Розробка складу препарату мелоксикам гель на підставі результатів біофармацевтичних досліджень

The meloxicam concentration and the composition of permeation enhancers in a gel for cutaneous application have been chosen on the basis of the results of pharmacological screening on the model of acute aseptic carrageenan inflammation of the rat’s paw. The efficiency of the analgesic effect of gels increases with increasing the meloxicam content, and the efficiency of the anti-inflammatory activity passes through a maximum at the meloxicam concentration of 1.0-1.5%. The efficiency of the anti-inflammatory and analgesic effects is the highest when gels contain 15% of N-methylpyrrolidone and 25% of ethanol (96%). Due to the content of hydrophilic non-aqueous solvents the gel base possesses the osmotic activity, whereby providing the moderate water absorption by the drug and prolonged release of meloxicam from the gel in experiments in vitro. Such properties of the gel, on the one hand, are an important prerequisite for transdermal absorption of meloxicam, on the other hand, the dehydrating action onto the skin is not expected. Pharmacokinetic studies of 1% gel of meloxicam were conducted compared to the intramuscular (i/m) administration of Movalis injection of 15 mg/1.5 ml. The analytical procedure (liquid chromatography with the solid phase extraction of the active substance from the plasma) has been developed and validated for analytical support of the pharmacokinetic studies. It has been found in the experiment on rabbits that in case of cutaneous application of the gel meloxicam is absorbed slowly and continuously into the systemic circulation at a relatively high extent of transdermal absorption and slow elimination. Therefore, it can be expected that the gel developed provides the effective prolonged therapeutic action.Выбрана концентрация мелоксикама и состав усилителей проникновения в геле для накожного применения по результатам фармакологического скрининга на модели острого асептического каррагенинового воспаления стопы крыс. Эффективность анальгетического действия гелей увеличивается с повышением содержания мелоксикама, а эффективность противовоспалительного действия проходит через максимум при концентрации мелоксикама 1,0-1,5 %. Эффективность противовоспалительного и анальгетического действия оказывается наиболее высокой при наличии в составе гелей 15 % N‑метилпирролидона и 25 % этанола (96 %). Гелевая основа обладает осмотической активностью, вследствие чего обеспечивает умеренную абсорбцию препаратом воды и пролонгированное высвобождение мелоксикама из гелей в опытах in vitro, которое является важной предпосылкой для его трансдермального всасывания. В эксперименте на кроликах установлено, что при накожном применении геля мелоксикам медленно и продолжительно всасывается в системный кровоток при относительно высокой степени трансдермальной абсорбции и медленной элиминации, что позволяет прогнозировать эффективное и пролонгированное терапевтическое действие.Обрано концентрацію мелоксикаму та склад підсилювачів проникнення в гелі для нашкірного застосування за результатами фармакологічного скринінгу на моделі гострого асептичного карагенінового запалення стопи щурів. Ефективність аналгетичної дії гелів зростає з підвищенням вмісту мелоксикаму, а ефективність протизапальної дії проходить через максимум при концентрації мелоксикаму 1,0-1,5%. Ефективність протизапальної та аналгетичної дії виявляється найбільшою при наявності у складі гелів 15% N-метилпіролідону та 25% етанолу (96%). Гелева основа володіє осмотичною активністю, внаслідок чого забезпечується помірна абсорбція препаратом води та пролонговане вивільнення мелоксикаму з гелів у дослідах in vitro, що є важливою передумовою для його трансдермального всмоктування. В експерименті на кроликах виявлено, що при нашкірному застосуванні гелю мелоксикам повільно й тривало всмоктується в системний кровообіг при відносно високому ступені трансдермальної абсорбції та повільній елімінації, що дозволяє прогнозувати ефективну та пролонговану терапевтичну дію

A Decline in p38 MAPK Signaling Underlies Immunosenescence in Caenorhabditis elegans

The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans

The Somatic Reproductive Tissues of C. elegans Promote Longevity through Steroid Hormone Signaling

Removal of the germ cells of C. elegans extends lifespan in part because signals from the somatic reproductive tissues activate the nuclear hormone receptor DAF-12

Variable Pathogenicity Determines Individual Lifespan in Caenorhabditis elegans

A common property of aging in all animals is that chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for Caenorhabditis elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with the superoxide dismutase gene sod-3 expression being the best single predictor of remaining lifespan. Correlation with remaining lifespan became stronger if expression from two aging markers was monitored simultaneously, accounting for up to 49% of the variation in individual lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability is different pathogenicity from individual to individual and that the mechanism involves variable activation of the insulin-signaling pathway

Co-Regulation of the DAF-16 Target Gene, cyp-35B1/dod-13, by HSF-1 in C. elegans Dauer Larvae and daf-2 Insulin Pathway Mutants

Insulin/IGF-I-like signaling (IIS) has both cell autonomous and non-autonomous functions. In some cases, targets through which IIS regulates cell-autonomous functions, such as cell growth and metabolism, have been identified. In contrast, targets for many non-autonomous IIS functions, such as C. elegans dauer morphogenesis, remain elusive. Here, we report the use of genomic and genetic approaches to identify potential non-autonomous targets of C. elegans IIS. First, we used transcriptional microarrays to identify target genes regulated non-autonomously by IIS in the intestine or in neurons. C. elegans IIS controls expression of a number of stress response genes, which were differentially regulated by tissue-restricted IIS. In particular, expression of sod-3, a MnSOD enzyme, was not regulated by tissue-restricted IIS on the microarrays, while expression of hsp-16 genes was rescued back to wildtype by tissue restricted IIS. One IIS target regulated non-autonomously by age-1 was cyp-35B1/dod-13, encoding a cytochrome P450. Genetic analysis of the cyp-35B1 promoter showed both DAF-16 and HSF-1 are direct regulators. Based on these findings, we propose that hsf-1 may participate in the pathways mediating non-autonomous activities of age-1 in C. elegans

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity

C. elegans Germline-Deficient Mutants Respond to Pathogen Infection Using Shared and Distinct Mechanisms

Reproduction extracts a cost in resources that organisms are then unable to utilize to deal with a multitude of environmental stressors. In the nematode C. elegans, development of the germline shortens the lifespan of the animal and increases its susceptibility to microbial pathogens. Prior studies have demonstrated germline-deficient nematodes to have increased resistance to Gram negative bacteria. We show that germline-deficient strains display increased resistance across a broad range of pathogens including Gram positive and Gram negative bacteria, and the fungal pathogen Cryptococcus neoformans. Furthermore, we show that the FOXO transcription factor DAF-16, which regulates longevity and immunity in C. elegans, appears to be crucial for maintaining longevity in both wild-type and germline-deficient backgrounds. Our studies indicate that germline-deficient mutants glp-1 and glp-4 respond to pathogen infection using common and different mechanisms that involve the activation of DAF-16

A Transcription Elongation Factor That Links Signals from the Reproductive System to Lifespan Extension in Caenorhabditis elegans

In Caenorhabditis elegans and Drosophila melanogaster, the aging of the soma is influenced by the germline. When germline-stem cells are removed, aging slows and lifespan is increased. The mechanism by which somatic tissues respond to loss of the germline is not well-understood. Surprisingly, we have found that a predicted transcription elongation factor, TCER-1, plays a key role in this process. TCER-1 is required for loss of the germ cells to increase C. elegans' lifespan, and it acts as a regulatory switch in the pathway. When the germ cells are removed, the levels of TCER-1 rise in somatic tissues. This increase is sufficient to trigger key downstream events, as overexpression of tcer-1 extends the lifespan of normal animals that have an intact reproductive system. Our findings suggest that TCER-1 extends lifespan by promoting the expression of a set of genes regulated by the conserved, life-extending transcription factor DAF-16/FOXO. Interestingly, TCER-1 is not required for DAF-16/FOXO to extend lifespan in animals with reduced insulin/IGF-1 signaling. Thus, TCER-1 specifically links the activity of a broadly deployed transcription factor, DAF-16/FOXO, to longevity signals from reproductive tissues

The Interplay between Protein L-Isoaspartyl Methyltransferase Activity and Insulin-Like Signaling to Extend Lifespan in Caenorhabditis elegans

The protein L-isoaspartyl-O-methyltransferase functions to initiate the repair of isomerized aspartyl and asparaginyl residues that spontaneously accumulate with age in a variety of organisms. Caenorhabditis elegans nematodes lacking the pcm-1 gene encoding this enzyme display a normal lifespan and phenotype under standard laboratory growth conditions. However, significant defects in development, egg laying, dauer survival, and autophagy have been observed in pcm-1 mutant nematodes when deprived of food and when exposed to oxidative stress. Interestingly, overexpression of this repair enzyme in both Drosophila and C. elegans extends adult lifespan under thermal stress. In this work, we show the involvement of the insulin/insulin-like growth factor-1 signaling (IIS) pathway in PCM-1-dependent lifespan extension in C. elegans. We demonstrate that reducing the levels of the DAF-16 downstream transcriptional effector of the IIS pathway by RNA interference reduces the lifespan extension resulting from PCM-1 overexpression. Using quantitative real-time PCR analysis, we show the up-regulation of DAF-16-dependent stress response genes in the PCM-1 overexpressor animals compared to wild-type and pcm-1 mutant nematodes under mild thermal stress conditions. Additionally, similar to other long-lived C. elegans mutants in the IIS pathway, including daf-2 and age-1 mutants, PCM-1 overexpressor adult animals display increased resistance to severe thermal stress, whereas pcm-1 mutant animals survive less long under these conditions. Although we observe a higher accumulation of damaged proteins in pcm-1 mutant nematodes, the basal level of isoaspartyl residues detected in wild-type animals was not reduced by PCM-1 overexpression. Our results support a signaling role for the protein L-isoaspartyl methyltransferase in lifespan extension that involves the IIS pathway, but that may be independent of its function in overall protein repair

Control of Metabolic Homeostasis by Stress Signaling Is Mediated by the Lipocalin NLaz

Metabolic homeostasis in metazoans is regulated by endocrine control of insulin/IGF signaling (IIS) activity. Stress and inflammatory signaling pathways—such as Jun-N-terminal Kinase (JNK) signaling—repress IIS, curtailing anabolic processes to promote stress tolerance and extend lifespan. While this interaction constitutes an adaptive response that allows managing energy resources under stress conditions, excessive JNK activity in adipose tissue of vertebrates has been found to cause insulin resistance, promoting type II diabetes. Thus, the interaction between JNK and IIS has to be tightly regulated to ensure proper metabolic adaptation to environmental challenges. Here, we identify a new regulatory mechanism by which JNK influences metabolism systemically. We show that JNK signaling is required for metabolic homeostasis in flies and that this function is mediated by the Drosophila Lipocalin family member Neural Lazarillo (NLaz), a homologue of vertebrate Apolipoprotein D (ApoD) and Retinol Binding Protein 4 (RBP4). Lipocalins are emerging as central regulators of peripheral insulin sensitivity and have been implicated in metabolic diseases. NLaz is transcriptionally regulated by JNK signaling and is required for JNK-mediated stress and starvation tolerance. Loss of NLaz function reduces stress resistance and lifespan, while its over-expression represses growth, promotes stress tolerance and extends lifespan—phenotypes that are consistent with reduced IIS activity. Accordingly, we find that NLaz represses IIS activity in larvae and adult flies. Our results show that JNK-NLaz signaling antagonizes IIS and is critical for metabolic adaptation of the organism to environmental challenges. The JNK pathway and Lipocalins are structurally and functionally conserved, suggesting that similar interactions represent an evolutionarily conserved system for the control of metabolic homeostasis