In Vitro and In Vivo Evaluations of PLGA Microspheres Containing Nalmefene

Poor patient compliance, untoward reactions and unstable blood drug levels after the bolus administration are impeding the pharmacotherapy for insobriety. A long-acting preparation may address these limitations. The aim of this paper was to further investigate the in vitro characteristics and in vivo performances of nalmefene microspheres. Nalmefene was blended with poly (lactide-co-glycolide) (PLGA) to prepare the target microspheres by an O/O emulsification solvent evaporation method. The prepared microspheres exhibited a controlled release profile of nalmefene in vitro over 4 weeks, which was well fitted with a first-order model. In vitro degradation study showed that the drug release in vitro was dominated by both drug diffusion and polymer degradation mechanisms. Pharmacokinetics study indicated that the prepared microspheres could provide a relatively constant of nalmefene plasma concentration for at least one month in rats. The in vivo pharmacokinetics profile was well correlated with the in vitro drug release. Pharmacodynamics studies revealed that the drug loaded microspheres could produce a long-acting antagonism efficacy on rats. These results demonstrated the promising application of injectable PLGA microspheres containing nalmefene for the long-term treatment of alcohol dependence.     

Effects of GABAergic Drugs In Vivo on High-Affinity Choline Uptake In Vitro in Mouse Hippocampal Synaptosomes

The effects of several gamma-aminobutyric acid (GABA)-ergic drugs on sodium-dependent high-affinity choline uptake (HACU) were investigated in the hippocampus. HACU was measured in vitro after in vivo administration of the drug to mice. HACU was inhibited by those drugs that enhance GABA transmission. The convulsant 3-mercaptopropionic acid, which decreases GABA levels, stimulated HACU. From these results and previous findings, it appears that GABA mediates a tonic inhibitory effect on the septal-hippocampal cholinergic system.     

In Vitro and In Vivo Models of Cerebral Ischemia Show Discrepancy in Therapeutic Effects of M2 Macrophages

The inflammatory response following ischemic stroke is dominated by innate immune cells: resident microglia and blood-derived macrophages. The ambivalent role of these cells in stroke outcome might be explained in part by the acquisition of distinct functional phenotypes: classically (M1) and alternatively activated (M2) macrophages. To shed light on the crosstalk between hypoxic neurons and macrophages, an in vitro model was set up in which bone marrow-derived macrophages were co-cultured with hippocampal slices subjected to oxygen and glucose deprivation. The results showed that macrophages provided potent protection against neuron cell loss through a paracrine mechanism, and that they expressed M2-type alternative polarization. These findings raised the possibility of using bone marrow-derived M2 macrophages in cellular therapy for stroke. Therefore, 2 million M2 macrophages (or vehicle) were intravenously administered during the subacute stage of ischemia (D4) in a model of transient middle cerebral artery occlusion. Functional neuroscores and magnetic resonance imaging endpoints (infarct volumes, blood-brain barrier integrity, phagocytic activity assessed by iron oxide uptake) were longitudinally monitored for 2 weeks. This cell-based treatment did not significantly improve any outcome measure compared with vehicle, suggesting that this strategy is not relevant to stroke therapy.     

Stage-Dependent Effects of Chloroquine on Plasmodium falciparum In Vitro1

The erythrocytic developmental cycle of Plasmodium falciparum can be conveniently divided into the ring, trophozoite, and schizont stages based on morphology and metabolism. Using highly synchronous cultures of P. falciparum, considerable variation was demonstrated among these stages in sensitivity to chloroquine. The effects of timed, sequential exposure to several clinically relevant concentrations of chloroquine were monitored by three techniques: morphological analysis, changes in the rate of glucose consumption, and changes in the incorporation of ³H-hypoxanthine into parasite nucleic acids. All three techniques gave essentially identical results. The trophozoite and schizont stages were considerably more sensitive to the drug than ring-stage parasites. Chloroquine sensitivity decreased as nuclear division neared completion. The increase in chloroquine sensitivity was coincident with a marked rise in the rate of glucose consumption and nucleic acid synthesis. The rate of nucleic acid synthesis decreased as schizogony progressed while glucose consumption continued at high rates during this process. The degree of chloroquine sensitivity was not highly correlated with either metabolic activity.     

Effects of Lead In Vivo and In Vitro on GABAergic Neurochemistry

Abstract: Alterations in aspects of neurotransmission utilizing -γ-aminobutyric acid (GABA) are associated with in vivo exposure of rats to lead at doses that do not produce convulsions, but sensitize animals to convulsant agents. These effects are observed regionally and include: decreased GABA levels in cerebellum; increased activity of glutamate decarboxylase (GAD) in caudate; and decreased GABA release (both resting and K⁺-stimulated) in cortex, caudate, cerebellum and substantia nigra. Sodium-dependent uptake of GABA by synaptosomes of cerebellum, substantia nigra and caudate was also affected: in these regions, affinity (K_m) was increased and maximal velocity (V_max) was reduced. Sodium-independent binding of GABA to synaptic membranes was increased in cerebellum, but was observed only when tissue was Tritonized and prepared without freezing and washing. No effects on GAD or on GABA uptake, release, or binding were observed when lead was added to brain tissue in vitro in concentrations as high as 100 μM. The results suggest that lead may produce chronic inhibition of presynaptic GABAergic function, notably in the cerebellum, which is associated with supersensitivity of postsynaptic GABA receptors. Failure of lead to affect GABAergic function in vitro may indicate that these effects are secondary to another neurotoxic action of lead in the CNS or are consequent to a nonneuronal metabolic action of lead.     

AMP-Activated Protein Kinase Suppresses the In Vitro and In Vivo Proliferation of Hepatocellular Carcinoma

AMP-activated protein kinase (AMPK) is a central metabolic sensor and plays an important role in regulating glucose, lipid and cholesterol metabolism. Therefore, AMPK is a key therapeutic target in diabetes. Recent pilot studies have suggested that diabetes drugs may reduce the risk of cancer by affecting the AMPK pathway. However, the association between AMPK and the proliferation of hepatocellular carcinoma (HCC) is unknown. In this study, we investigated the relationship between AMPK activity and the proliferation of HCC in cell lines, nude mice and human clinic samples. We first investigated the relationship between AMPK activity and cell proliferation in two HCC cell lines, PLC/PRF/5 and HepG2, by two AMPK activators, 5-aminoimidazole-4-carboxamide-1-h-D-ribofuranoside (AICAR) and metformain. AICAR and metformin treatment significantly inhibited the proliferation of HCC cells and induced cell cycle arrest at G1-S checkpoint. We then observed that metformin abrogated the growth of HCC xenografts in nude mice. The clinical pathology of AMPK activity in HCC, including cell proliferation, differential grade, tumor size and microvessel density, was studied by using 30 clinical tissue samples. In HCC tissue samples, phosphorylated AMPK was expressed mainly in cytoplasm. AMPK activity decreased significantly in HCC in comparison with paracancerous liver tissues (P<0.05). AMPK activity was negatively correlated with the level of Ki-67 (a marker of cell proliferation), differential degradation and tumor size (P<0.05), but not with microvessel density, hemorrhage or necrosis in HCC. Our findings suggest that AMPK activity inhibits the proliferation of HCC and AMPK might be an effective target for prevention and treatment of HCC.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)     

In Vitro and in Vivo Effects of Nitrofurantoin on Experimental Toxoplasmosis

Toxoplasma gondii is an important opportunistic pathogen that causes toxoplasmosis, which has very few therapeutic treatment options. The most effective therapy is a combination of pyrimethamine and sulfadiazine; however, their utility is limited because of drug toxicity and serious side effects. For these reasons, new drugs with lower toxicity are urgently needed. In this study, the compound, (Z)-1-[(5-nitrofuran-2-yl)methyleneamino]-imidazolidine-2,4-dione (nitrofurantoin), showed anti-T. gondii effects in vitro and in vivo. In HeLa cells, the selectivity of nitrofurantoin was 2.3, which was greater than that of pyrimethamine (0.9). In T. gondii-infected female ICR mice, the inhibition rate of T. gondii growth in the peritoneal cavity was 44.7% compared to the negative control group after 4-day treatment with 100 mg/kg of nitrofurantoin. In addition, hematology indicators showed that T. gondii infection-induced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, biochemical parameters involved in liver injury, were reduced by nitrofurantoin significantly. Moreover, nitrofurantoin exerted significant effects on the index of antioxidant status, i.e., malondialdehyde (MDA) and glutathione (GSH). The nitrofurantoin-treated group inhibited the T. gondii-induced MDA levels while alleviating the decrease in GSH levels. Thus, nitrofurantoin is a potential anti-T. gondii candidate for clinical application.     

Preparation, In Vitro Characterization and Preliminary In Vivo Evaluation of Buccal Polymeric Films Containing Chlorhexidine

The aim of this work was to investigate the suitability of some polymeric films as buccal systems for the delivery of the antiseptic drug chlorhexidine diacetate, considered as a valid adjunct in the treatment of oral candidiasis. Six different film formulations, mono- or double-layered, containing 5 or 10 mg of chlorhexidine diacetate, respectively, and alginate and/or hydroxypropylmethylcellulose and/or chitosan as excipients, were prepared by a casting-solvent evaporation technique and characterized in terms of drug content, morphology (scanning electron microscopy), drug release behavior, and swelling properties. Moreover, the in vivo concentrations of chlorhexidine diacetate in saliva were evaluated after application of a selected formulation on the oral mucosa of healthy volunteers. The casting-solvent evaporation proved to be a suitable technique for preparing soft, flexible, and easily handy mono- or double-layered chlorhexidine-loaded films. Some prepared formulations showed favorable in vitro drug release rates and swelling properties. The behavior of a selected formulation, chosen on the basis of its in vitro release results, was preliminarily investigated in vivo after application in the oral cavity of healthy volunteers. The films were well tolerated and the salivary chlorhexidine concentrations were maintained above the minimum inhibitory concentration for Candida albicans for almost 3 h. These preliminary results indicate that polymeric films can represent a valid vehicle for buccal delivery of antifungal/antimicrobial drugs.     

Genetic and Biochemical Approaches for In Vivo and In Vitro Assessment of Protein Oligomerization: The Ryanodine Receptor Case Study

Oligomerization is often a structural requirement for proteins to accomplish their specific cellular function. For instance, tetramerization of the ryanodine receptor (RyR) is necessary for the formation of a functional Ca²⁺ release channel pore. Here, we describe detailed protocols for the assessment of protein self-association, including yeast two-hybrid (Y2H), co-immunoprecipitation (co-IP) and chemical cross-linking assays. In the Y2H system, protein self-interaction is detected by β-galactosidase assay in yeast co-expressing GAL4 bait and target fusions of the test protein. Protein self-interaction is further assessed by co-IP using HA- and cMyc-tagged fusions of the test protein co-expressed in mammalian HEK293 cells. The precise stoichiometry of the protein homo-oligomer is examined by cross-linking and SDS-PAGE analysis following expression in HEK293 cells. Using these different but complementary techniques, we have consistently observed the self-association of the RyR N-terminal domain and demonstrated its intrinsic ability to form tetramers. These methods can be applied to protein-protein interaction and homo-oligomerization studies of other mammalian integral membrane proteins.     

The Effects of Piroxicam in the Attenuation of MPP+/MPTP Toxicity In Vitro and In Vivo

Several lines of evidence support the neuroprotective action of cyclooxygenase-2 (COX-2) inhibitors in various models of Parkinson’s disease (PD). In the current study, we investigated the neuroprotective properties of several COX inhibitors against 1-methyl-4-phenylpyridinium (MPP+) in neuroblastoma Neuro 2A (N-2A) cells in vitro and the protection against degeneration of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons after the administration of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) in C57/BL6 male mice. The data obtained demonstrate a lack of protective effects observed by COX 1-2 inhibitors ibuprofen and acetylsalicylic acid against MPP+ toxicity in N-2A, where piroxicam was protective in a dose dependent manner (MPP+ control: 15 ± 2% MPP+ piroxicam: 5 mM 89 ± 4%). The data also indicate a drop in mitochondrial oxygen (O₂) consumption and ATP during MPP+ toxicity with no restoration of mitochondrial function concurrent to a heightened concentration of somatic ATP during piroxicam rescue. These findings indicate that the neuroprotective effects of COX inhibitors against MPP+ are not consistent, but that piroxicam may work through an unique mechanism to propel anaerobic energy metabolism. On the other hand, using mice, piroxicam (20 mg/kg) was effective against MPTP-induced dopaminergic degeneration in the (SNc) and loss of locomotive function in mice. Administering a 3 day pre-treatment of piroxicam (20 mg/kg) was effective in antagonizing the losses in SNc tyrosine hydroxylase protein expression, SNc DA concentration and associated anomaly in ambulatory locomotor activity. It was concluded from these findings that piroxicam is unique among COX inhibitors in providing very significant neuroprotection against MPP+ in vitro and in vivo.     

Effects of Dexamethasone In Vivo and In Vitro on Hexose Transport in Brain Microvasculature

Glucocorticoids induce hyperinsulinemia, hyperglycemia, and depress glucose transport by aortic endothelium. High glucocorticoid doses are used for many diseases, but with unknown effects on brain glucose transport or metabolism. This study tested the hypothesis that glucocorticoids affect glucose transport or metabolism by brain microvascular endothelium. Male rats received dexamethasone (DEX) sc with sucrose feeding for up to seven days. Cerebral microvessels from rats treated with DEX/sucrose demonstrated increased GLUT1 and brain glucose extraction compared to controls. Glucose transport in vivo correlated with hyperinsulinemia. Pre-treatment with low doses of strep-tozotocin blunted hyperinsulinemia and prevented increased glucose extraction induced by DEX. In contrast, isolated brain microvessels exposed to DEX in vitro demonstrated suppression of 2-deox-yglucose uptake and glucose oxidation. We conclude that DEX/sucrose treatment in vivo increases blood-brain glucose transport in a manner that requires the effects of chronic hyperinsulinemia. These effects override any direct inhibitory effects of either hyperglycemia or DEX.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

The Effects of Mechanical Loading on Tendons - An In Vivo and In Vitro Model Study

Mechanical loading constantly acts on tendons, and a better understanding of its effects on the tendons is essential to gain more insights into tendon patho-physiology. This study aims to investigate tendon mechanobiological responses through the use of mouse treadmill running as an in vivo model and mechanical stretching of tendon cells as an in vitro model. In the in vivo study, mice underwent moderate treadmill running (MTR) and intensive treadmill running (ITR) regimens. Treadmill running elevated the expression of mechanical growth factors (MGF) and enhanced the proliferative potential of tendon stem cells (TSCs) in both patellar and Achilles tendons. In both tendons, MTR upregulated tenocyte-related genes: collagen type I (Coll. I ∼10 fold) and tenomodulin (∼3–4 fold), but did not affect non-tenocyte-related genes: LPL (adipocyte), Sox9 (chondrocyte), Runx2 and Osterix (both osteocyte). However, ITR upregulated both tenocyte (Coll. I ∼7–11 fold; tenomodulin ∼4–5 fold) and non-tenocyte-related genes (∼3–8 fold). In the in vitro study, TSCs and tenocytes were stretched to 4% and 8% using a custom made mechanical loading system. Low mechanical stretching (4%) of TSCs from both patellar and Achilles tendons increased the expression of only the tenocyte-related genes (Coll. I ∼5–6 fold; tenomodulin ∼6–13 fold), but high mechanical stretching (8%) increased the expression of both tenocyte (Coll. I ∼28–50 fold; tenomodulin ∼14–48 fold) and non-tenocyte-related genes (2–5-fold). However, in tenocytes, non-tenocyte related gene expression was not altered by the application of either low or high mechanical stretching. These findings indicate that appropriate mechanical loading could be beneficial to tendons because of their potential to induce anabolic changes in tendon cells. However, while excessive mechanical loading caused anabolic changes in tendons, it also induced differentiation of TSCs into non-tenocytes, which may lead to the development of degenerative tendinopathy frequently seen in clinical settings.     

Topical Formulations Containing Pimenta pseudocaryophyllus Extract: In Vitro Antioxidant Activity and In Vivo Efficacy Against UV-B-Induced Oxidative Stress

Pimenta pseudocaryophyllus is a Brazilian native plant that presents high concentrations of flavonoids and other polyphenolic compounds. Herein, we evaluated: (1) the chemical properties of P. pseudocaryophyllus ethanolic extract (PPE), (2) the in vitro antioxidant activity (AA) of PPE and of two different topical formulations (F1 and F2) containing PPE, (3) physico-chemical and functional stability, (4) in vitro release of PPE, and (5) in vivo capacity of formulations to prevent UV-B irradiation-induced skin damage. Results show that the polyphenol and flavonoid contents in PPE were 199.33 and 28.32 mg/g, respectively, and HPLC results show the presence of eugenol, tannic acid, and rutin. Evaluation of the in vitro AA of PPE demonstrated a dose-dependent effect and an IC₅₀ of 4.75 μg/mL in 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 3.0 μg/mL in 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The ferric-reducing antioxidant power (FRAP assay) was 0.046 μmol/L trolox equivalent/μg/mL of extract. Among the AA, only the capacity to scavenge DPPH radical of PPE was maintained in F1 and F2. In addition, both formulations satisfactorily released the extract. The evaluation of the functional stability of F1 and F2 did not demonstrate loss of activity by storage at room temperature and at 4°C/6 months. In irradiated mice, treatment with F1 and F2 added with PPE significantly increased the capacity to scavenge ABTS radical and the FRAP of skin compared to vehicle-treated mice. In conclusion, the present results suggest that formulations containing PPE may be a topical source of antioxidant compounds to decrease oxidative damages of the skin.     

Effects of Sertraline and Fluoxetine on P-Glycoprotein at Barrier Sites: In Vivo and In Vitro Approaches

Background and Purpose

Retention of substances from systemic circulation in the brain and testes are limited due to high levels of P-glycoprotein (P-gp) in the luminal membranes of brain and testes capillary endothelial cells. From a clinical perspective, P-gp rapidly extrudes lipophilic therapeutic agents, which then fail to reach efficacious levels. Recent studies have demonstrated that acute administration of selective serotonin reuptake inhibitors (SSRI) can affect P-gp function, in vitro and in vivo. However, little is known concerning the time-course of these effects or the effects of different SSRI in vivo.

Experimental Approach

The P-gp substrate, tritiated digoxin ([³H] digoxin), was co-administered with fluoxetine or sertraline to determine if either compound increased drug accumulation within the brains and testes of mice due to inhibition of P-gp activity. We undertook parallel studies in endothelial cells derived from brain microvessels to determine the dose-response and time-course of effects.

Key Results

In vitro, sertraline resulted in rapid and potent inhibition of P-gp function in brain endothelial cells, as determined by cellular calcein accumulation. In vivo, a biphasic effect was demonstrated. Brain accumulation of [³H] digoxin was increased 5 minutes after treatment with sertraline, but by 60 minutes after sertraline treatment, brain accumulation of digoxin was reduced compared to control. By 240 minutes after sertraline treatment brain digoxin accumulation was elevated compared to control. A similar pattern of results was obtained in the testes. There was no significant effect of fluoxetine on P-gp function, in vitro or in vivo.

Conclusions and Implications

Acute sertraline administration can modulate P-gp activity in the blood-brain barrier and blood-testes barrier. This clearly has implications for the ability of therapeutic agents that are P-gp substrates, to enter the brain when co-administered with SSRI.     

Effects of Kainic Acid on Brain Calcium Fluxes Studied In Vivo and In Vitro

The effect of in vivo administration of kainic acid into the rabbit hippocampus was studied with brain dialysis and subsequent determination of the Ca2+ concentration in the dialysate. When included in the perfusing medium, kainic acid as well as veratridine induced a decrease in extracellular Ca2+. The effect of kainic acid (but not of veratridine) was insensitive to tetrodotoxin. In vitro studies revealed no effect of kainic acid on 45Ca2+ uptake by isolated astrocytes, but showed an enhancement of synaptosomal 45Ca2+ accumulation. This was, however, only 25% of the stimulatory effect of high K+ depolarization. Glutamate activated synaptosomal Ca2+ uptake, whereas dihydrokainate had no effect. The uptake evoked by kainate and glutamate was independent of the K+ level in the medium which indicates the involvement of other than voltage-sensitive Ca2+ channels. The results confirm previous finding that kainic acid promotes the uptake of Ca2+ in brain cells. Kainate affects Ca2+ fluxes pre- and postsynaptically. Presynaptic Ca2+ influx may be mediated by chemically gated mechanisms.     

Radiation-Induced DNA Methylation Disorders: In Vitro and In Vivo Studies

Biology Bulletin - Phenomenological aspects and mechanisms of DNA methylation disorders (changes in the total level of DNA methylation and in genome repetitive elements, locus-specific methylation...     

In Vitro and In Vivo Effects of Palmaria palmata Derived Peptides on Glucose Metabolism

International Journal of Peptide Research and Therapeutics - Three synthetic peptides, ILAP, LLAP and MAGVDHI, derived from a Palmaria palmata protein hydrolysate were assessed for their...