In vivo and in vitro genotoxic evaluation of indorenate

5-Methoxytryptamine, beta-methylcarboxylate hydrochloride (indorenate) is a new antihypertensive serotonin derivative. We evaluated its genotoxic activity using the mouse bone marrow and cytogenetic test and the human lymphocyte culture cytogenetic assay. As endpoints we measured chromosomal aberrations, sister-chromatid exchanges and cellular proliferation kinetics. Our results agree in both systems showing that indorenate is a non-genotoxic agent in these assays.     

In vivo antibody-mediated modulation of aminopeptidase A in mouse proximal tubular epithelial cells.

Aminopeptidase A (APA) is one of the many renal hydrolases. In mouse kidney, APA is predominantly expressed on the brush borders and sparsely on the basolateral membranes of proximal tubular epithelial cells. However, when large amounts of monoclonal antibodies (MAbs) against APA were injected into mice, we observed strong binding of the MAbs to the basolateral membranes, whereas the MAbs bound only transiently to the brush borders of the proximal tubular epithelial cells. In parallel, APA itself disappeared from the brush borders by both endocytosis and shedding, whereas it was increasingly expressed on the basolateral sides. Using ultrastructural immunohistology, we found no evidence for transcellular transport of endocytosed APA to the basolateral side of the proximal tubular epithelial cells. The absence of transcellular transport was confirmed by experiments in which we used a low dose of the MAbs. Such a low dose did not result in binding of the MAbs to the brush borders and had no effect on the presence of APA in the brush borders of the proximal tubular epithelial cells. In these experiments we still could observe binding of the MAbs to the basolateral membranes in parallel with the local appearance of APA. In addition, treatment of mice with chlorpromazine, a calmodulin antagonist that interferes with cytoskeletal function, largely inhibited the MAb-induced modulation of APA. Our studies suggest that injection of MAbs to APA specifically interrupts the normal intracellular traffic of this enzyme in proximal tubular epithelial cells. This intracellular transport is dependent on the action of cytoskeletal proteins.     

In vitro and in vivo evaluation of O-alkyl derivatives of tramadol

Tramadol is a centrally acting opioid analgesic structurally related to codeine and morphine. O-Alkyl, N-desmethyl, and non-phenol containing derivatives of tramadol were synthesized to probe their effect on metabolic stability and both in vitro and in vivo potency.     

In vitro and in vivo evaluation of hydrophilic dendronized linear polymers

Rigid-rod dendronized linear polymers consisting of a poly(4-hydroxystyrene) backbone and fourth-generation polyester dendrons were evaluated in vitro and in vivo to determine their suitability as drug delivery vectors. Cytotoxicity assays indicated that the polymers were well tolerated by cells in vitro. Biodistribution studies of the polymers in both nontumored and tumored mice revealed that as for random coil linear polymers, renal clearance was a function of polymer size, with significant urinary excretion observed for a 67 kDa dendronized polymer. High accumulation in organs of the reticuloendothelial system was exhibited by a dendronized polymer with a very high molecular weight (M(n) = 1740 kDa), but was not as significant for smaller polymers with M(n) = 67 kDa and M(n) = 251 kDa. The rank order for tumor accumulation of the polymers on a percent injected dose per gram tumor basis was 251 kDa approximately 1740 kDa > 67 kDa. These data will help guide the selection of highly functionalizable rigid-rod dendronized polymers with pharmacokinetic properties appropriate for use as drug carriers.     

In vitro and in vivo evaluation of topical formulations of Spantide II

The purpose of this study was to develop and evaluate topical formulations of Spantide II, a neurokinin-1 receptor (NK-1R) antagonist, for the treatment of inflammatory skin disorders. Spantide II lotion and gel was formulated with and without n-methyl-2-pyrrolidone (NMP) as a penetration enhancer. The release of Spantide II from gels was evaluated using microporous polyethylene and polypropylene membranes in a Franz Diffusion cell setup. In vitro percutaneous absorption of Spantide II from lotion and gel formulations was evaluated using the above setup by replacing the membranes with hairless rat skin. The in vivo anti-inflammatory activity of Spantide II formulations was evaluated in an allergic contact dermatitis (ACD) mouse model. Among different gels studied, PF127 gel showed highest (70-fold) release of Spantide II compared with hydroxypropyl methylcellulose (HPMC) and hydroxypropyl cellulose (HPC) gels. Lotion and gel formulations with or without NMP showed no detectable levels of Spantide II in the receiver compartment of the Franz diffusion cell until 24 hours. However, Spantide II showed significant retention in epidermis and dermis from lotion and gel formulations at 24 hours. The dermal levels increased ≈3.5- and 2-fold when the lotion and gel formulations contained NMP as compared with the formulation with no NMP (P<.05). The in vivo studies indicated that Spantide II formulations with NMP were effective in significantly reducing ACD response, similar to dexamethasone (0.5 mM). In conclusion, Spantide II was stable as a topical formulation and delivered to target skin tissue (epidermis and dermis) for the treatment of ACD. In addition this study supports the role of cutaneous neurosensory system in modulating inflammatory responses in the skin. Published: October 31, 2005     

In vitro and in vivo antimalarial evaluation of semi-synthetic derivatives of gomphostenin

A novel series of semi-synthetic gomphostenin derivatives (1–9) were prepared utilizing C-14 hydroxyl group for the first time and studied for their antimalarial properties. In vitro antiplasmodial activity was evaluated against both the chloroquine sensitive and resistant strains of Plasmodium falciparum. Most of the compounds exhibited superior or comparable antiplasmodial activity compared to parent compound, that is, gomphostenin (GN). Based upon in vitro antiplasmodial activity, compounds with IC₅₀ values less than 10 μM were selected for in vivo antiplasmodial evaluation against Plasmodium berghei infection in mice model. GN derivatives 3 and 5 were found to have curative activity with moderate chemosuppression of 65% and 69%, respectively, at the dose level of 150 mg/kg/day.     

In vivo and in vitro aging is detrimental to mouse spermatogonial stem cell function

The development of techniques to maintain the spermatogonial stem cell (SSC) in vivo and in vitro for extended periods essentially allows for the indefinite continuation of an individual germline. Recent evidence indicates that the aging of male reproductive function is due to failure of the SSC niche. SSCs are routinely cultured for 6 mo, and no apparent effect of culture over this period has been observed. To determine the effects of SSC aging, we utilized an in vitro culture system, followed by quantitative transplantation experiments. After culture for 6 mo, SSCs that had been aged in vivo for 1500 days had a slower proliferation rate than SSCs that were aged in vivo to 8 or 300 days. Examination of methylation patterns revealed no apparent difference in DNA methylation between SSCs that were aged 8, 300, or 1500 days before culture. Long-term culture periods resulted in a loss of stem cell potential without an obvious change in the visual appearance of the culture. DNA microarray analysis of in vivo- and in vitro-aged SSCs identified the differential expression of several genes important for SSC function, including B-cell CLL/lymphoma 6, member B (Bcl6b), Lim homeobox protein 1 (Lhx1), and thymus cell antigen 1, theta (Thy1). Collectively, these data indicate that, although both in vitro and in vivo aging are detrimental to SSC function, in vitro aging results in greater loss of function, potentially due to a decrease in core SSC self-renewal gene expression and an increase in germ cell differentiation gene expression.     

In vitro aging of erythrocytes

Various in vitro-aging procedures are introduced as an alternative to the difficult isolation of physiologically aged erythrocytes standing immediately before elimination. N-acetylneuraminic acid, ATP- and glutathione contents of these model cells are estimated and the kind and degree of membrane damage were characterized by the rates of phagocytosis and exovesiculation. Conclusions concerning molecular biological mechanisms of age-related processes were drawn by correlating biochemical data with the degree of membrane lesion. Alterations of spectrin (cross-linking, aggregation, rearrangement) seem to play the main role in all in vitro-ageing processes described here.     

In vivo and in vitro studies of hypocholesterolemic effects of diosgenin in rats

There is evidence that diosgenin when given orally or parenterally decreases cholesterol plasma levels in rat, chicken and rabbits that have had a diet-induced hypercholesterolemia. 2. The per-oral administration of [3H]diosgenin yielded 12% of the given dose distributed throughout: liver, spleen, epididymal fat, brain and carcass of the rat. 3. In everted gut sacs, [3H]diosgenin was better absorbed than cholesterol. 4. In these tests diosgenin was recovered esterified from the tissues and the recovered cholesterol showed less esterification in the presence of diosgenin than in its absence.     

In vivo and in vitro evaluation of cell-mediated immunity in patients with paracoccidioidomycosis

The cellular immune response to specific and nonspecific agents was investigated. both in vivo and in vitro, in 19 patients with paracoceidioidomycosis. In addition, the immunologic study of an investigator aceidentally inoculated with P. brasiliensis was included in this study. Nearly half of the patients showed depressed cell-mediated immune responses, as evaluated by intradermal tests with an antigenic preparation from P. brasiliensis (P.b.Ag.), ubiquitous antigens, and by the ability to develop sensitization to 2,4-dinitrochlorobenzene. A similar proportion of impaired responses was observed when the patients' lymphocytes were cultured with phytohemagglutinin (PHA). C'. albicans antigen and P.h.Ag. A factor was detected in the plasma of some patients which reduced the ability of patients' and normal lymphocytes to undergo blastic transformation. A positive correlation was found between the ability to develop delayed cutaneous hypersensitivity reactions to P.b.Ag. and other ubiquitous antigens, normal in vitro responsiveness to PHA and the absence of humoral blastogenic inhibitory factor. The inhibition of leukocyte migration, but not lymphocyte transformation, correlated positively with delayed hypersensitivity. The percentage of T lymphocytes was significantly reduced in the group of patients, being the absolute number and percentage of B cells bearing receptors tor complement normal. Two polar immunological patterns emerged. One characterized by positiveness in the skin test to P.b.Ag. and lack of significant abnormalities in cellular immunity, and another anergic to P.b.Ag., with cell mediated immunity severely depressed. Between the two polar groups, there were patients with intermediary patterns of immune response. This paper also includes the results obtained with the administration of transfer factor and levamisole to some of the patients.     

In vivo and in vitro aging of collagen examined using an isometric melting technique.

1. In vivo and in vitro aging of tendon from rat tail, kangaroo tail and human wrist tendon was examined by the technique of isometric melting, in physiological saline. 2. For all these collagens, two mechanisms of structure stabilisation can be distinguished in the melting curves. One of these involves co-valent cross-linking as judged by its increasing stability to heat and acid pH, while the second appears to involve only secondary interactions. 3. The time rate of the first process is slow in vivo; rat tendon up to 2 years does not show it, but it is present in 6-year-old human tendon. However, its in vitro rate is markedly dependent upon the free oxygen content of the physiological saline. At an oxygen concentration of 300 nmol/ml, the in vitro aging rate is about 30 times the in vivo rate for rat tail tendon, and about 20 times for both kangaroo tail tendon and human wrist tendon. At a concentration of 60 nmol/ml (which is about the same as normal arteriovenous blood difference) in vitro aging proceeds close to the in vivo rate.     

In vitro study on effect of bardoxolone methyl on cisplatin-induced cellular senescence in human proximal tubular cells

Molecular and Cellular Biochemistry - Bardoxolone methyl [methyl-2-cyano-3, 12-dioxooleana-1, 9(11)dien-28-oate (CDDO-Me)], an activator of the nuclear factor erythroid-derived 2-related factor2...     

In vitro and in vivo toxicity evaluation of the freshwater cyanobacterium Heteroleiblenia kuetzingii

Cyanobacteria are prokaryotic organisms characterized by their ability to produce secondary metabolites with different biological activities. The aim of this work was to evaluate the in vitro and in vivo toxicity of the cosmopolitan freshwater cyanobacterium H. kuetzingii. An extract from H. kuetzingii and cyanobacterial growth media were assessed for presence of intracellular and extracellular toxins by in vitro tests using primary cell cultures from mouse kidney and fibroblasts, cell lines A549 and 3T3, a fish cell line RTgill-W1 as well as by a traditional in vivo mouse bioassay. The presence of toxicity was compared with the ELISA and HPLC data for corresponding cyanotoxins. In vitro tests showed pronounced cytotoxicity of the cyanobacterium extract and growth medium in which H. kuetzingii released potential extracellular toxic compounds as the mammalian cells were significantly more sensitive to exposure compared to the fish cells. Histopathological analyses of the liver and kidneys of treated mice showed pathological changes such as leukocyte infiltration and necrosis, changes in the proximal and distal convoluted tubules, lack of differentiation of Bowman’s space, enlarged Bowman’s capsules and massive hemorrhages. ELISA and HPLC analyses confirmed the presence of saxitoxins and microcystins at low concentrations. In addition, the histological analyses suggest that H. kuetzingii produces other, yet unknown toxic metabolites. Monitoring efforts are therefore required to evaluate the potential hazard for the freshwater aquatic systems and possible public health implications associated with this cyanobacterium.     

In vitro and in vivo evaluation of oridonin-loaded long circulating nanostructured lipid carriers

The purpose of this study was to develop poly(ethylene glycol)-coated nanostructured lipid carriers (PEG-NLC) for parenteral delivery of oridonin (ORI) to prolong drug circulation time in blood. Oridonin-loaded PEG-NLC (ORI-PEG-NLC) consisting of PEG(2000)-stearate, glycerol monostearate and medium chain triglycerides were prepared by emulsion-evaporation and low temperature-solidification technique. Oridonin-loaded NLC (ORI-NLC) were also prepared as control. ORI-PEG-NLC were observed by transmission election microscope and the morphology was in rotiform shape. The mean particle size of ORI-PEG-NLC was 329.2 nm and entrapment efficacy was 71.18%. The results of differential scanning calorimetry and X-ray diffraction revealed a low-crystalline structure of ORI and verified the incorporation of ORI into the nanoparticles. In vitro drug release of ORI-PEG-NLC exhibited biphasic drug release patterns with burst release initially and prolonged release afterwards. Pharmacokinetic analysis showed that the mean residence time of ORI-PEG-NLC was prolonged and AUC (area under tissue concentration-time curve) value was also improved compared with ORI-NLC and ORI solution. In conclusion, ORI-PEG-NLC could be a potential carrier to get prolonged retention time of oridonin in blood.     

In vitro and in vivo evaluation of the subtype-selective muscarinic agonist PD 151832

PD 151832 is a potent partial muscarinic agonist that displays a high level of functional selectivity for the muscarinic m1 receptor subtype, as evidenced by its selective stimulation of PI turnover and cellular metabolic activity in transfected Hm1-CHO cells at concentrations that produce minimal stimulation of other cloned human muscarinic receptors. PD 151832 enhanced the amplification of Hm1-transfected NIH-3T3 cells at concentrations lower than those required to produce similar effects in Hm2 or Hm3-transfected cells. The functional m1 selectivity of PD 151832 is consistent with its improvement of mouse water maze performance at doses far lower than those required to produce peripheral parasympathetic side effects.     

In vitro and in vivo evaluation of antioxidant activity of Trichosanthes cucumerina aerial parts

The present study was conducted to determine whether aerial parts of Trichosanthes cucumerina extracts can exert significant antioxidant activity. The antioxidant activity of a hot water extract (HWE) and a cold ethanolic extract (CE) of T. cucumerina aerial parts was evaluated by assessing its (a) radical scavenging ability and prevention effect of lipid peroxidation in vitro, and (b) effects on lipid peroxidation and antioxidant enzyme activities, in vivo.In vitro antioxidant assays (DPPH, TBARS and carotene-linoleic acid assays) clearly demonstrated the antioxidant potential of HWE and CEE. Moreover, HWE increased SOD: by 91.2% and GPX by 104.4% while CEE increased SOD: by 115.5% and GPX by 96.4%) in CCl4-induced rats. Treatments with HWE and CE prevented the accumulation of lipid peroxidation products by 30.5% and 33.8%, respectively, in liver tissues compared to the rats exposed only to CCl4. In conclusion, the present investigation demonstrates for the first time that components in T. cucumerina aerial parts can exert significant antioxidant activity in vivo and in vitro.     

In vitro and in vivo evaluation of the prebiotic activity of water-soluble blueberry extracts

The prebiotic effects of water extracts of two blueberry (BBE) cultivars (‘Centurion’ and ‘Maru’) were studied using pure and mixed cultures of human faecal bacteria. The results demonstrated for the first time that addition of BBE from both cultivars to broth media containing pure cultures of Lactobacillus rhamnosus and Bifidobacterium breve resulted in a significant increase (P < 0.05–0.0001) in the population size of these strains. Batch fermentation system was used to monitor the effect of BBE addition on the mixed faecal bacterial populations (obtained from healthy human donors). Addition of BBE from both cultivars to batch cultures inoculated with mixed human faecal cultures resulted in a significant increase in the number of lactobacilli (P < 0.01–0.0001) and bifidobacteria (P < 0.05–0.0001). Furthermore, a significant influence on the population size of lactobacilli and bifidobacteria was observed after administration of extracts from both cultivars to rats daily for 6 days in comparison with the control group. In rats gavaged orally with 4 ml kg⁻¹ day⁻¹ of BBE for 6 days, the population size of lactobacilli (P < 0.05) and bifidobacteria (P < 0.05–0.01) was increased significantly. We hypothesize that BBE could modify the bacterial profile by increasing the numbers of beneficial bacteria and thereby improving gut health.     

Sex-related incidence of tubular metaplasia in Bowman's capsule of aging rats

Although chronic progressive nephropathy and proteinuria are well-known to affect old laboratory rats, the occurrence of tubular metaplasia of Bowman's capsule (TM) in aging rats has received little attention. We report here that old (24-26 months) male, but not female Sprague-Dawley rats show a high incidence of TM which is significantly (P less than 0.01) correlated with the levels of glomerular sclerosis and intracellular deposits of iron in the tubular epithelium. The incidence of these changes was not correlated with serum testosterone levels, which showed a significant age-related reduction in males. The reported findings suggest that the aging male Sprague-Dawley rat is a useful animal model to investigate the pathogenesis of TM and related morphologic changes in hematuric humans.     

Formulation and development of floating capsules of celecoxib: In vitro and in vivo evaluation

The objective of the present study was to develop a hydrodynamically balanced system for celecoxib as single-unit floating capsules. Various grades of low-density polymers were used for formulation of these capsules. The capsules were prepared by physical blending of celecoxib and the polymer in varying ratios. The formulation was optimized on the basis of in vitro buoyancy and in vitro release in citrate phosphate buffer pH 3.0 (with 1% sodium lauryl sulfate). Capsules prepared with polyethylene oxide 60K and Eudragit RL100 gave the best in vitro percentage release and were used as the optimized formulation. By fitting the data into zero-order, first-order, and Higuchi models, we concluded that the release followed zero-order kinetics, as the correlation coefficient (R value) was higher for zero-order release. For gamma scintigraphy studies, celecoxib was radiolabeled with technetium-99m by the stannous reduction method. To achieve the maximum labeling efficiency the process was optimized by studying the reaction at various pH conditions and stannous concentration levels. The radiolabeled complex was added to the optimized capsule, and dissolution studies were performed to ensure that there was no leaching of radioactivity from the capsules. Gamma imaging was performed in rabbits to assess the buoyancy of the optimized formulation. The optimized formulation remained buoyant during 5 hours of gamma scintigraphic studies in rabbits.     

The protective role of autophagy against aging and acute ischemic injury in kidney proximal tubular cells

In kidney, proximal tubules consume a large amount of energy in the process of electrolyte reabsorption. These tubules contain large quantities of mitochondria which provide the energy for this reabsorption. Proximal tubules are susceptible to many kinds of insults such as ischemia-reperfusion injury and nephrotoxic substrates, but little is known of the factors that counteract cellular stress signaling pathways. Autophagy mediates bulk degradation and recycling of cytoplasmic constituents to maintain cellular homeostasis. We demonstrated the critical role of autophagy in normal proximal tubule function and protection against acute tubular injury.