Combination of Antiretroviral Drugs Zidovudine and Efavirenz Impairs Tumor Growths in a Mouse Model of Cancer.

LINE1 retrotransposons, which are thought to be the remnants of ancient integrations of retrovirus-like elements, are aberrantly (re)activated in many cancer cells. Due to LINE1-induced alterations in target gene expression and/or chromosomal rearrangements, they may be important drivers of tumorigenesis. Moreover, LINE1 encoded proteins, Open Reading Frame (ORF)1 and ORF2, may have pro-oncogenic potential through inductors of oncogenic transcription factors or inhibitors of cell cycle suppressors. The current study therefore aimed to investigate in vitro and in vivo anti-tumorigenic effects of two well-known antiretroviral drugs, zidovudine, a nucleoside analogue inhibitor of RT (NRTI), and efavirenz, a non-nucleoside RT inhibitor (NNRTI). Our data demonstrate that both drugs in clinically relevant doses significantly reduced the proliferation of murine and human cancer cell lines, as well as growth of tumors in a murine subcutaneous model. Intriguingly, we found that the combination of both zidovudine and efavirenz almost entirely blocked tumorigenesis in vivo. Because both drugs are FDA-approved agents and the combination was very well tolerated in mice, the combination therapy as presented in our paper might be an opportunity to treat colorectal tumors and metastasis to the liver in an inexpensive way.

Point-of-Care Detection of Nonadherence to Antiretroviral Treatment for HIV-1 in Resource-Limited Settings Using Drug Level Testing for Efavirenz, Lopinavir, and Dolutegravir: A Validation and Pharmacokinetic Simulation Study.

BACKGROUND: Virological failure during antiretroviral treatment (ART) may indicate the presence of drug resistance, but may also originate from nonadherence. Qualitative detection of ART components using drug level testing may be used to differentiate between these scenarios. We aimed to validate and implement qualitative point-of-care drug level tests for efavirenz (EFV), lopinavir (LPV), and dolutegravir (DTG) in rural South Africa. METHODS: Qualitative performance of immunoassays for EFV, LPV, and DTG was assessed by calculating limit of detection (LoD), region of uncertainty, and qualitative agreement with a reference test. Minimum duration of nonadherence resulting in a negative drug level test was assessed by simulation of treatment cessation using validated population pharmacokinetic models. RESULTS: LoD was 0.05 mg/L for EFV, 0.06 mg/L for LPV, and 0.02 mg/L for DTG. Region of uncertainty was 0.01-0.06 mg/L for EFV, 0.01-0.07 mg/L for LPV, and 0.01-0.02 mg/L for DTG. Qualitative agreement with reference testing at the LoD in patient samples was 95.2% (79/83) for EFV, 99.3% (140/141) for LPV, and 100% (118/118) for DTG. After simulated treatment cessation, median time to undetectability below LoD was 7 days [interquartile range (IQR) 4-13] for EFV, 30 hours (IQR 24-36) for LPV, and 6 days (IQR 4-7) for DTG. CONCLUSIONS: We demonstrate that qualitative ART drug level testing using immunoassays is feasible in a rural resource-limited setting. Implementation of this technology enables reliable detection of recent nonadherence and may allow for rapid and cost-effective differentiation between patients in need for adherence counseling and patients who require drug resistance testing or alternative treatment.

Layer-by-Layer Assembled Nanostructured Lipid Carriers for CD-44 Receptor-Based Targeting in HIV-Infected Macrophages for Efficient HIV-1 Inhibition.

Macrophages act as a cellular reservoir in HIV infection. Elimination of HIV from macrophages has been an unfulfilled dream due to the failure of drugs to reach them. To address this, we developed CD44 receptor-targeted, novel hyaluronic acid (HA)-coated nanostructured lipid carriers (NLCs) of efavirenz via washless layer-by-layer (LbL) assembly of HA and polyallylamine hydrochloride (PAH). NLCs were subjected to TEM analysis, size and zeta potential, in vitro release and encapsulation efficiency studies. The uptake of NLCs in THP-1 cells was studied using fluorescence microscopy and flow cytometry. The anti-HIV efficacy was evaluated using p24 antigen inhibition assay. NLCs were found to be spherical in shape with anionic zeta potential (-23.66 ± 0.87 mV) and 241.83 ± 5.38 nm particle size. NLCs exhibited prolonged release of efavirenz during in vitro drug release studies. Flow cytometry revealed 1.73-fold higher uptake of HA-coated NLCs in THP-1 cells. Cytotoxicity studies showed no significant change in cell viability in presence of NLCs as compared with the control. HA-coated NLCs distributed throughout the cell including cytoplasm, plasma membrane and nucleus, as observed during fluorescence microscopy. HA-coated NLCs demonstrated consistent and significantly higher inhibition (81.26 ± 1.70%) of p24 antigen which was 2.08-fold higher than plain NLCs. The obtained results suggested preferential uptake of HA-coated NLCs via CD44-mediated uptake. The present finding demonstrates that HA-based CD44 receptor targeting in HIV infection is an attractive strategy for maximising the drug delivery to macrophages and achieve effective viral inhibition.

Clinically relevant enantiomer specific R- and S-praziquantel pharmacokinetic drug-drug interactions with efavirenz and ritonavir.

We conducted a clinical study to determine the effect of efavirenz and ritonavir on the pharmacokinetics of R- and S-PZQ in healthy male participants. This was toward evaluating the risk of drug-drug interactions, which may occur after PZQ administration to HIV patients on efavirenz or ritonavir containing regimens. A non-randomized, open-label, single-dose, one sequence crossover study with 2 arms was conducted. We gave 26 healthy volunteers a single oral dose of 40 mg/kg PZQ followed by a daily oral dose of either 400 mg efavirenz or 100 mg ritonavir for 14 consecutive days. On day 14, they ingested a single 40 mg/kg dose of PZQ. We measured plasma levels up to 12 h on day 1 and day 14. Samples were analyzed by LC-MS. Pharmacokinetic analysis was conducted in WinNonlin to determine the primary endpoints (plasma T1/2 , Cmin , and AUC). Efavirenz had a significant effect on the pharmacokinetics of PZQ (p < .05), reducing the AUC by 4-fold (1213.15 vs. 281.35 h·ng/ml for R-PZQ and 5669 vs. 871.84 h·ng/ml for S-PZQ). Ritonavir had no significant effect on R-PZQ but increased the AUC 2-fold for S-PZQ (p < .05) (4154.79 vs. 7291.05 h·ng/ml). Using PZQ in HIV patients needs investigation, as there is a risk of both treatment failure and adverse effects because of induction and inhibition, respectively.

Longitudinal trends and determinants of patient-reported side effects on ART-a Swedish national registry study.

INTRODUCTION: The use of patient-reported outcomes (PROs) to systematically quantify adverse events (AE) will assist in the improvement of medical care and the QoL of patients living with HIV (PLWH). The aim of this study was to investigate the associations between self-reported side effects and other PROs, demographics and laboratory data, and further evaluate the Health Questionnaire (HQ) as a tool for following trends in patient-reported side effects over time in relation to trends in prescribed third agent in ART. MATERIALS AND METHODS: The Swedish National Registry InfCareHiv includes an annual self-reported nine-item HQwhich is used in patient-centered HIV care in all Swedish HIV units. In this study, the experience of side effects was addressed. We analyzed 9,476 HQs completed by 4,186 PLWH together with details about their prescribed ART and relevant biomarkers collected during 2011-2017. Data were analyzed using descriptive statistics, Pearson's correlation coefficient and mixed logistic regression. RESULTS: The cross-sectional analysis of the HQs showed that the frequency of reported side effects decreased from 32% (2011) to 15% (2017). During the same period, there was a shift in ART prescription from efavirenz (EFV) to dolutegravir (DTG) (positive correlation coefficient r = 0.94, p = 0.0016). Further, PLWH who reported being satisfied with their physical health (OR: 0.47, p = <0.001) or psychological health (OR: 0.70, p = 0.001) were less likely to report side effects than those less satisfied. CONCLUSIONS: Self-reported side effects were found to have a close relationship with the patient's ratings of their overall health situation and demonstrated a strong correlation with the sharp decline in use of EFV and rise in use of DTG, with reported side effects being halved. This study supports the feasibility of using the HQ as a tool for longitudinal follow up of trends in PROs.

DL-propargylglycine administration inhibits TET2 and FOXP3 expression and alleviates symptoms of neonatal Cows' milk allergy in mouse model.

BACKGROUND: Cows' milk allergy (CMA) is a hypersensitivity immune reaction brought on by specific immunologic mechanisms to cow's milk proteins. As one of the most common food allergies in infants, the incidence of CMA during the first year of life is estimated to be nearly 7.5%. Due to the limitation in the knowledge of the pathological mechanism underlying CMA, however, the clinical interventions and therapies remain very unsatisfactory. AIM OF THE STUDY: The transcriptional factor FOXP3 possesses crucial roles in CMA, and increased FOXP3 mRNA expression has a predictive function in faster acquisition of tolerance in infants with CMA. But the exact mechanism remains not fully elucidated. METHODS: For PAG treatment, PAG (dissolved in saline 30 mg/mL, 0, 5, 10, 20 mg/kg BW) was administered daily intraperitoneally (ip) for one week at the time that 6 weeks after the CMP sensitisation. RESULTS: In the present study, we revealed that the expression of FOXP3 is significantly up-regulated in PBMCs from CMA patients and CMA mice on mRNA and protein level. Furthermore, a dramatic reduction in the FOXP3 TSDR methylation and a significant increase in the expression of TET2 are observed in CMA patients and CMA mice. More importantly, we found that propargylglycine (PAG) significantly alleviates symptoms of CMA in mice by suppressing the expression of FOXP3 through restoring TET2 expression. CONCLUSIONS: Our work revealed a novel function of PAG on CMA, which may provide a deeper insight into the pathomechanism of CMA and a novel therapy target for CMA clinical interventions.

Inhibition of endogenous hydrogen sulfide biosynthesis enhances the anti-cancer effect of 3,3'-diindolylmethane in human gastric cancer cells.

AIMS: 3,3'-Diindolylmethane (DIM) has limited anti-cancer effects in gastric cancer. Hydrogen sulfide (H2S) plays an important role in the tumor development and therapy, cystathionine-ß-synthase (CBS) and cystathionine-γ-lyase (CSE), two key endogenous H2S biosynthesis enzymes, can affect endogenous H2S levels and alter cancer treatment. Our main objective was to investigate whether the aminooxyacetic acid (AOAA) and DL-Propargylglycine (PAG), two specific inhibitors of CBS and CSE, could assist DIM to exert a stronger anti-cancer effects in gastric cancer BGC-823 and SGC-7901 cells. MATERIALS AND METHODS: Cell proliferation was assayed by MTT and cell colony-forming assay. Apoptosis and migration were detected by Hoechst staining and scratch test respectively. Western blot was used to evaluate the expression of proteins related to proliferation, apoptosis and migration. KEY FINDINGS: Combination of AOAA or PAG with DIM synergistically inhibited proliferation and migration, increased apoptosis in gastric cancer cells. The p38-p53 axis was also further activated by the combination of AOAA or PAG with DIM. Exogenous H2S from sodium hydrosulfide, attenuated the efficacy of DIM in cancer cells by reducing the activation level of p38-p53 axis. Taken together, AOAA or PAG inhibited the expression of endogenous H2S biosynthesis enzymes and effectively enhanced susceptibility of gastric cancer to DIM through activating p38-p53 axis. SIGNIFICANCE: The current study highlight more precise requirements for the clinical application of sulfur-containing anti-cancer drugs, and open a new way to enhance the sensitivity of DIM in chemotherapy of gastric cancer.

Long-Acting Efavirenz and HIV-1 Fusion Inhibitor Peptide Co-loaded Polymer-Lipid Hybrid Nanoparticles: Statistical Optimization, Cellular Uptake, and In Vivo Biodistribution.

The objective of the present study was to develop long-acting efavirenz (Efa)-enfuvirtide (Enf) Co-loaded polymer-lipid hybrid nanoparticles (PLN) with improved intracellular delivery to target T-cells and macrophage cells located in multiple human immunodeficiency virus sanctuaries. The Box-Behnken design was utilized to optimize three high-risk factors, namely, Efa amount, sonication time for primary emulsion, and sonication time for aqueous nanodispersion obtained from preliminary studies. Lyophilized Efa-Enf Co-loaded PLN using trehalose elicited spherical morphology, drug amorphization on incorporation, and absence of drug-excipient interaction. In vitro release studies revealed an sustained release of both the drugs from PLN with the differential release profile. Efa-Enf Co-loaded PLN exhibited low hemolytic, platelet and leukocyte aggregation as well as low cytotoxicity in Jurkat E6.1 T-cells and U937 macrophage cells. Circular dichroism spectra confirmed the presence of an α-helix form of Enf after encapsulation in PLN. Coumarin-6-loaded PLN exhibited enhanced cellular uptake in Jurkat E6.1 T-cells and U937 macrophage cells in comparison to free coumarin-6, as evidenced by fluorescence microscopy and flow cytometry. In vivo biodistribution studies after intravenous administration of near-infrared dye-loaded PLN (surrogate for Efa-Enf PLN) revealed non-uniform distribution within 2 h in the order of spleen ≥ liver > lymph node > thymus > lungs > female reproductive tract (FRT) > heart > kidneys > brain. However, subcutaneous administration caused non-uniform biodistribution after 3 days, eliciting a long-acting slow release from the injection site depot until day 5 in the infection-spread site (lymph nodes and FRT), reservoir sites (liver and spleen) and the difficult-to-access site (brain). Furthermore, it presents a vital illustration of the available tissue-specific drug concentration prediction from simulated surrogate PLN.

Long-term NaHS administration reduces oxidative stress and apoptosis in a rat model of left-side varicocele.

The main aim of this study was to assay the testicular H2 S levels in the varicocele rat model and then to investigate the protective effects of NaHS on morphometric changes, sperm parameters, oxidative stress and apoptosis markers in rat's testis. D,L-propargylglycine (PAG) was administrated to show the effects of cystathionine γ-lyase enzyme (CSE) inhibition in the varicocele. Rats were assigned to four groups: (a) Sham, (b) varicocele, (c) varicocele + PAG and (d) varicocele + NaHS. Animals in varicocele + NaHS group received 30 µmol/L NaHS in drinking water for 56 days. In the varicocele + PAG group, animals received PAG 19 mg/kg twice a week. Morphometric assessment, oxidative stress markers, testicular H2 S levels, sperm parameters, TUNEL assay and expression of Bax/Bcl2 were evaluated at the end of experiment. Testicular H2 S levels were significantly decreased in varicocele group. NaHS significantly improved sperm parameters, morphometric characteristics and oxidative stress compared to varicocele group. Oxidative stress status deteriorated in the PAG group compared to the varicocele group. This study showed that a low testicular H2 S level might play a critical role in male infertility. Thus, NaHS administration may be a promising treatment strategy for male infertility in varicocele. In addition, CSE may not be the only important enzyme in testicular H2 S production.

ALDH1A3 is epigenetically regulated during melanocyte transformation and is a target for melanoma treatment.

Despite the promising targeted and immune-based interventions in melanoma treatment, long-lasting responses are limited. Melanoma cells present an aberrant redox state that leads to the production of toxic aldehydes that must be converted into less reactive molecules. Targeting the detoxification machinery constitutes a novel therapeutic avenue for melanoma. Here, using 56 cell lines representing nine different tumor types, we demonstrate that melanoma cells exhibit a strong correlation between reactive oxygen species amounts and aldehyde dehydrogenase 1 (ALDH1) activity. We found that ALDH1A3 is upregulated by epigenetic mechanisms in melanoma cells compared with normal melanocytes. Furthermore, it is highly expressed in a large percentage of human nevi and melanomas during melanocyte transformation, which is consistent with the data from the TCGA, CCLE and protein atlas databases. Melanoma treatment with the novel irreversible isoform-specific ALDH1 inhibitor [4-dimethylamino-4-methyl-pent-2-ynthioic acid-S methylester] di-methyl-ampal-thio-ester (DIMATE) or depletion of ALDH1A1 and/or ALDH1A3, promoted the accumulation of apoptogenic aldehydes leading to apoptosis and tumor growth inhibition in immunocompetent, immunosuppressed and patient-derived xenograft mouse models. Interestingly, DIMATE also targeted the slow cycling label-retaining tumor cell population containing the tumorigenic and chemoresistant cells. Our findings suggest that aldehyde detoxification is relevant metabolic mechanism in melanoma cells, which can be used as a novel approach for melanoma treatment.

Study of the Cytotoxic and Bactericidal Effects of Sila-substituted Thioalkyne and Mercapto-thione Compounds based on 1,2,3-Triazole Scaffold.

A series of sila-organosulphur compounds containing 1,2,3-triazole cores were screened for their cytotoxic activity on human breast cancer cell line MCF-7. Most of the tested compounds exhibited moderate-to-good activity against the cancer cells. Especially, the compound 4-((2-(trimethylsilyl)ethynylthio)methyl)-1-benzyl-1H-1,2,3-triazole (3a) from series of sila-substituted thioalkyne 1,2,3-triazoles (STATs) and the compounds 3-(1-benzyl-1H-1,2,3-triazol-4-yl)-1-mercapto-1,1-bis(trimethylsilyl)propane-2-thione (4a) and 1-mercapto-1,1-bis(trimethylsilyl)-3-(1-phenethyl-1H-1,2,3-triazol-4-yl)propane-2-thione (4e) from series of sila-substituted mercapto-thione 1,2,3-triazoles (SMTTs) exhibited promising cytotoxicity against MCF-7 with IC50 values of 35.17, 32.63 and 30.3 µg/mL, respectively. In addition, the possible mechanisms for inhibition of cell growth and induction of apoptotic cell death were explored by DAPI staining, cell cycle analysis and qRT-PCR. The synthetic compounds were evaluated for their in vitro antibacterial activities, and as a result, the most prominent effects were observed for 3e and 4e. Especially, 3e was found to be quite active against all the tested strains with the MIC values ranging from 15 to 62 µg/mL, except P. aeruginosa. The results of the time-kill assay suggested that the compound of 3e completely inhibited the growth of both gram-negative bacteria, A. baumannii, and gram-positive bacteria, S. aureus. In addition, SEM analysis confirmed morphostructural damage of the bacteria. Our findings could be applicable for developing dual-targeting anticancer/antibacterial therapeutics.

Caspase-8 activation by TRAIL monotherapy predicts responses to IAPi and TRAIL combination treatment in breast cancer cell lines.

The discovery of cancer cell-selective tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis generated broad excitement and development of TRAIL receptor agonists (TRA) as potential cancer therapy. Studies demonstrating the synergistic combination effect of SMAC mimetics and TRA further suggested potentially effective treatment in multiple tumour settings. However, predictive biomarkers allowing identification of patients that could respond to treatment are lacking. Here, we described a high throughput combination screen conducted across a panel of 31 breast cancer cell lines in which we observed highly synergistic activity between TRAIL and the inhibitors of apoptosis proteins (IAP) inhibitor (IAPi) AZD5582 in ~30% of cell lines. We detected no difference in the expression levels of the IAPi or TRAIL-targeted proteins or common modulators of the apoptotic pathway between the sensitive and resistant cell lines. Synergistic combination effect of AZD5582 and TRAIL correlated with sensitivity to TRAIL, but not to AZD5582 as a single agent. TRAIL treatment led to significantly greater activity of Caspase-8 in sensitive than in resistant cell lines (P=0.002). The majority (12/14) of AZD5582+TRAIL-resistant cell lines retained a functional cell death pathway, as they were sensitive to AZD5582+TNFα combination treatment. This suggested that failure of the TRAIL receptor complex to transduce the death signal to Caspase-8 underlies AZD5582+TRAIL resistance. We developed a 3D spheroid assay and demonstrated its suitability for the ex vivo analysis of the Caspase-8 activity as a predictive biomarker. Altogether, our study demonstrated a link between the functionality of the TRAIL receptor pathway and the synergistic activity of the IAPi+TRA combination treatment. It also provided a rationale for development of the Caspase-8 activity assay as a functional predictive biomarker that could allow better prediction of the response to IAPi+TRA-based therapies than the analysis of expression levels of protein biomarkers.

DL-propargylglycine reduces blood pressure and renal injury but increases kidney weight in angiotensin-II infused rats.

Hydrogen sulfide (H2S), carbon monoxide (CO) and nitric oxide (NO) share signaling and vasorelaxant properties and are involved in proliferation and apoptosis. Inhibiting NO production or availability induces hypertension and proteinuria, which is prevented by concomitant blockade of the H2S producing enzyme cystathionine γ-lyase (CSE) by d,l-propargylglycine (PAG). We hypothesized that blocking H2S production ameliorates Angiotensin II (AngII)-induced hypertension and renal injury in a rodent model. Effects of concomitant administration of PAG or saline were therefore studied in healthy (CON) and AngII hypertensive rats. In CON rats, PAG did not affect systolic blood pressure (SBP), but slightly increased proteinuria. In AngII rats PAG reduced SBP, proteinuria and plasma creatinine (180 ± 12 vs. 211 ± 19 mmHg; 66 ± 35 vs. 346 ± 92 mg/24 h; 24 ± 6 vs. 47 ± 15 µmol/L, respectively; p < 0.01). Unexpectedly, kidney to body weight ratio was increased in all groups by PAG (p < 0.05). Renal injury induced by AngII was reduced by PAG (p < 0.001). HO-1 gene expression was increased by PAG alone (p < 0.05). PAG increased inner cortical tubular cell proliferation after 1 week and decreased outer cortical tubular nucleus number/field after 4 weeks. In vitro proximal tubular cell size increased after exposure to PAG. In summary, blocking H2S production with PAG reduced SBP and renal injury in AngII infused rats. Independent of the cardiovascular and renal effects, PAG increased HO-1 gene expression and kidney weight. PAG alone increased tubular cell size and proliferation in-vivo and in-vitro. Our results are indicative of a complex interplay of gasotransmitter signaling/action of mutually compensatory nature in the kidney.

In vivo targeting through click chemistry.

Targeting small molecules to diseased tissues as therapy or diagnosis is a significant challenge in drug delivery. Drug-eluting devices implanted during invasive surgery allow the controlled presentation of drugs at the disease site, but cannot be modified once the surgery is complete. We demonstrate that bioorthogonal click chemistry can be used to target circulating small molecules to hydrogels resident intramuscularly in diseased tissues. We also demonstrate that small molecules can be repeatedly targeted to the diseased area over the course of at least one month. Finally, two bioorthogonal reactions were used to segregate two small molecules injected as a mixture to two separate locations in a mouse disease model. These results demonstrate that click chemistry can be used for pharmacological drug delivery, and this concept is expected to have applications in refilling drug depots in cancer therapy, wound healing, and drug-eluting vascular grafts and stents.

Phase I safety and pharmacodynamic of inecalcitol, a novel VDR agonist with docetaxel in metastatic castration-resistant prostate cancer patients.

PURPOSE: We conducted a phase I multicenter trial in naïve metastatic castrate-resistant prostate cancer patients with escalating inecalcitol dosages, combined with docetaxel-based chemotherapy. Inecalcitol is a novel vitamin D receptor agonist with higher antiproliferative effects and a 100-fold lower hypercalcemic activity than calcitriol. EXPERIMENTAL DESIGN: Safety and efficacy were evaluated in groups of three to six patients receiving inecalcitol during a 21-day cycle in combination with docetaxel (75 mg/m2 every 3 weeks) and oral prednisone (5 mg twice a day) up to six cycles. Primary endpoint was dose-limiting toxicity (DLT) defined as grade 3 hypercalcemia within the first cycle. Efficacy endpoint was ≥30% PSA decline within 3 months. RESULTS: Eight dose levels (40-8,000 µg) were evaluated in 54 patients. DLT occurred in two of four patients receiving 8,000 µg/day after one and two weeks of inecalcitol. Calcemia normalized a few days after interruption of inecalcitol. Two other patients reached grade 2, and the dose level was reduced to 4,000 µg. After dose reduction, calcemia remained within normal range and grade 1 hypercalcemia. The maximum tolerated dose was 4,000 µg daily. Respectively, 85% and 76% of the patients had ≥30% PSA decline within 3 months and ≥50% PSA decline at any time during the study. Median time to PSA progression was 169 days. CONCLUSION: High antiproliferative daily inecalcitol dose has been safely used in combination with docetaxel and shows encouraging PSA response (≥30% PSA response: 85%; ≥50% PSA response: 76%). A randomized phase II study is planned.

Evaluation of propargyl alcohol toxicity and carcinogenicity in F344/N rats and B6C3F1/N mice following whole-body inhalation exposure.

Propargyl alcohol (PA) is a high production volume chemical used in synthesis of many industrial chemicals and agricultural products. Despite the potential for prolonged or accidental exposure to PA in industrial settings, the toxicity potential of PA was not well characterized. To address the knowledge gaps relevant to the toxicity profile of PA, the National Toxicology Program (NTP) conducted 2-week, 14-week and 2-year studies in male and female F344/N rats and B6C3F1/N mice. For the 2-week inhalation study, the rats and mice were exposed to 0, 31.3, 62.5, 125, 250 or 500ppm. Significant mortality was observed in both rats and mice exposed to ≥125ppm of PA. The major target organ of toxicity in both mice and rats was the liver with exposure-related histopathological changes (250 and 500ppm). Based on the decreased survival in the 2-week study, the rats and mice were exposed to 0, 4, 8, 16, 32 or 64ppm of PA in the 14-week study. No treatment-related mortality was observed. Mean body weights of male (≥8ppm) and female mice (32 and 64ppm) were significantly decreased (7-16%). Histopathological changes were noted in the nasal cavity, and included suppurative inflammation, squamous metaplasia, hyaline droplet accumulation, olfactory epithelium atrophy, and necrosis. In the 2-year inhalation studies, the rats were exposed to 0, 16, 32 and 64ppm of PA and the mice were exposed to 0, 8, 16 and 32ppm of PA. Survival of male rats was significantly reduced (32 and 64ppm). Mean body weights of 64ppm male rats were significantly decreased relative to the controls. Both mice and rats showed a spectrum of non-neoplastic changes in the nose. Increased neoplastic incidences of nasal respiratory/transitional epithelial adenoma were observed in both rats and mice. The incidence of mononuclear cell leukemia was significantly increased in male rats and was considered to be treatment-related. In conclusion, the key findings from this study indicated that the nose was the primary target organ of toxicity for PA. Long term inhalation exposure to PA led to nonneoplastic changes in the nose, and increased incidences of respiratory/transitional epithelial adenomas in both mice and rats. Increased incidences of harderian gland adenoma may also have been related to exposure to PA in male mice.

Design, synthesis, biological evaluation and molecular modeling studies of 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes as a new class of potent antitumor agents.

A series of novel enediyne-containing molecules, 1-aryl-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diynes, were synthesized and displayed significant IC50 values of 10(-7) to 10(-6) M against various cancer cell lines. Of these compounds, 1-(2-pyridinyl)-6-(3,4,5-trimethoxyphenyl)-3(Z)-hexen-1,5-diyne (8) demonstrated the greatest growth inhibition activity. Compound 8 also arrested cancer cells in the G2/M phase and induced apoptosis via activation of Caspase-3. In addition to the G2/M block, compound 8 caused microtubule depolymerization at low concentrations and markedly decreased tumor size in xenographic studies.

A multicenter, randomized, double-blind study comparing different FK778 doses (manitimus) with tacrolimus and steroids vs. MMF with tacrolimus and steroids in renal transplantation.

BACKGROUND: This multicenter phase II study in renal transplantation compared 3 concentration-controlled ranges of FK778 (manitimus) with mycophenolate mofetil (MMF) both given in combination with tacrolimus and corticosteroids. METHODS: 364 patients were randomized to 12-month treatment: high-level FK778 group (H, N=87) received 4 x 600 mg/day (4 days) followed by 120 mg/day; mid-level FK778 group (M, N=92) received 3 x 600 mg/day (3 days) followed by 110 mg/day, low-level FK778 group (L, N=92) received 2 x 600 mg/day (2 days) followed by 100 mg/day, and control group received MMF 1 g/day (MMF, N=93). After week 6, FK778 doses were adjusted to trough ranges of 75-125 µg/mL (H), 50-100 µg/mL (M) and 25-75 µg/mL (L). Tacrolimus and steroids were administered at the same dose in each of the 4 groups. RESULTS: Biopsy proven acute rejection (BPAR) at 24 weeks, the primary study endpoint, was comparable in the L (22.8%) and MMF (17.2%) groups but higher in the H (34.5%) and M (29.3%) groups. BPAR at 12 months was comparable in the L (23.9%) and MMF (19.4%) groups but higher in the H (34.5%) and M (31.5%) groups. Graft and patient survival were lowest in the H group and renal function was poorest in the H and M groups. Premature study withdrawal was highest in the H group. CONCLUSIONS: Efficacy was similar between the low-level FK778 and MMF groups. Increased FK778 exposure was poorly tolerated and did not improve efficacy.

Role of hydrogen sulfide in severe burn injury-induced inflammation in mice.

Endogenous hydrogen sulfide (H(2)S) is naturally synthesized in many types of mammalian cells from L-cysteine in the reactions catalyzed by cystathionine-ß-synthase and cystathionine-γ-lyase (CSE). H(2)S has been demonstrated to play a proinflammatory role in various animal models of hindpaw edema, acute pancreatitis, lipopolysaccharide-induced endotoxemia and cecal ligation, and puncture-induced sepsis. Full-thickness burns that exceed 25% of the total body surface area (TBSA) produce a profound systemic inflammatory reaction characterized by leukocyte activation and plasma leakage in the microvasculature of tissues and organs remote from the wound. The aim of this study was to investigate the effect of local burn injury on induced distant organ endogenous H(2)S release and expression of CSE. Male BALB/c mice were subjected to 30% TBSA full-thickness burn and treated with saline (administered intraperitoneally [i.p.]); DL-propargylglycine (PAG, 50 mg/kg i.p.), which is a CSE inhibitor; or sodium hydrosulfide (NaHS, 10 mg/kg i.p.), which is an H(2)S donor. PAG was administered either 1 h before or 1 h after the burn injury, whereas NaHS was given at the same time as the burn injury. Measurements of liver myeloperoxidase (MPO) activities, liver H(2)S-synthesizing activity, plasma H(2)S level and liver and lung CSE mRNA expression and histological examination of tissues were performed after burn injury. Burn injury significantly increased the plasma H(2)S level and liver H(2)S synthesis 8 h after burn compared with the sham group. Burn injury also resulted in a significant upregulation of CSE mRNA in liver and lung. Prophylactic as well as therapeutic administration of PAG significantly reduced burn-associated systemic inflammation, as evidenced by MPO activity and histological changes in liver and lung. Injection of NaHS significantly aggravated burn-associated systemic inflammation. Therefore, our findings show for the first time the role of H(2)S in contributing to inflammatory damage after burn injury.

Pancreatic islet overproduction of H2S and suppressed insulin release in Zucker diabetic rats.

Hydrogen sulfide (H(2)S) has been traditionally known for its toxic effects on living organisms. The role of H(2)S in the homeostatic regulation of pancreatic insulin metabolism has been unclear. The present study is aimed at elucidating the effect of endogenously produced H(2)S on pancreatic insulin release and its role in diabetes development. Diabetes development in Zucker diabetic fatty (ZDF) rats was evaluated in comparison with Zucker fatty (ZF) and Zucker lean (ZL) rats. Pancreatic H(2)S production and insulin release were also assayed. It was found that H(2)S was generated in rat pancreas islets, catalyzed predominantly by cystathionine gamma-lyase (CSE). Pancreatic CSE expression and H(2)S production were greater in ZDF rats than in ZF or ZL rats. ZDF rats exhibited reduced serum insulin level, hyperglycemia, and insulin resistance. Inhibition of pancreatic H(2)S production in ZDF rats by intraperitoneal injection of DL-propargylglycine (PPG) for 4 weeks increased serum insulin level, lowered hyperglycemia, and reduced hemoglobin A1c level (P<0.05). Although in ZF rats it also reduced pancreatic H(2)S production and serum H(2)S level, PPG treatment did not alter serum insulin and glucose level. Finally, H(2)S significantly increased K(ATP) channel activity in freshly isolated rat pancreatic beta-cells. It appears that insulin release is impaired in ZDF because of abnormally high pancreatic production of H(2)S. New therapeutic approach for diabetes management can be devised based on our observation by inhibiting endogenous H(2)S production from pancreas.