Design of potent GlyT1 inhibitors: In vitro and in vivo profiles

Inhibitors of Gly transporter type-1 (GlyT1) for the treatment of schizophrenia have been pursued on the basis of the NMDA receptor (R) hypofunction hypothesis, which stems largely from the observation that NMDAR antagonists induce symptoms that more closely mimic those characteristic of schizophrenia than do other classes of psychotic agents. GlyT1 is responsible for uptake of synaptic Gly, an NMDAR co-agonist amino acid, in neuronal populations throughout the forebrain. GlyT1 inhibition thereby potentiates NMDAR activity by increasing synaptic Gly levels. Correspondingly, a large body of data suggests that GlyT1 inhibitors likely confer more comprehensive symptom alleviation than current antipsychotics. To date, a number of small-molecule GlyT1 inhibitors have been reported by the pharmaceutical industry. Developments in the discovery and characterization of GlyT1 inhibitors are discussed in this review.

EFFECT OF IN-VIVO AND IN-VITRO LOVASTATIN TREATMENT ON MAST-CELL ACTIVATION

The hydroxymethylglutaryl coenzmye A (HMG CoA) reductase inhibitor lovastatin is used to treat hyperlipidaemia. This agent prevents the isoprenylation of some proteins involved in signal transduction processes and inhibits IgE-receptor-linked mediator release from RBL-2H3 cells. In this study the effect of in vivo and in vitro administration of lovastatin on histamine release from rat peritoneal mast cells was examined. Lovastatin (4 mg/kg/day for 2 weeks) inhibited histamine release induced by concanavalin A (con A) from rat peritoneal mast cells of Hooded-Lister rats and both homozygous lean and obese Zucker rats. In contrast, release induced by antirat IgE (anti-IgE) was only significantly inhibited in cells derived from Hooded-Lister rats and that induced by compound 48/ 80 was not altered. Lovastatin (20 mu M, 24 h, in vitro) caused a significant inhibition of the subsequent histamine release to con A, anti-IgE and compound 48/80 but not to the calcium ionophore A 23187. It is important to determine whether such inhibitory effects are also observed after the chronic, clinical administration of lovastatin and other HMG CoA reductase inhibitors.

Adverse reactions to drugs: in vitro studies with isolated cells

Objective and design: Drug-induced adverse reactions can be allergic or pseudoallergic in nature, in this study the histamine releasing ability of 4 radiographic contrast media and 2 opioid analgesics was tested on a variety of mast cell containing cell suspensions.

An in vitro screen of bacterial lipopolysaccharide biosynthetic enzymes identifies an inhibitor of ADP-heptose biosynthesis

The lipopolysaccharide (LPS)-rich outer membrane of gram-negative bacteria provides a protective barrier that insulates these organisms from the action of numerous antibiotics. Breach of the LPS layer can therefore provide access to the cell interior to otherwise impermeant toxic molecules and can expose vulnerable binding sites for immune system components such as complement. Inhibition of LPS biosynthesis, leading to a truncated LPS molecule, is an alternative strategy for antibacterial drug development in which this vital cellular structure is weakened. A significant challenge for in vitro screens of small molecules for inhibition of LPS biosynthesis is the difficulty in accessing the complex carbohydrate substrates. We have optimized an assay of the enzymes required for LPS heptose biosynthesis that simultaneously surveys five enzyme activities by using commercially available substrates and report its use in a small-molecule screen that identifies an inhibitor of heptose synthesis.

Novel macrocyclic and acyclic cationic lipids for gene transfer: Synthesis and in vitro evaluation

The synthesis and in vitro evaluation of four cationic lipid gene delivery vectors, characterized by acyclic or macrocyclic, and saturated or unsaturated hydrophobic regions, is described. The synthesis employed standard protocols, including ring-closing metathesis for macrocyclic lipid construction. All lipoplexes studied, formulated from plasmid DNA and a liposome composed of a synthesized lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC), and either 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) or cholesterol as co-lipid, exhibited plasmid DNA binding and protection from DNase I degradation, and concentration dependent cytotoxicity using Chinese hamster ovary-K1 cells. The transfection efficiency of formulations with cholesterol outperformed those with DOPE, and in many cases the EPC/cholesterol control, and formulations with a macrocyclic lipid (+/- 10:1) outperformed their acyclic counterparts (+/- 3:1).

A modified SILCS contraceptive diaphragm for long-term controlled release of the HIV microbicide dapivirine

Background: There is considerable interest in developing new multipurpose prevention technologies to address women's reproductive health needs. This study describes an innovative barrier contraceptive device--based on the SILCS diaphragm--that also provides long-term controlled release of the lead candidate anti-HIV microbicide dapivirine.

Study design: Diaphragm devices comprising various dapivirine-loaded polymer spring cores overmolded with a nonmedicated silicone elastomer sheath were fabricated by injection molding processes. In vitro release testing, thermal analysis and mechanical characterization were performed on the devices.

Results: A diaphragm device containing a polyoxymethylene spring core loaded with 10% w/w dapivirine provided continuous and controlled release of dapivirine over a 6-month period, with a mean in vitro daily release rate of 174 mcg/day. The mechanical properties of the new diaphragm were closely matched to the SILCS diaphragm.

Conclusions: The study demonstrates proof of concept for a dapivirine-releasing diaphragm with daily release quantities potentially capable of preventing HIV transmission. In discontinuous clinical use, release of dapivirine may be readily extended over 1 or more years. © 2013 Elsevier Inc. All rights reserved.

In vitro mesenchymal stem cell responses on laser-welded NiTi alloy

The biocompatibility of NiTi after laser welding was studied by examining the in vitro (mesenchymal stem cell) MSC responses at different sets of time varying from early (4 to 12 h) to intermediate phases (1 and 4 days) of cell culture. The effects of physical (surface roughness and topography) and chemical (surface Ti/Ni ratio) changes as a consequence of laser welding in different regions (WZ, HAZ, and BM) on the cell morphology and cell coverage were studied. The results in this research indicated that the morphology of MSCs was affected primarily by the topographical factors in the WZ: the well-defined and directional dendritic pattern and the presence of deeper grooves. The morphology of MSCs was not significantly modulated by surface roughness. Despite the possible initial Ni release in the medium during the cell culture, no toxic effect seemed to cause to MSCs as evidenced by the success of adhesion and spreading of the cells onto different regions in the laser weldment. The good biocompatibility of the NiTi laser weldment has been firstly reported in this study.

Increased susceptibility of a triclabendazole (TCBZ)-resistant isolate of Fasciola hepatica to TCBZ following co-incubation in vitro with the P-glycoprotein inhibitor, R(+)-verapamil

SUMMARY A study was carried out to investigate whether the action of triclabendazole sulphoxide (TCBZ.SO) against the liver fluke, Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Oberon TCBZ-resistant and Cullompton TCBZ-susceptible fluke isolates were used for this in vitro study and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. For experiments with the Oberon isolate, flukes were incubated for 24 h with either R(+)-VPL (1×10-4 m) on its own, TCBZ.SO (15 µg mL-1) alone, a combination of R(+)-VPL (1×10-4 m) plus TCBZ.SO (15 µg mL-1), TCBZ.SO (50 µg mL-1) on its own, or a combination of TCBZ.SO (50 µg mL-1) plus R(+)-VPL (1×10-4 m). They were also incubated in TCBZ.SO (50 µg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive; and in TCBZ.SO (50 µg mL-1) alone for a time to match that of the combination inactivity time. Flukes from the Cullompton isolate were treated with either TCBZ.SO (50 µg mL-1) alone or in combination with R(+)-VPL (1×10-4 m) until they became inactive, or with TCBZ.SO (50 µg mL-1) alone time-matched to the combination inactivity time. Morphological changes resulting from drug treatment and following Pgp inhibition were assessed by means of scanning electron microscopy. Incubation in R(+)-VPL alone had a minimal effect on either isolate. TCBZ.SO treatment had a relatively greater impact on the TCBZ-susceptible Cullompton isolate. When R(+)-VPL was combined with TCBZ.SO in the incubation medium, however, the surface disruption to both isolates was more severe than that seen after TCBZ.SO treatment alone; also, the time taken to reach inactivity was shorter. More significantly, though, the potentiation of drug activity was greater in the Oberon isolate; also, it was more distinct at the higher concentration of TCBZ.SO. So, the Oberon isolate appears to be particularly sensitive to efflux pump inhibition. The results of this study suggest that enhanced drug efflux in the Oberon isolate may be involved in the mechanism of resistance to TCBZ.

Modified Silicone Elastomer Vaginal Gels for Sustained Release of Antiretroviral HIV Microbicides

We previously reported nonaqueous silicone elastomer gels (SEGs) for sustained vaginal administration of the CCR5-targeted entry inhibitor maraviroc (MVC). Here, we describe chemically modified SEGs (h-SEGs) in which the hydrophobic cyclomethicone component was partially replaced with relatively hydrophilic silanol-terminated polydimethylsiloxanes (st-PDMS). MVC and emtricitabine (a nucleoside reverse transcriptase inhibitor), both currently under evaluation as topical microbicides to counter sexual transmission of human immunodeficiency virus type 1 (HIV-1), were used as model antiretroviral (ARV) drugs. Gel viscosity and in vitro ARV release were significantly influenced by st-PDMS molecular weight and concentration in the h-SEGs. Unexpectedly, gels prepared with lower molecular weight grades of st-PDMS showed higher viscosities. h-SEGs provided enhanced release over 24 h compared with aqueous hydroxyethylcellulose (HEC) gels, did not modify the pH of simulated vaginal fluid (SVF), and were shown to less cytotoxic than standard HEC vaginal gel. ARV solubility increased as st-PDMS molecular weight decreased (i.e., as percentage hydroxyl content increased), helping to explain the in vitro release trends. Dye ingression and SVF dilution studies confirmed the increased hydrophilicity of the h-SEGs. h-SEGs have potential for use in vaginal drug delivery, particularly for ARV-based HIV-1 microbicides.

Disruption of vitellogenesis and spermatogenesis by triclabendazole (TCBZ) in a TCBZ-resistant isolate of Fasciola hepatica following incubation in vitro with a P-glycoprotein inhibitor

A study has been carried out to investigate whether the action of triclabendazole (TCBZ) against Fasciola hepatica is altered by inhibition of P-glycoprotein (Pgp)-linked drug efflux pumps. The Sligo TCBZ-resistant fluke isolate was used for these experiments and the Pgp inhibitor selected was R(+)-verapamil [R(+)-VPL]. In the first experiment, flukes were initially incubated for 2 h in R(+)-VPL (100 μ m), then incubated in R(+)-VPL+triclabendazole sulphoxide (TCBZ.SO) (50 μg mL-1, or 133·1 μ m) until flukes ceased movement (at 9 h post-treatment). In a second experiment, flukes were incubated in TCBZ.SO alone and removed from the incubation medium following cessation of motility (after 15 h). In the third experiment, flukes were incubated for 24 h in R(+)-VPL on its own. Changes to the testis tubules and vitelline follicles following drug treatment and following Pgp inhibition were assessed by means of light microscope histology and transmission electron microscopy. Incubation of the Sligo isolate in either R(+)-VPL or TCBZ.SO on their own had a limited impact on the morphology of the two tissues. Greater disruption was observed when the drugs were combined, in terms of the block in development of the spermatogenic and vitelline cells and the apoptotic breakdown of the remaining cells. Sperm formation was severely affected and abnormal. Large spaces appeared in the vitelline follicles and synthesis of shell protein was disrupted. The results of this study support the concept of altered drug efflux in TCBZ-resistant flukes and indicate that drug transporters may play a role in the development of drug resistance.

In Vitro and In Vivo Anti-Schistosomal Activity of the Alkylphospholipid Analog Edelfosine

Background: Schistosomiasis is a parasitic disease caused by trematodes of the genus Schistosoma. Five species of Schistosoma are known to infect humans, out of which S. haematobium is the most prevalent, causing the chronic parasitic disease schistosomiasis that still represents a major problem of public health in many regions of the world and especially in tropical areas, leading to serious manifestations and mortality in developing countries. Since the 1970s, praziquantel (PZQ) is the drug of choice for the treatment of schistosomiasis, but concerns about relying on a single drug to treat millions of people, and the potential appearance of drug resistance, make identification of alternative schistosomiasis chemotherapies a high priority. Alkylphospholipid analogs (APLs), together with their prototypic molecule edelfosine (EDLF), are a family of synthetic antineoplastic compounds that show additional pharmacological actions, including antiparasitic activities against several protozoan parasites.

Methodology/Principal Findings: We found APLs ranked edelfosine> perifosine> erucylphosphocholine> miltefosine for their in vitro schistosomicidal activity against adult S. mansoni worms. Edelfosine accumulated mainly in the worm tegument, and led to tegumental alterations, membrane permeabilization, motility impairment, blockade of male-female pairing as well as induction of apoptosis-like processes in cells in the close vicinity to the tegument. Edelfosine oral treatment also showed in vivo schistosomicidal activity and decreased significantly the egg burden in the liver, a key event in schistosomiasis.

Conclusions/Significance: Our data show that edelfosine is the most potent APL in killing S. mansoni adult worms in vitro. Edelfosine schistosomicidal activity seems to depend on its action on the tegumental structure, leading to tegumental damage, membrane permeabilization and apoptosis-like cell death. Oral administration of edelfosine diminished worm and egg burdens in S. mansoni-infected CD1 mice. Here we report that edelfosine showed promising antischistosomal properties in vitro and in vivo.

Novel microtubule-targeting agents, pyrrolo-1,5-benzoxazepines, induce apoptosis in multi-drug-resistant cancer cells

PURPOSE: The development of multi-drug resistance (MDR) due to the expression of members of the ATP binding cassette (ABC) transporter family is a major obstacle in cancer treatment. The broad range of substrate specificities associated with these transporters leads to the efflux of many anti-cancer drugs from tumour cells. Therefore, the development of new chemotherapeutic agents that are not substrates of these transporters is important. We have recently demonstrated that some members of a novel series of pyrrolo-1,5-benzoxazepine (PBOX) compounds are microtubule-depolymerising agents that potently induce apoptosis in several cancer cell lines and impair growth of mouse breast tumours. The aim of this current study was to establish whether PBOXs were capable of inducing apoptosis in cancer cells expressing either P-glycoprotein or breast cancer resistance protein (BCRP), two of the main ABC transporters associated with MDR.

METHODS: We performed in vitro studies to assess the effects of PBOXs on cell proliferation, cell cycle and apoptosis in human cancer cell lines and their drug-resistant substrains expressing either P-glycoprotein or BCRP. In addition, we performed a preliminary molecular docking study to examine interactions between PBOXs and P-glycoprotein.

RESULTS: We established that three representative PBOXs, PBOX-6, -15 and -16 were capable of inducing apoptosis in drug-resistant HL60-MDR1 cells (expressing P-glycoprotein) and HL60-ABCG2 cells (expressing BCRP) with similar potencies as in parental human promyelocytic leukaemia HL60 cells. Likewise, resistance to PBOX-6 and -16 was not evident in P-glycoprotein-expressing A2780-ADR cells in comparison with parent human ovarian carcinoma A2780 cells. Finally, we deduced by molecular docking that PBOX-6 is not likely to form favourable interactions with the substrate binding site of P-glycoprotein.

CONCLUSION: Our results suggest that pro-apoptotic PBOX compounds may be potential candidates for the treatment of P-glycoprotein- or BCRP-associated MDR cancers.

Interleukin-8 is associated with proliferation, migration, angiogenesis and chemosensitivity in vitro and in vivo in colon cancer cell line models

Interleukin-8 (IL-8), a chemokine with a defining CXC amino acid motif, is known to possess tumorigenic and proangiogenic properties. Overexpression of IL-8 has been detected in many human tumors, including colorectal cancer (CRC), and is associated with poor prognosis. The goal of our study was to determine the role of IL-8 overexpression in CRC cells in vitro and in vivo. We stably transfected the IL-8 cDNA into two human colon cancer cell lines, HCT116 and Caco2, and selected IL-8-secreting transfectants. Real-time RT-PCR confirmed that IL-8 mRNA was overexpressed in IL-8 transfectants with 45- to 85-fold higher than parental cells. The IL-8-transfected clones secreted 19- to 28-fold more IL-8 protein than control and parental cells as detected by ELISA. The IL-8 transfectants demonstrated increased cellular proliferation, cell migration and invasion based on functional assays. Growth inhibition studies showed that IL-8 overexpression lead to a significant resistance to oxaliplatin (p < 0.0001). Inhibition of IL-8 overexpression with small interfering RNA reversed the observed increases in tumorigenic functions and oxaliplatin resistance, suggesting that IL-8 not only provides a proliferative advantage but also promotes the metastatic potential of colon cancer cells. Using a tumor xenograft model, IL-8-expressing cells formed significantly larger tumors than the control cells with increased microvessel density. Together, these findings indicate that overexpression of IL-8 promotes tumor growth, metastasis, chemoresistance and angiogenesis, implying IL-8 to be an important therapeutic target in CRC.

In vitro and in vivo insulinotropic properties of the multifunctional frog skin peptide hymenochirin-1B: a structure–activity study

Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro5, Glu6, and Asp9 by either l-lysine or d-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal β cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca2+. The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.