Showing papers on "In vivo published in 2005"

Chemical imaging of tissue in vivo with video-rate coherent anti-Stokes Raman scattering microscopy.

Abstract: Imaging living organisms with molecular selectivity typically requires the introduction of specific labels. Many applications in biology and medicine, however, would significantly benefit from a noninvasive imaging technique that circumvents such exogenous probes. In vivo microscopy based on vibrational spectroscopic contrast offers a unique approach for visualizing tissue architecture with molecular specificity. We have developed a sensitive technique for vibrational imaging of tissues by combining coherent anti-Stokes Raman scattering (CARS) with video-rate microscopy. Backscattering of the intense forward-propagating CARS radiation in tissue gives rise to a strong epi-CARS signal that makes in vivo imaging possible. This substantially large signal allows for real-time monitoring of dynamic processes, such as the diffusion of chemical compounds, in tissues. By tuning into the CH2 stretching vibrational band, we demonstrate CARS imaging and spectroscopy of lipid-rich tissue structures in the skin of a live mouse, including sebaceous glands, corneocytes, and adipocytes, with unprecedented contrast at subcellular resolution.

Predicting in vivo drug interactions from in vitro drug discovery data

Abstract: In vitro screening for drugs that inhibit cytochrome P450 enzymes is well established as a means for predicting potential metabolism-mediated drug interactions in vivo. Given that these predictions are based on enzyme kinetic parameters observed from in vitro experiments, the miscalculation of the inhibitory potency of a compound can lead to an inaccurate prediction of an in vivo drug interaction, potentially precluding a safe drug from advancing in development or allowing a potent inhibitor to 'slip' into the patient population. Here, we describe the principles underlying the generation of in vitro drug metabolism data and highlight commonly encountered uncertainties and sources of bias and error that can affect extrapolation of drug-drug interaction information to the clinical setting.

Therapeutic EphA2 Gene Targeting In vivo Using Neutral Liposomal Small Interfering RNA Delivery

Abstract: Inducing destruction of specific mRNA using small interfering RNA (siRNA) is a powerful tool in analysis of protein function, but its use as a therapeutic modality has been limited by inefficient or impractical delivery systems. We have used siRNA incorporated into the neutral liposome 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) for efficient in vivo siRNA delivery. In nude mice bearing i.p. ovarian tumors, nonsilencing siRNA tagged with the fluorochrome Alexa 555 was encapsulated into DOPC liposomes and shown to be taken up by the tumor as well as many major organs. Furthermore, DOPC-encapsulated siRNA targeting the oncoprotein EphA2 was highly effective in reducing in vivo EphA2 expression 48 hours after a single dose as measured by both Western blot and immunohistochemistry. Therapy experiments in an orthotopic mouse model of ovarian cancer were initiated 1 week after injection of either HeyA8 or SKOV3ip1 cell lines. Three weeks of treatment with EphA2-targeting siRNA-DOPC (150 microg/kg twice weekly) reduced tumor growth when compared with a nonsilencing siRNA (SKOV3ip1: 0.35 versus 0.70 g; P = 0.020; HeyA8: 0.98 versus 1.51 g; P = 0.16). When EphA2-targeting siRNA-DOPC was combined with paclitaxel, tumor growth was dramatically reduced compared with treatment with paclitaxel and a nonsilencing siRNA (SKOV3ip1: 0.04 versus 0.22 g; P < 0.001; HeyA8: 0.21 versus 0.84 g; P = 0.0027). These studies show the feasibility of siRNA as a clinically applicable therapeutic modality.

Metabolism and bioavailability of trans‐resveratrol

Abstract: Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a polyphenolic compound accounting to the stilbene class. Most stilbenes in plants act as antifungal phytoalexins, compounds that are usually synthesized only in response to infection or injury. Resveratrol has been detected in trees, in a few flowering plants, in peanuts, and in grapevines. The major dietary sources of resveratrol include grapes, wine, peanuts, and peanut products. Numerous in vitro studies describe different biological effects of resveratrol. The major impacts are the antioxidative, anti-inflammatory, and estrogenic effects as well as anticancer and chemopreventive activities. In order to reveal information on absorption, metabolism, and the consequent bioavailability of resveratrol, different research approaches were performed, including in vitro, ex vivo, and in vivo models, all of which are considered in this review. Summarizing the data, resveratrol is absorbed and metabolized. Around 75% of this polyphenol are excreted via feces and urine. The oral bioavailability of resveratrol is almost zero due to rapid and extensive metabolism and the consequent formation of various metabolites as resveratrol glucuronides and resveratrol sulfates. The potential biologic activity of resveratrol conjugates should be considered in future investigations.

Organically modified silica nanoparticles: a nonviral vector for in vivo gene delivery and expression in the brain.

Abstract: This article reports on the application of organically modified silica (ORMOSIL) nanoparticles as a nonviral vector for efficient in vivo gene delivery. Highly monodispersed, stable aqueous suspension of nanoparticles, surface-functionalized with amino groups for binding of DNA, were prepared and characterized. Stereotaxic injections of nanoparticles, complexed with plasmid DNA encoding for EGFP, into the mouse ventral midbrain and into lateral ventricle, allowed us to fluorescently visualize the extensive transfection of neuronal-like cells in substantia nigra and areas surrounding the lateral ventricle. No ORMOSIL-based toxicity was observed 4 weeks after transfection. The efficiency of transfection equaled or exceeded that obtained in studies using a viral vector. An in vivo optical imaging technique (a fiber-based confocal fluorescent imaging system) provided an effective means to show the retention of viability of the transfected cells. The ORMOSIL-mediated transfections also were used to manipulate the biology of the neural stem/progenitor cells in vivo. Transfection of a plasmid expressing the nucleus-targeting fibroblast growth factor receptor type 1 resulted in significant inhibition of the in vivo incorporation of bromodeoxyuridine into the DNA of the cells in the subventricular zone and the adjacent rostral migratory stream. This in vivo approach shows that the nuclear receptor can control the proliferation of the stem/progenitor cells in this region of the brain. The results of this nanomedicine approach using ORMOSIL nanoparticles as a nonviral gene delivery platform have a promising future direction for effective therapeutic manipulation of the neural stem/progenitor cells as well as in vivo targeted brain therapy.

Liposome-encapsulated curcumin: in vitro and in vivo effects on proliferation, apoptosis, signaling, and angiogenesis.

Abstract: BACKGROUND Because a role for nuclear factor-κB (NF-κB) has been implicated in the pathogenesis of pancreatic carcinoma, this transcription factor is a potential target for the treatment of this devastating disease. Curcumin (diferuloylmethane) is a phytochemical with potent NF-κB-inhibitory activity. It is pharmacologically safe, but its bioavailability is poor after oral administration. METHODS The authors encapsulated curcumin in a liposomal delivery system that would allow intravenous administration. They studied the in vitro and in vivo effects of this compound on proliferation, apoptosis, signaling, and angiogenesis using human pancreatic carcinoma cells. NF-κB was constitutively active in all human pancreatic carcinoma cell lines evaluated and liposomal curcumin consistently suppressed NF-κB binding (electrophoretic mobility gel shift assay) and decreased the expression of NF-κB-regulated gene products, including cyclooxygenase-2 (immunoblots) and interleukin-8 (enzyme-linked immunoassay), both of which have been implicated in tumor growth/invasiveness. These in vitro changes were associated with concentration and time-dependent antiproliferative activity (3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide assay [MTT assay]) and proapoptotic effects (annexin V/propidium iodide staining [fluorescence-activated cell sorting] and polyadenosine-5′-diphosphate-ribose-polymerase cleavage). RESULTS The activity of liposomal curcumin was equal to or better than that of free curcumin at equimolar concentrations. In vivo, curcumin suppressed pancreatic carcinoma growth in murine xenograft models and inhibited tumor angiogenesis. CONCLUSIONS Liposomal curcumin down-regulated the NF-κB machinery, suppressed growth, and induced apoptosis of human pancreatic cells in vitro. Antitumor and antiangiogenesis effects were observed in vivo. The experiments in the current study provide a biologic rationale for treatment of patients suffering from pancreatic carcinoma with this nontoxic phytochemical encapsulated in liposomes for systemic delivery. Cancer 2005. © 2005 American Cancer Society.

In vivo quercitrin anti-inflammatory effect involves release of quercetin, which inhibits inflammation through down-regulation of the NF-kappaB pathway.

Abstract: Quercetin is a common antioxidant flavonoid found in vegetables, which is usually present in glycosylated forms, such as quercitrin (3-rhamnosylquercetin). Previous in vitro experiments have shown that quercetin exerts a bigger effect than quercitrin in the down-regulation of the inflammatory response. However, such results have not been reproduced in in vivo experimental models of intestinal inflammation, in which quercetin did not show beneficial effects while its glycosides, quercitrin or rutin, have demonstrated their effectiveness. In this study, we have reported that the in vivo effects of quercitrin in the experimental model of rat colitis induced by dextran sulfate sodium can be mediated by the release of quercetin generated after glycoside's cleavage by the intestinal microbiota. This is supported by the fact that quercetin, but not quercitrin, is able to down-regulate the inflammatory response of bone marrow-derived macrophages in vitro. Moreover, we have demonstrated that quercetin inhibits cytokine and inducible nitric oxide synthase expression through inhibition of the NF-jB pathway without modification of c-Jun N-terminal kinase activity (both in vitro and in vivo). As a conclusion, our report suggests that quercitrin releases quercetin in order to perform its anti-inflammatory effect which is mediated through the inhibition of the NF-jB pathway.

Dynamic Imaging of Allogeneic Mesenchymal Stem Cells Trafficking to Myocardial Infarction

Abstract: Background— Recent results from animal studies suggest that stem cells may be able to home to sites of myocardial injury to assist in tissue regeneration. However, the histological interpretation of postmortem tissue, on which many of these studies are based, has recently been widely debated. Methods and Results— With the use of the high sensitivity of a combined single-photon emission CT (SPECT)/CT scanner, the in vivo trafficking of allogeneic mesenchymal stem cells (MSCs) colabeled with a radiotracer and MR contrast agent to acute myocardial infarction was dynamically determined. Redistribution of the labeled MSCs after intravenous injection from initial localization in the lungs to nontarget organs such as the liver, kidney, and spleen was observed within 24 to 48 hours after injection. Focal and diffuse uptake of MSCs in the infarcted myocardium was already visible in SPECT/CT images in the first 24 hours after injection and persisted until 7 days after injection and was validated by tissue counts of...

An in vivo microanalytical technique for measuring the local biochemical milieu of human skeletal muscle

Abstract: Myofascial pain associated with myofascial trigger points (MTrPs) is a common cause of nonarticular musculoskeletal pain. Although the presence of MTrPs can be determined by soft tissue palpation, ...

In vivo imaging platform for tracking immunotherapeutic cells

Abstract: Cellular therapeutics show great promise for the treatment of disease, but few noninvasive techniques exist for monitoring the cells after administration. Here we present a magnetic resonance imaging (MRI) technology that uses perfluoropolyether (PFPE) agents to track cells in vivo. Fluorine MRI selectively images only the labeled cells, and a 'conventional' (1)H image places the cells in their anatomical context. We labeled phenotypically defined dendritic cells (DCs) with PFPE ex vivo and observed efficient intracellular uptake of the PFPE with little effect on DC function. We injected labeled DCs into tissue or intravenously in mice and then tracked the cells in vivo using (19)F MRI. Although we focused on DCs, which are being developed as immunotherapeutics for cancer and autoimmune diseases, this technology should be useful for monitoring a wide range of cell types in vivo.

Telomere end-binding proteins control the formation of G-quadruplex DNA structures in vivo

Abstract: Telomere end-binding proteins (TEBPs) bind to the guanine-rich overhang (G-overhang) of telomeres. Although the DNA binding properties of TEBPs have been investigated in vitro, little is known about their functions in vivo. Here we use RNA interference to explore in vivo functions of two ciliate TEBPs, TEBPα and TEBPβ. Silencing the expression of genes encoding both TEBPs shows that they cooperate to control the formation of an antiparallel guanine quadruplex (G-quadruplex) DNA structure at telomeres in vivo. This function seems to depend on the role of TEBPα in attaching telomeres in the nucleus and in recruiting TEBPβ to these sites. In vitro DNA binding and footprinting studies confirm the in vivo observations and highlight the role of the C terminus of TEBPβ in G-quadruplex formation. We have also found that G-quadruplex formation in vivo is regulated by the cell cycle–dependent phosphorylation of TEBPβ.

In Vivo Quantitative Measurement of Intact Fibrous Cap and Lipid-Rich Necrotic Core Size in Atherosclerotic Carotid Plaque: Comparison of High-Resolution, Contrast-Enhanced Magnetic Resonance Imaging and Histology

Abstract: Background— Previous studies with contrast-enhanced magnetic resonance imaging (CEMRI) have shown that the fibrous cap (FC) in atherosclerotic carotid plaques enhances with gadolinium-based contrast agents. Conversely, the lipid-rich necrotic core (LR-NC), lacking both vasculature and matrix, shows no or only slight enhancement. The goal of this study was to assess whether CEMRI can be used to accurately measure the dimensions of the intact FC and LR-NC. Methods and Results— Twenty-one patients scheduled for carotid endarterectomy were imaged with a 1.5-T scanner. Precontrast images and CEMRI were obtained. One hundred eight locations with an intact FC were matched between MRI and the excised histology specimens. Quantitative measurements of FC length along the lumen circumference, FC area, and LR-NC area were collected from CEMRI images and histology sections. Blinded comparison of corresponding MR images and histology slices showed moderate to good correlation for length (r=0.73, P<0.001) and area (r=0....

Mechanism-based inhibition of cytochrome P450 3A4 by therapeutic drugs.

Abstract: Consistent with its highest abundance in humans, cytochrome P450 (CYP) 3A is responsible for the metabolism of about 60% of currently known drugs. However, this unusual low substrate specificity also makes CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Mechanism-based inhibition of CYP3A4 is characterised by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-, time- and concentration-dependent enzyme inactivation, occurring when some drugs are converted by CYP isoenzymes to reactive metabolites capable of irreversibly binding covalently to CYP3A4. Approaches using in vitro, in silico and in vivo models can be used to study CYP3A4 inactivation by drugs. Human liver microsomes are always used to estimate inactivation kinetic parameters including the concentration required for half-maximal inactivation (K I) and the maximal rate of inactivation at saturation (k inact). Clinically important mechanism-based CYP3A4 inhibitors include antibacterials (e.g. clarithromycin, erythromycin and isoniazid), anticancer agents (e.g. tamoxifen and irinotecan), anti-HIV agents (e.g. ritonavir and delavirdine), anti-hypertensives (e.g. dihydralazine, verapamil and diltiazem), sex steroids and their receptor modulators (e.g. gestodene and raloxifene), and several herbal constituents (e.g. bergamottin and glabridin). Drugs inactivating CYP3A4 often possess several common moieties such as a tertiary amine function, furan ring, and acetylene function. It appears that the chemical properties of a drug critical to CYP3A4 inactivation include formation of reactive metabolites by CYP isoenzymes, preponderance of CYP inducers and P-glycoprotein (P-gp) substrate, and occurrence of clinically significant pharmacokinetic interactions with coadministered drugs. Compared with reversible inhibition of CYP3A4, mechanism-based inhibition of CYP3A4 more frequently cause pharmacokinetic-pharmacodynamic drug-drug interactions, as the inactivated CYP3A4 has to be replaced by newly synthesised CYP3A4 protein. The resultant drug interactions may lead to adverse drug effects, including some fatal events. For example, when aforementioned CYP3A4 inhibitors are coadministered with terfenadine, cisapride or astemizole (all CYP3A4 substrates), torsades de pointes (a life-threatening ventricular arrhythmia associated with QT prolongation) may occur. However, predicting drug-drug interactions involving CYP3A4 inactivation is difficult, since the clinical outcomes depend on a number of factors that are associated with drugs and patients. The apparent pharmacokinetic effect of a mechanism-based inhibitor of CYP3A4 would be a function of its K I, k inact and partition ratio and the zero-order synthesis rate of new or replacement enzyme. The inactivators for CYP3A4 can be inducers and P-gp substrates/inhibitors, confounding in vitro-in vivo extrapolation. The clinical significance of CYP3A inhibition for drug safety and efficacy warrants closer understanding of the mechanisms for each inhibitor. Furthermore, such inactivation may be exploited for therapeutic gain in certain circumstances.

Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium

Abstract: Here we report a genetically modified bacteria strain, Salmonella typhimurium A1, selected for anticancer activity in vivo. The strain grows in tumor xenografts. In sharp contrast, normal tissue is cleared of these bacteria even in immunodeficient athymic mice. S. typhimurium A1 is auxotrophic (Leu/Arg-dependent) but apparently receives sufficient support from the neoplastic tissue to grow locally. Whether additional genetic lesions are present is not known. In in vitro infection, the GFP-expressing bacteria grew in the cytoplasm of PC-3 human prostate cancer cells and caused nuclear destruction. These effects were visualized in cells labeled with GFP in the nucleus and red fluorescent protein in the cytoplasm. In vivo, the bacteria caused tumor inhibition and regression of xenografts visualized by whole-body imaging. The bacteria, introduced i.v. or intratumorally, invaded and replicated intracellularly in PC-3 prostate cancer cells labeled with red fluorescent protein grafted into nude mice. By day 15, S. typhimurium A1 was undetectable in the liver, lung, spleen, and kidney, but it continued to proliferate in the PC-3 tumor, which stopped growing. When the bacteria were injected intratumorally, the tumor completely regressed by day 20. There were no obvious adverse effects on the host when the bacteria were injected by either route. The S. typhimurium A1 strain grew throughout the tumor, including viable malignant tissue. This result is in marked contrast to bacteria previously tried for cancer therapy that were confined to necrotic areas of the tumor, which may account, in part, for the strain's unique antitumor efficacy.

Adenovirus Binding to Blood Factors Results in Liver Cell Infection and Hepatotoxicity

Abstract: Adenoviruses (Ad) are efficient vehicles for gene delivery in vitro and in vivo. Therefore, they are a promising tool in gene therapy, particularly in the treatment of cancer and cardiovascular diseases. However, preclinical and clinical studies undertaken during the last decade have revealed a series of problems that limit both the safety and efficacy of Ad vectors, specifically after intravenous application. Major obstacles to clinical use include innate toxicity and Ad sequestration by nontarget tissues. The factors and mechanisms underlying these processes are poorly understood. The majority of intravenously injected Ad particles are sequestered by the liver, which in turn causes an inflammatory response characterized by acute transaminitis and vascular damage. Here, we describe a novel pathway that is used by Ad for infection of hepatocytes and Kupffer cells upon intravenous virus application in mice. We found that blood factors play a major role in targeting Ad vectors to hepatic cells. We demonstrated that coagulation factor IX and complement component C4-binding protein can bind the Ad fiber knob domain and provide a bridge for virus uptake through cell surface heparan sulfate proteoglycans and low-density lipoprotein receptor-related protein. An Ad vector, Ad5mut, which contained mutations in the fiber knob domain ablating blood factor binding, demonstrated significantly reduced infection of liver cells and liver toxicity in vivo. This study contributes to a better understanding of adenovirus-host interactions for intravenously applied vectors. It also provides a rationale for novel strategies to target adenovirus vector to specific tissues and to reduce virus-associated toxicity after systemic application.

The influence of surgical operations on components of the human immune system

Abstract: Surgical operations have been shown to cause a variety of immunological disturbances in man both in vivo and in vitro. With few exceptions the overall picture is one of a generalized state of immunodepression in the postoperative period. The implications of these observations are that host defences may be compromised by surgical procedures, thus providing a 'fertile soil' for bacterial invasion and tumour cell metastasis at the very time when risks from invading pathogens and viable tumour cells are maximal. We have studied the effects of surgical operations on the immune system in 35 patients with benign disease. Surgical procedures were classified as either minor (n = 15) or major (n = 20). A panel of monoclonal antibodies was used to identify peripheral blood lymphocyte subpopulations and analysis was performed using flow cytometry. Simultaneous estimations of plasma alpha-1 proteinase inhibitor (alpha-1-PI), alpha-2-macroglobulin (alpha-2-M), alpha-2-pregnancy-associated glycoprotein (alpha-2-PAG) and plasma suppressive activity (PSA) on stimulated allogeneic lymphocytes were performed before operation and on postoperative days 1, 3, 7, 17 and 21. Circulating numbers of all lymphocyte subpopulations fell significantly following surgery, except for B lymphocytes which did not change. The magnitude, and duration of the reduction in cell numbers and the subpopulation affected was significantly related to the degree of surgical trauma, and returned to pre-operative values by postoperative day 7. Changes in alpha-1-PI, alpha-2-M, alpha-2-PAG and PSA were also significantly related to the degree of surgical trauma, and these plasma changes persisted longer than the cellular disturbances. Surgical operations induce a reversible depression of cellular immunity which precedes plasma suppressive activity in its return to pre-operative levels. Immunostimulating agents such as interferon and the interleukins deserve evaluation as prophylactic agents pre-operatively.

A Small-Molecule-Inducible Nrf2-Mediated Antioxidant Response Provides Effective Prophylaxis against Cerebral Ischemia In Vivo

Abstract: The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) coordinates expression of genes required for free radical scavenging, detoxification of xenobiotics, and maintenance of redox potential. Previously, activation of this pleiotropic response was neuroprotective in cell culture models that simulate components of stroke damage. However, the role of Nrf2 in limiting stroke damage in vivo remained unclear. We report that Nrf2 activation protects the brain from cerebral ischemia in vivo . Acute (1-3 d) intracerebroventricular or intraperitoneal pretreatment with tert -butylhydroquinone (tBHQ), an Nrf2 activity inducer, reduced cortical damage and sensorimotor deficit at 24 h and even 1 month after ischemia-reperfusion in rats. Cortical glutathione levels robustly increased with tBHQ administration to rats and Nrf2-expressing mice, but not Nrf2 -/- mice. Basal and inducible activities of antioxidant/detoxification enzymes in Nrf2 -/- mice were reduced when compared with Nrf2 +/+ controls. Interestingly, larger infarcts were observed in Nrf2 -/- mice at 7 d after stroke, but not at 24 h, suggesting that Nrf2 may play a role in shaping the penumbra well after the onset of ischemia. Neuronal death caused by a “penumbral” model of stroke, using intracortical endothelin-1 microinjection, was attenuated by tBHQ administration to Nrf2 +/+ , but not to Nrf2 -/- mice, confirming the Nrf2-specific action of tBHQ in vivo . We conclude that Nrf2 plays a role in modulating ischemic injury in vivo . Accordingly, Nrf2 activation by small molecule inducers may be a practical preventative treatment for stroke-prone patients.

Epidermal Growth Factor Receptor–Targeted Immunoliposomes Significantly Enhance the Efficacy of Multiple Anticancer Drugs In vivo

Abstract: We previously reported the development of epidermal growth factor receptor (EGFR)-targeted immunoliposomes that bind and internalize in tumor cells which overexpress EGFR and/or mutant EGFR variant III (EGFRvIII), enabling intracellular delivery of potent anticancer agents in vitro. We now describe in vivo proof-of-concept for this approach for the delivery of multiple anticancer drugs in EGFR-overexpressing tumor models. Anti-EGFR immunoliposomes were constructed modularly with Fab' fragments of cetuximab (IMC-C225), covalently linked to liposomes containing probes and/or anticancer drugs. Pharmacokinetic and biodistribution studies confirmed long circulation times (t(1/2) = 21 hours) and efficient accumulation in tumors (up to 15% ID/g) irrespective of the presence of the targeting ligand. Although total accumulations of anti-EGFR immunoliposomes and nontargeted liposomes in EGFR-overexpressing tumors were comparable, only immunoliposomes internalized extensively within tumor cells (92% of analyzed cells versus <5% for nontargeted liposomes), indicating different mechanisms of delivery at the cellular level. In vivo therapy studies in a series of xenograft models featuring overexpression of EGFR and/or EGFRvIII showed the superiority of immunoliposomal delivery of encapsulated drugs, which included doxorubicin, epirubicin, and vinorelbine. For each of these drugs, anti-EGFR immunoliposome delivery showed significant antitumor effects and was significantly superior to all other treatments, including the corresponding free or liposomal drug (P < 0.001-0.003). We conclude that anti-EGFR immunoliposomes provide efficient and targeted drug delivery of anticancer compounds and may represent a useful new treatment approach for tumors that overexpress the EGFR.

Synthesis and antiinflammatory activity of coumarin derivatives

Abstract: The synthesis of several coumarin Mannich bases is described. The structures of the synthesized compounds were confirmed by spectral and elemental analysis. Their lipophilicity was determined experimentally by RPTLC method. All compounds were evaluated for their antiinflammatory and antioxidant activity and for their ability to inhibit in vitro lipoxygenase. The derivatives were found to present antioxidant and antiinflammatory activities. The tested derivatives inhibited carraggeenin-induced hind paw edema. They also significantly suppressed the arthritis induced by Freund's adjuvant. Compound 10, the most active in vivo, was found to possess protective properties against adjuvant-induced arthritis in rats. The biological in vitro activities were concentration dependent. Hydrophilicity, the presence of a free 7-OH, and steric requirements for the substituent at position 8 are the most important factors in terms of SAR. An attempt was made to correlate several physicochemical properties of the molecules with their in vivo/in vitro activity.

AMG 9810 [(E)-3-(4-t-Butylphenyl)-N-(2,3-dihydrobenzo[b][1,4] dioxin-6-yl)acrylamide], a Novel Vanilloid Receptor 1 (TRPV1) Antagonist with Antihyperalgesic Properties

Abstract: The vanilloid receptor 1 (VR1 or TRPV1) is a membrane-bound, nonselective cation channel expressed by peripheral sensory neurons. TRPV1 antagonists produce antihyperalgesic effects in animal models of inflammatory and neuropathic pain. Here, we describe the in vitro and in vivo pharmacology of a novel TRPV1 antagonist, AMG 9810, (E)-3-(4-t-butylphenyl)-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)acrylamide. AMG 9810 is a competitive antagonist of capsaicin activation (IC50 value for human TRPV1, 24.5 +/- 15.7 nM; rat TRPV1, 85.6 +/- 39.4 nM) and blocks all known modes of TRPV1 activation, including protons (IC50 value for rat TRPV1, 294 +/- 192 nM; human TRPV1, 92.7 +/- 72.8 nM), heat (IC50 value for rat TRPV1, 21 +/- 17 nM; human TRPV1, 15.8 +/- 10.8 nM), and endogenous ligands, such as anandamide, N-arachidonyl dopamine, and oleoyldopamine. AMG 9810 blocks capsaicin-evoked depolarization and calcitonin gene-related peptide release in cultures of rat dorsal root ganglion primary neurons. Screening of AMG 9810 against a panel of G protein-coupled receptors and ion channels indicated selectivity toward TRPV1. In vivo, AMG 9810 is effective at preventing capsaicin-induced eye wiping in a dose-dependent manner, and it reverses thermal and mechanical hyperalgesia in a model of inflammatory pain induced by intraplantar injection of complete Freund's adjuvant. At effective doses, AMG 9810 did not show any significant effects on motor function, as measured by open field locomotor activity and motor coordination tests. AMG 9810 is the first cinnamide TRPV1 antagonist reported to block capsaicin-induced eye wiping behavior and reverse hyperalgesia in an animal model of inflammatory pain.

Two-Dimensional Strain–A Doppler-Independent Ultrasound Method for Quantitation of Regional Deformation: Validation In Vitro and In Vivo

Abstract: Background A new 2-dimensional strain echocardiography (2DSE) method has been introduced that measures myocardial deformations by tracking localized acoustic markers. We compared strains measured in vitro and in vivo by 2DSE with those obtained by sonomicrometry. Methods For the in vitro study, a tissue-mimicking gelatin block was cyclically compressed and longitudinal strains obtained by 2DSE and sonomicrometry crystals. For the in vivo study, arrays of crystals were implanted into the apical anteroseptal (test region) and midposterior (control region) in 16 open-chest pigs and strains measured by 2DSE and crystals at baseline and after acute ischemia. Results In vitro, pooled data demonstrated good correlation ( r = 0.99, P r = 0.94, P Conclusion The 2DSE demonstrated good overall correlation and agreement with sonomicrometry for the tested in vitro and in vivo values. Some caution with 2DSE measurements is needed for combinations of low strains and strain rates.

Platelet-derived microparticles induce angiogenesis and stimulate post-ischemic revascularization.

Abstract: Objective : Platelet activation is accompanied by the release of microparticles. However, little is known about the role of platelet-derived microparticles (PMP) in the regulation of angiogenesis and related clinical situations. The aim of our study was to evaluate the effect of PMP on angiogenesis and to analyze its mechanisms. Methods : Both in vitro (rat aortic ring model, cell invasion test) and in vivo (agarose bead transplantation, artificial cardiac ischemia in Sabra rats) approaches were used in the study. Results : A dose-dependent pro-angiogenic effect of PMP was observed in the rat aortic ring model. This effect could be eliminated by inhibition of VEGF, bFGF, and PDGF, but not heparanase. PMP exerted their effect via PI 3-kinase, Src kinase, and ERK, whereas protein kinase C and p38 were not involved. Moreover, PMP induced invasion of endothelial cells through a layer of matrigel. This effect was mediated by VEGF, heparanase, and PDGF, but not bFGF. Furthermore, PMP induced angiogenesis in an in vivo model in which agarose beads containing PMP were transplanted subcutaneously into mice. In addition, the effect of PMP on angiogenesis was evaluated in the model of in vivo chronic myocardial ischemia in rats. Ischemia induced a decrease in the number of functioning capillaries (34 ± 21.5 vs. 157 ± 42.0 per view field), but their amount increased after injection of PMP into the myocarium (97 ± 27.3; p <0.001 vs. ischemia without PMP). Conclusions : PMP induce angiogenesis both in vitro and in vivo. Injection of PMP into the ischemic myocardium might improve the process of revascularization after chronic ischemia.

Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

Abstract: Pulmonary arterial hypertension (PAH) is characterized by genetic and acquired abnormalities that suppress apoptosis and enhance cell proliferation in the vascular wall, including downregulation of the bone morphogenetic protein axis and voltage-gated K+ (Kv) channels. Survivin is an "inhibitor of apoptosis" protein, previously thought to be expressed primarily in cancer cells. We found that survivin was expressed in the pulmonary arteries (PAs) of 6 patients with PAH and rats with monocrotaline-induced PAH, but not in the PAs of 3 patients and rats without PAH. Gene therapy with inhalation of an adenovirus carrying a phosphorylation-deficient survivin mutant with dominant-negative properties reversed established monocrotaline-induced PAH and prolonged survival by 25%. The survivin mutant lowered pulmonary vascular resistance, RV hypertrophy, and PA medial hypertrophy. Both in vitro and in vivo, inhibition of survivin induced PA smooth muscle cell apoptosis, decreased proliferation, depolarized mitochondria, caused efflux of cytochrome c in the cytoplasm and translocation of apoptosis-inducing factor into the nucleus, and increased Kv channel current; the opposite effects were observed with gene transfer of WT survivin, both in vivo and in vitro. Inhibition of the inappropriate expression of survivin that accompanies human and experimental PAH is a novel therapeutic strategy that acts by inducing vascular mitochondria-dependent apoptosis.

Tumor-specific targeting of an anticancer drug delivery system by LHRH peptide

Abstract: The central problem in cancer chemotherapy is the severe toxic side effects of anticancer drugs on healthy tissues. Invariably the side effects impose dose reduction, treatment delay, or discontinuance of therapy. To limit the adverse side effects of cancer chemotherapy on healthy organs, we proposed a drug delivery system (DDS) with specific targeting ligands for cancer cells. The proposed DDS minimizes the uptake of the drug by normal cells and enhances the influx and retention of the drug in cancer cells. This delivery system includes three main components: (i) an apoptosis-inducing agent (anticancer drug), (ii) a targeting moiety-penetration enhancer, and (iii) a carrier. We describe one of the variants of such a system, which utilizes camptothecin as an apoptosis-inducing agent and poly(ethylene glycol) as a carrier. Luteinizing hormone-releasing hormone (LHRH) was used as a targeting moiety (ligand) to LHRH receptors that are overexpressed in the plasma membrane of several types of cancer cells and are not expressed detectably in normal visceral organs. The results showed that the use of LHRH peptide as a targeting moiety in the anticancer DDS substantially enhanced the efficacy of chemotherapy, led to amplified apoptosis induction in the tumor, and minimized the side effects of the anticancer drug on healthy organs. The LHRH receptor targeting DDS did not show in vivo pituitary toxicity and did not significantly influence the time course or the plasma concentration of luteinizing hormone and its physiological effects on the reproductive functions of mice.