (NPs) of acetylated cashew gum (ACG). NPs synthesized by dialysis showed greater average size compared to those synthesized by nanoprecipitation, but they presented improved stability and yield. NPs were loaded with diclofenac diethylamine and the efficiency of the drug incorporation was over 60% for both methods, for an ACG:NP a weight ratio of 10:1. The cytotoxicity assay demonstrated that the NPs had no significant effect on the cell viability, verifying their biocompatibility. The release profile for the diclofenac diethylamine associated with the ACG-NPs showed a more controlled release compared to the free drug and a Fickian diffusion mechanism was observed. Transdermal permeation reached 90% penetration of the drug.

Improved Skin Penetration Using In Situ Nanoparticulate Diclofenac Diethylamine in Hydrogel Systems: In Vitro and In Vivo Studies.

Delivering diclofenac diethylamine transdermally by means of a hydrogel is an approach to reduce or avoid systemic toxicity of the drug while providing local action for a prolonged period. In the present investigation, a process was developed to produce nanosize particles (about 10 nm) of diclofenac diethylamine in situ during the development of hydrogel, using simple mixing technique. Hydrogel was developed with polyvinyl alcohol (PVA) (5.8% w/w) and carbopol 71G (1.5% w/w). The formulations were evaluated on the basis of field emission scanning electron microscopy, texture analysis, and the assessment of various physiochemical properties. Viscosity (163-165 cps for hydrogel containing microsize drug particles and 171-173 cps for hydrogel containing nanosize drug particles, respectively) and swelling index (varied between 0.62 and 0.68) data favor the hydrogels for satisfactory topical applications. The measured hardness of the different hydrogels was uniform indicating a uniform spreadability. Data of in vitro skin (cadaver) permeation for 10 h showed that the enhancement ratios of the flux of the formulation containing nanosize drug (without the permeation enhancer) were 9.72 and 1.30 compared to the formulation containing microsized drug and the marketed formulations, respectively. In vivo plasma level of the drug increased predominantly for the hydrogel containing nanosize drug-clusters. The study depicts a simple technique for preparing hydrogel containing nanosize diclofenac diethylamine particles in situ, which can be commercially viable. The study also shows the advantage of the experimental transdermal hydrogel with nanosize drug particles over the hydrogel with microsize drug particles.

Assessment of chronic inhalation non-cancer toxicity for diethylamine.

A non-cancer inhalation chronic toxicity assessment for diethylamine (DEA, CAS number 109-89-7) was conducted by the Texas Commission on Environmental Quality. A chronic Reference Value (ReV) was determined based on a high-quality study conducted in mice and rats by the National Toxicology Program. Chronic inhalation ReVs are health-based exposure concentrations used in assessing health risks of long-term (i.e. lifetime) chemical exposure. DEA is used industrially as an organic intermediate to produce corrosion inhibitors, and is widely used in rubber, pharmaceuticals, resins, pesticides, insect repellants, dye processing and as a polymerization inhibitor. Although systemic effects have been noted at higher concentrations, DEA acts primarily as a respiratory irritant with effects occurring in the upper respiratory tract. Rats were exposed to 0, 31, 62.5 and 125 ppm DEA and mice to 0, 16, 31 and 62.5 ppm DEA for 6 h/day, 5 days/week for 105 weeks. Mice were slightly more sensitive than rats. The critical effect identified in mice was hyperostosis in the turbinates although DEA caused a number of other non-neoplatic lesions. Dose-response data were suitable to benchmark concentration (BMC) modeling. The human equivalent point of departure (PODHEC) was calculated from the 95% lower limit of the BMC(10) using default duration and animal-to-human dosimetric adjustments. Total uncertainty factors of 90 were applied to the PODHEC to account for variation in sensitivity within the human population, toxicodynamic differences between mice and humans, and database uncertainty. The chronic ReV for DEA is 11 ppb (33 µg/m(3)).

In vitro antimicrobial activity of nanoconjugated vancomycin against drug resistant Staphylococcus aureus.

The mounting problem of antibiotic resistance of Staphylococcus aureus has prompted renewed efforts toward the discovery of novel antimicrobial agents. The present study was aimed to evaluate the in vitro antimicrobial activity of nanoconjugated vancomycin against vancomycin sensitive and resistant S. aureus strains. Folic acid tagged chitosan nanoparticles are used as Trojan horse to deliver vancomycin into bacterial cells. In vitro antimicrobial activity of nanoconjugated vancomycin against VSSA and VRSA strains was determined by minimum inhibitory concentration, minimum bactericidal concentration, tolerance and disc agar diffusion test. Cell viability and biofilm formation was assessed as indicators of pathogenicity. To establish the possible antimicrobial mechanism of nanoconjugated vancomycin, the cell wall thickness was studied by TEM study. The result of the present study reveals that nano-sized vehicles enhance the transport of vancomycin across epithelial surfaces, and exhibits its efficient drug-action which has been understood from studies of MIC, MBC, DAD of chitosan derivative nanoparticle loaded with vancomycin. Tolerance values distinctly showed that vancomycin loaded into nano-conjugate is very effective and has strong bactericidal effect on VRSA. These findings strongly enhanced our understanding of the molecular mechanism of nanoconjugated vancomycin and provide additional rationale for application of antimicrobial therapeutic approaches for treatment of staphylococcal pathogenesis.

Effect of rubbing on the in vitro skin permeation of diclofenac-diethylamine 1.16% gel.

BACKGROUND: Rubbing a topical NSAID (non steroidal anti-inflammatory drug) on the skin may increase local drug permeation, affecting its distribution to the site of pain and inflammation. The present study evaluates this hypothesis, by assessing in vitro the effect on skin permeation of applying diclofenac-dieythylamine 1.16% gel with or without rubbing. METHODS: A single dose of 5 mg/cm2 diclofenac-diethylamine 1.16% gel was applied on excised human skin mounted in Franz-type diffusion cells without or with rubbing for 45 s. Drug penetration into the skin layers was determined after 1 h using the tape stripping technique. In vitro cutaneous permeation into the receptor fluid of the diffusion chamber was measured up to 24 h. Skin electrical resistance was also recorded. RESULTS: Application of diclofenac-diethylamine 1.16% gel with rubbing resulted to a 5-fold higher flux of diclofenac through the skin than when applied without rubbing at 8 h (P = 0.04). Skin rubbing for 45 s decreased by 2-fold skin electrical resistance when compared to the standard application. Application of diclofenac-diethylamine 1.16% gel with rubbing tended to result in higher accumulation in the stripped skin vs. the superficial skin layers when applied without rubbing (P = 0.2). CONCLUSION: These results suggest that rubbing may alter the superficial skin layer resulting in a transient faster initial diffusion of topically applied diclofenac through the stratum corneum into the deeper skin layer of the dermis to the tissue target.

Discovery of a calcimimetic with differential effects on parathyroid hormone and calcitonin secretion.

Calcimimetics are positive allosteric modulators to the calcium-sensing receptor (CaSR). Activation of the CaSR inhibits the secretion of parathyroid hormone (PTH), stimulates the secretion of calcitonin, and decreases serum calcium (Ca(2+)). Cinacalcet, a second-generation calcimimetic, is used therapeutically to control PTH in patients with chronic kidney disease who are on dialysis with secondary hyperparathyroidism. A calcimimetic that displays increased separation of PTH versus Ca(2+) lowering in patients would potentially allow the use of calcimimetics to treat patients in earlier stages of renal disease because hypocalcemia can develop in this population. Toward this end, we developed a third-generation calcimimetic, determined the molecular pharmacological properties of it using an operation model of allosteric modulation/agonism, and measured the compound effects on PTH, serum ionized Ca(2+), and calcitonin levels in 5/6 nephrectomized rats. We found the new molecule effectively reduced PTH levels without promoting calcitonin secretion or hypocalcemia. Furthermore, our third-generation molecule was less efficacious at promoting calcitonin secretion from human thyroid carcinoma cells compared with 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (R-568), a first-generation calcimimetic. These data provide evidence that calcimimetics with increased potency can be used to lower PTH without production of significant hypocalcemia because the threshold for inhibition of PTH secretion is much lower than the threshold for calcitonin secretion.

Bicellar systems for in vitro percutaneous absorption of diclofenac.

This work evaluates the effect of different bicellar systems on the percutaneous absorption of diclofenac diethylamine (DDEA) using two different approaches. In the first case, the drug was included in bicellar systems, which were applied on the skin and, in the second case, the skin was treated by applying bicellar systems without drug before to the application of a DDEA aqueous solution. The characterization of bicellar systems showed that the particle size decreased when DDEA was encapsulated. Percutaneous absorption studies demonstrated a lower penetration of DDEA when the drug was included in bicellar systems than when the drug was applied in an aqueous solution. This effect was possibly due to a certain rigidity of the bicellar systems caused by the incorporation of DDEA. The absorption of DDEA on skin pretreated with bicelles increased compared to the absorption of DDEA on intact skin. Bicelles without DDEA could cause certain disorganization of the SC barrier function, thereby facilitating the percutaneous penetration of DDEA subsequently applied. Thus, depending on their physicochemical parameters and on the application conditions, these systems have potential enhancement or retardant effects on percutaneous absorption that result in an interesting strategy, which may be used in future drug delivery applications.

The relationship between circulating natural killer cells after reduced intensity conditioning hematopoietic stem cell transplantation and relapse-free survival and graft-versus-host disease.

BACKGROUND: Natural killer cells are known to have anti-tumor activity in haploidentical hematopoietic stem cell transplantation. We hypothesized that reconstituted circulating natural killer cells may be associated with improved relapse-free survival after HLA-matched hematopoietic stem cell transplantation. DESIGN AND METHODS: Serial peripheral blood absolute natural killer cell counts were prospectively measured by flow cytometry of lymphocytes expressing CD56 and CD16 in 167 patients. Cluster analysis was used at engraftment and 60 days post-transplant to distinguish patients with high and low absolute natural killer cell counts. At engraftment 80 patients had high counts (> 22.2/mm3) and 43 had low counts. At 60 days post-transplant 84 patients had high counts (> 18.2/mm3) and 38 had low counts. The primary study end-points were death, relapse and acute graft-versus-host disease. The median follow-up was 373 days (range, 67-1767). RESULTS: Among patients given reduced intensity conditioning, a low absolute natural killer cell count at 60 days post-transplant was independently associated with relapse [adjusted hazard ratio (AHR) = 28.4, 95% confidence interval (CI) 4.3-186.4] and death (AHR = 17.5, 95% CI 4.3-71.3). Furthermore, patients given reduced intensity conditioning who had a high absolute natural killer cell count at 60 days had a significantly better 1-year survival than those with a low count by Kaplan-Meier analysis (83% vs. 11%, p<0.001). Multivariate analysis confirmed that low 60-day absolute natural killer count in patients given reduced intensity conditioning was independently associated with an increase in relapse or death (AHR = 20.22, 95% CI 4.76-85.40). In contrast, there was no significant association between 60-day absolute natural killer cell counts and clinical outcomes in patients receiving myeloablative conditioning. There was no significant association between absolute natural killer cell count and graft-versus-host disease. CONCLUSIONS: High natural killer cell reconstitution is associated with reduced relapse and death without an increased incidence of graft-versus-host-disease after reduced intensity conditioning allogeneic hematopoietic stem cell transplantation. Measuring reconstituted natural killer cells expressing CD56(+)/CD16(+) post-transplant may have novel prognostic and therapeutic implications.

Functional studies of the effect of NO donor on human CLCN1 polymorphism/mutants expressed in Xenopus laevis oocytes.

In this study, we investigated the effect of NO donor, diethylamine/nitric oxide (DEA/NO), on the electrophysiological behavior of human skeletal muscle chloride channel (CLCN1). The wild-type and variants of CLCN1, including one polymorphism (P727L) and four mutants (T631I, D644G, G482R, and S471F), were expressed in Xenopus oocytes and the ionic current was measured by two-electrode voltage-clamp method. Our results revealed that there is no significant difference in the current-voltage relationships and half-voltage values of open probability between wild-type and variants of CLCN1 except for G482R. Application of the DEA-NO (0.1mM) significantly increases the channel conductance of wild-type, T631I, D644G, and S471F, but not P727L. This indicates that P727L polymorphism causes loss of sensitivity of CLCN1 to the DEA/NO treatment, which could be due to a conformational change caused by proline substitution. The data suggest that the polymorphic changes may affect the function of CLCN1 in response to the treatment of chemical compounds.

The physicochemical characterization and in vitro/in vivo evaluation of natural surfactants-based emulsions as vehicles for diclofenac diethylamine.

Two sugar-based emulsifiers, cetearyl alcohol & cetearyl glycoside and sorbitan stearate & sucrose cocoate, known as potential promoters of lamellar liquid crystals/gel phases, were investigated in order to formulate an optimal vehicle for amphiphilic drug - diclofenac diethylamine (DDA). Physico-chemical characterization and study of vehicle's physical stability were performed. Then, the in vitro DDA liberation profile, dependent on the mode of drug incorporation to the system, and the in vivo, short-term effects of chosen samples on skin parameters were examined. Droplets size distribution and rheological behavior indicated satisfying physical stability of both types of vehicles. Unexpectedly, the manner of DDA incorporation to the system had no significant influence on DDA release. In vivo study pointed to emulsion's favorable potential for skin hydration and barrier improvement, particularly in cetearyl glycoside-based vehicle.

Peripheral nerve avulsion injuries as experimental models for adult motoneuron degeneration.

We have used adult rat peripheral nerve avulsion models to evaluate the effects of neuroprotective molecules on motoneuron degeneration. The right facial nerves of adult Fischer 344 male rats were avulsed and adenoviral vectors encoding glial cell line-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor (BDNF), transforming growth factor-beta2 (TGFbeta2), and growth inhibitory factor (GIF) were injected into the facial canal. The treatment with the vectors significantly prevented the loss of lesioned facial motoneurons, improved choline acetyltransferase (ChAT) immunoreactivity and suppressed the induction of nitric oxide synthase activity in these neurons. In separate experiments, animals were orally administered a solution of a neuroprotective compound T-588 after avulsion. Both free oral administration and oral tube administration of T-588 improved the survival of injured motoneurons and ameliorated their ChAT immunoreactivity. These results indicate that the gene transfer of GDNF, BDNF, TGFbeta2, and GIF and oral administration of T-588 may prevent the degeneration of motoneurons in adult humans with motoneuron injury and motor neuron diseases.

T-588 protects motor neuron death following axotomy.

R(-)-1-(benzo [b] thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy] ethanol hydrochloride) (T-588) enhances acetylcholine release. This compound slows the motor deterioration of wobbler mouse motor neuron disease and enhances neurite outgrowth and choline acetyltransferase activity in cultured rat spinal motor neurons. We examined the ability of T-588 on axotomized spinal motor neuron death in the rat spinal cord. After the postnatal unilateral section of sciatic nerve, there was approximately a 50% survival of motor neurons in the fourth lumbar segment. In comparison with vehicle, intraperitoneal injection of T-588 for 14 consecutive days rescued spinal motor neuron death. Our results showing in vivo neurotrophic activity of T-588 for motor neurons support the applicability of T-588 for the treatment of motor neuron diseases, such as amyotrophic lateral sclerosis and motor neuropathies.

T-588, a novel neuroprotective agent, delays progression of neuromuscular dysfunction in wobbler mouse motoneuron disease.

R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588) enhances acetylcholine release from the frontal cortex and hippocampus in rats, and can ameliorate cognitive dysfunction in various amnesia models of rodents. T-588 protects rat cerebellar granule cells from glutamate neurotoxicity in culture. This agent also inhibits facilitation in the crayfish neuromuscular junction and mammalian cerebellum. Clinical trials of T-588 are underway in patients with Alzheimer's disease. We attempted to determine whether T-588 treatment ameliorates neuromuscular dysfunction in the wobbler mouse, an animal model of motoneuron disease (MND). After the initial diagnosis of MND at the age of 3-4 weeks, wobbler mice were orally administered T-588 (3, 10, 30 mg/kg) or vehicle daily for 4 weeks in a blinded fashion. We compared symptomatic, pathological and biochemical changes among the groups. In comparison with vehicle, T-588 administration potentiated grip strength, attenuated forelimb contracture and increased the weight of the biceps muscles. T-588-treated mice had retarded denervation muscle atrophy and elevated activities of choline acetyltransferase (ChAT) or lactate dehydrogenase in the biceps muscles. T-588 treatment also enhanced ChAT activities and promoted formation of cyclic adenosine monophosphate in the cervical cord. Pharmacokinetic study also showed that T-588 was transported efficiently into the cerebrum and spinal cord following oral administration. Thus, T-588 treatment delayed the progression of wobbler murine MND. Our findings suggest that this agent has therapeutic potential in human motor neuropathy or MND.

The effects of T-588, a novel cognitive enhancer, on noradrenaline uptake and release in rat cerebral cortical slices.

Previously, we reported that (R)-(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino)-ethoxy]e thanol hydrochloride (T-588), a novel cognitive enhancer, stimulated noradrenaline (NA) release from rat cerebral cortical slices. In this study, we investigated the effects of T-588 on NA uptake and release, compared to the effects of desipramine, a blocker of the NA carrier on the plasma membrane. Both T-588 and desipramine caused dose-dependent inhibition of [3H]NA uptake into the slices. Addition of 3 mM T-588 stimulated [3H]NA release from the prelabeled slices even in the presence of 10 microM desipramine, which inhibited NA uptake completely. Tyramine, which accelerates NA carrier-mediated release, also stimulated [3H]NA release, and tyramine-stimulated release was inhibited by desipramine. These findings indicated that T-588-stimulated NA release was not mediated by 1) inhibition of reuptake or 2) reverse transport mediated by NA carriers. Reserpine, which interacts with the intracellular vesicular transport system, increased [3H]NA efflux from slices. High K+-, not T-588-, stimulated [3H]NA release was shifted upward by reserpine. These findings suggest that T-588 evokes NA release by a mechanism similar to that induced by reserpine. T-588 might act as a cognitive enhancer via neurotransmitter release in the brain.

[Effects of iron deficiency on toxic action of nitrites and endogenous synthesis of N-nitrosamines]. / Vliianie defitsita zheleza na toksicheskoe deistvie nitritov i éndogennyi sintez N-nitrozoaminov.

Separate or joint ingestions of water solutions of sodium nitrite and/or N-diethylamine by rats fed iron deficiency diet caused sharp increasing of N-nitrosodiethylamine level in stomach, a decreasing of Hb and increasing of MetHb concentrations in blood of animal. These effects were greater on iron deficient diet than on control diet. The nitrite ingestion caused a decline level of cytochrome P-450 and inhibition of lipid peroxidation in rat liver.

Inhalation of diethylamine--acute nasal effects and subjective response.

Adult volunteers were exposed to 25 ppm (75 mg/m3) diethylamine in a climate chamber for 15 min in order to study the acute nasal reactions to an exposure equivalent to the present threshold limit value-short-term exposure limit. Changes in nasal volume and nasal resistance were measured by acoustic rhinometry and by rhinomanometry. Acute change in nasal volume, usually seen as acute nasal mucosa response to thermal stimuli, was not observed, nor was an acute change in nasal airway resistance. In a subsequent experiment, the aim was to measure acute sensory effects. Exposure to a concentration increasing from 0 to 12 ppm took place for 60 min, equal to an average concentration of 10 ppm (30 mg/m3). A moderate to strong olfactory response and distinct nasal and eye irritation were observed. In spite of considerable individual variation, the results were in agreement with sensory effect estimates obtained from animal studies.

Effects of administration of N-nitrosodialkylamines and N-nitrodiethylamine on hepatic UDP-glucuronosyltransferase activity in Wistar rats.

N-Nitrosodiethylamine (NEN) and N-nitrodiethylamine (NEA) are carcinogens and in vitro activators of hepatic UDP-glucuronosyltransferase (GT) toward 2-aminophenol (AP) and 4-nitrophenol (NP). In this communication, they were intraperitoneally administered to male Wistar rats for 7 days and GT activities were determined towards AP, NP, phenolphthalein (PH) and testosterone (TS). Administration of 30 or 20 mg/kg dose of NEN caused marked decrease of liver and body weights, and did not affect hepatic GT activities. Injection of 10 mg/kg dose of NEN did not diminish liver and body weights, and increased the maximally activated GT activities toward AP and NP. In contrast, 30 mg/kg dose of NEA, did not affect either liver and body weights or GT activities. N-Nitrosodimethylamine (NMN), which is a carcinogen and a weak in vitro AP GT activator, was more toxic than NEN, and 3.6 mg/kg dose of NMN appears to induce GT toward NP and AP. Administration of 46.5 mg/kg N-nitrosodibutylamine (NBN), which is a carcinogen but not a GT activator, did not affect GT activities or liver body weights.