Physiologically Based Pharmacokinetic Modeling and Tissue Distribution Characteristics of SHetA2 in Tumor-Bearing Mice.

The orally available novel small molecule SHetA2 is the lead sulfur-containing heteroarotinoid that selectively inhibits cancer cells over normal cells, and is currently under clinical development for anticancer treatment and cancer prevention. The objective of this study was to assess and characterize the tissue distribution of SHetA2 in tumor-bearing mice by developing a physiologically based pharmacokinetic (PBPK) model. An orthotopic SKOV3 ovarian cancer xenograft mouse model was used to most accurately mimic the ovarian cancer tumor microenvironment in the peritoneal cavity. SHetA2 concentrations in plasma and 14 different tissues were measured at various time points after a single intravenous dose of 10 mg/kg and oral dose of 60 mg/kg, and these data were used to develop a whole-body PBPK model. SHetA2 exhibited a multi-exponential plasma concentration decline with an elimination half-life of 4.5 h. Rapid and extensive tissue distribution, which was best described by a perfusion rate-limited model, was observed with the tissue-to-plasma partition coefficients (kp = 1.4-21.2). The PBPK modeling estimated the systemic clearance (76.4 mL/h) from circulation as a main elimination pathway of SHetA2. It also indicated that the amount absorbed into intestine was the major determining factor for the oral bioavailability (22.3%), while the first-pass loss from liver and intestine contributed minimally (< 1%). Our results provide an insight into SHetA2 tissue distribution characteristics. The developed PBPK model can be used to predict the drug exposure at tumors or local sites of action for different dosing regimens and scaled up to humans to correlate with efficacy.

Dual-Pharmacophore Pyrithione-Containing Cephalosporins Kill Both Replicating and Nonreplicating Mycobacterium tuberculosis.

The historical view of ß-lactams as ineffective antimycobacterials has given way to growing interest in the activity of this class against Mycobacterium tuberculosis (Mtb) in the presence of a ß-lactamase inhibitor. However, most antimycobacterial ß-lactams kill Mtb only or best when the bacilli are replicating. Here, a screen of 1904 ß-lactams led to the identification of cephalosporins substituted with a pyrithione moiety at C3' that are active against Mtb under both replicating and nonreplicating conditions, neither activity requiring a ß-lactamase inhibitor. Studies showed that activity against nonreplicating Mtb required the in situ release of the pyrithione, independent of the known class A ß-lactamase, BlaC. In contrast, replicating Mtb could be killed both by released pyrithione and by the parent ß-lactam. Thus, the antimycobacterial activity of pyrithione-containing cephalosporins arises from two mechanisms that kill mycobacteria in different metabolic states.

Cryogenic Fabrication of Dry Powders to Enhance the Solubility of a Promising Anticancer Drug, SHetA2, for Oral Administration.

SHetA2 is a novel anticancer drug with poor aqueous solubility. In formal toxicological studies, Kolliphor HS 15 was used as a solubilizing agent to increase the oral bioavailability of SHetA2. The purpose of this study was to formulate SHetA2 and Kolliphor HS 15 as solid powders to facilitate their filling in hard gelatin capsules for clinical trials. Two manufacturing processes, ultra-rapid freeze-drying (URFD) and spray freeze drying (SFD), were employed to fabricate solid powders of SHetA2-Kolliphor HS 15 and trehalose. The morphology, size, flowability, and compressibility of URFD-SHetA2 and SFD-SHetA2 powders were characterized. The crystallinity and apparent maximum solubility of SHetA2 in both powders were also determined. SFD-SHetA2 powders were spherical in shape, small, and with a wide size distribution while the URFD-SHetA2 powders were irregularly shaped and big but with a narrower distribution. DSC and XRD analyses indicated that SHetA2 was mostly amorphous in both powders. The flow of both powders was categorized as "good" (angle of repose < 35°). The uniformity of drug content in URFD-SHetA2 powders was more variable than that in SFD-SHetA2 powders. The solubility profile of SHetA2 in both powders SGF exhibited a transient supersaturation "spring effect" due to the drug's amorphousness followed by extended supersaturation "parachute effect" at approximately 6 µg/ml for both powders compared to 0.02 ± 0.01 µg/ml for unprocessed drug. In conclusion, both URFD and SFD formed solid SHetA2 Kolliphor powders that are possible formulation candidates to be filled in hard gelatin capsules for clinical trials.

Pharmacokinetics and Pharmacodynamics of Escalating Doses of SHetA2 After Vaginal Administration to Mice.

SHetA2 is a novel compound with strong potential to treat cervical dysplasia, but its low aqueous solubility limits its oral bioavailability. A vaginal suppository achieved SHetA2 cervix concentrations that were severalfold above the predicted therapeutic levels. Thus, we aimed at determining the minimum dose that would achieve SHetA2 therapeutic levels while reducing cyclin D1 levels, the pharmacodynamic end point. The disposition of SHetA2 after vaginal administration of escalating SHetA2 doses and the corresponding reduction in cyclin D1 levels was compared to that after the conventional oral treatment. Vaginal administration of a 15-mg/kg dose achieved an area under the cervix concentration versus time curve (AUCcervix) that was ∼120 times larger than that after a 60 mg/kg administered orally. AUCcervix and Cmax-cervix did not increase proportionally with respect to the dose, with the 30-mg/kg dose resulting in higher AUCcervix and Cmax-cervix (1368.53 µg.mL/h and 155.38 µg/g, respectively) compared to the 15 mg/kg (334.98 µg.mL/h and 121.78 µg/g, respectively) or 60 mg/kg (1178.55 µg.mL/h and 410.38 µg/g, respectively). Likewise, the 30-mg/kg dose caused a larger reduction in cyclin D1 levels than the other doses. Thus, the 30-mg/kg dose was selected for future efficacy studies in a mouse model of cervical neoplasia.

Influence of the estrus cycle of the mouse on the disposition of SHetA2 after vaginal administration.

SHetA2 is a novel compound with the potential to treat cervical dysplasia, but has poor water solubility. A vaginal suppository formulation was able to achieve therapeutic concentrations in the cervix of mice, but these concentrations were variable. Histological analysis indicated that mice in the same group were in different stages of their estrous cycle, which is known to induce anatomical changes in their gynecological tissues. We investigated the effects of these changes on the pharmacokinetics and pharmacodynamics of SHetA2 when administered vaginally. Mice were synchronized to be either in estrous or diestrus stage for administration of the SHetA2 suppository. Pharmacokinetic parameters were calculated from the SHetA2 concentrations vs. time data. The reduction in the expression of cyclin D1 protein in the cervix was used as pharmacodynamic endpoint. Mice dosed during diestrus had a larger AUCcervix (335 µg mL h-1), higher Cmax (121.8 ±â€¯38.7 µg/g) and longer t1/2-cervix (30.3 h) compared to mice dosed during estrus (120 µg mL h-1, 44.6 ±â€¯29.5 µg/g and 3.6 h respectively). Therapeutic concentrations of SHetA2 were maintained for 48 h in the cervix of mice dosed during diestrus and for only 12 h in the estrus group. The treatment reduced the expression of cyclin D1 protein in the cervix of mice in the estrus to a larger extent. These results indicate that the estrous cycle of mice influences significantly the disposition of SHetA2 after vaginal administration and may also influence its efficacy.

Pharmacokinetics and interspecies scaling of a novel, orally-bioavailable anti-cancer drug, SHetA2.

SHetA2 is a small molecule drug with promising cancer prevention and therapeutic activity and a high preclinical safety profile. The study objectives were to perform interspecies scaling and pharmacokinetic (PK) modeling of SHetA2 for human PK prediction. The PK data obtained from mice, rats, and dogs after intravenous and oral doses were used for simultaneous fitting to PK models. The disposition of SHetA2 was best described by a two-compartment model. The absorption kinetics was well characterized with a first-order absorption model for mice and rats, and a gastrointestinal transit model for dogs. Oral administration of SHetA2 showed a relatively fast absorption in mice, prolonged absorption (i.e., flip-flop kinetics) toward high doses in rats, and an early peak followed by a secondary peak at high doses in dogs. The oral bioavailability was 17.7-19.5% at 20-60 mg/kg doses in mice, <1.6% at 100-2000 mg/kg in rats, and 11.2% at 100 mg/kg decreasing to 3.45% at 400 mg/kg and 1.11% at 1500 mg/kg in dogs. The disposition parameters were well correlated with the body weight for all species using the allometric equation, which predicted values of CL (17.3 L/h), V1 (36.2 L), V2 (68.5 L) and CLD (15.2 L/h) for a 70-kg human. The oral absorption rate and bioavailability of SHetA2 was highly dependent on species, doses, formulations, and possibly other factors. The limited bioavailability at high doses was taken into consideration for the suggested first-in-human dose, which was much lower than the dose estimated based on toxicology studies. In summary, the present study provided the PK model for SHetA2 that depicted the disposition and absorption kinetics in preclinical species, and computational tools for human PK prediction.

Optimization of a Vaginal Suppository Formulation to Deliver SHetA2 as a Novel Treatment for Cervical Dysplasia.

Cervical dysplasia induced by the human papilloma virus unpredictably progresses to cervical cancer. Therapeutic options are invasive and affect the patient's quality of life. SHetA2 has demonstrated therapeutic efficacy against human and murine human papilloma virus-induced tumors, but its oral bioavailability is <1%. An optimized vaginal suppository formulation can deliver SHetA2 in sufficient doses to prevent cervical dysplasia. The quality by design approach was employed to optimize the suppository formulation consisting of cocoa butter as base with 5% Kolliphor and 40% SHetA2. The suppository had a content uniformity of 105.44 ± 0.42%, melted in <8 min, and had a complete release of SHetA2 in water. Administration of the suppository to mice-achieved cervix concentrations that were significantly higher than the SHetA2 therapeutic concentration, with the maximum concentration (Cmax-cervix = 336.78 µg/g) being more than 100-fold the therapeutic SHetA2 concentration. Furthermore, the levels of cyclin D1 protein decreased 9-fold indicating a correlation of drug concentrations with the pharmacodynamic endpoint. These proof-of-concept studies suggest that the SHetA2 optimized vaginal suppository formulation may have a potential use in the prevention of cervical dysplasia, but detailed efficacy studies are required to confirm this assumption.

Molecular mechanism of action and safety of 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione - a novel anticonvulsant drug candidate.

Previously, it was found that 5-(3-chlorophenyl)-4-hexyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (TP-315) effectively protects mice from maximal electroshock-induced seizures. The aim of this study was to determine possible interactions between TP-315 and different molecular targets, i.e. GABAA receptors, voltage-gated sodium channels, and human neuronal α7 and α4ß2 nicotinic acetylcholine receptors. The influence of TP-315 on the viability of human hepatic HepG2 cells was also established using PrestoBlue and ToxiLight assays. It was found that the anticonvulsant activity of TP-315 results (at least partially) from its influence on voltage-gated sodium channels (VGSCs). Moreover, the title compound slightly affected the viability of human hepatic cells.

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.

Enhancement of Cytotoxicity of Three Apoptosis-inducing Agents Against Human Oral Squamous Cell Carcinoma Cell Line by Benzoxazinotropone.

Tumor-specificity (TS) and anti-inflammatory activity of benzo[b]cyclohept[e][1,4]oxazin-6(11H)-one, generally known as benzoxazinotropone (BOT), have been reported. In order to find a new biological activity, the combination effect of BOT and three apoptosis-inducing agents was investigated. Cytotoxicity against four human oral squamous cell carcinoma (OSCC) cell lines and five human oral normal cells (gingival fibroblasts, periodontal ligament fibroblasts, pulp cells, oral keratinocytes and primary gingival epithelial cells) was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method. TS was evaluated by the ratio of the mean 50% cytotoxic concentration (CC50) against normal oral cells to the one against OSCC cell lines. Synergy was evaluated by CompuSyn software program. Expression of cleaved forms of poly ADP-ribose polymerase and caspsase-3 was evaluated by western blot analysis. BOT induced activation of caspase 3, suggesting the apoptosis induction in HSC-2 OSCC cells. BOT enhanced the cytotoxicity of doxorubicin (DXR) additively and that of curcumin and resveratrol synergistically. On the other hand, BOT did not enhance, but rather inhibit the cytotoxicity of DXR against normal keratinocytes. The present study suggests that BOT may enhance the anti-tumor activity of apoptosis-inducing agents, while reducing its cytotoxicity against normal cells.

Inhibition of sphingomyelin synthase 1 affects ceramide accumulation and hydrogen peroxide-induced apoptosis in Neuro-2a cells.

Oxidative stress plays a key role in brain injury after cerebral ischemia-reperfusion, which contributes toward excessive apoptosis of nerve cells. Therefore, it would be beneficial to identify a therapy that could interfere with the progression of apoptosis and protect the brain from ischemia-reperfusion injury. As ceramide, a well-known second messenger of apoptosis, can be metabolized by sphingomyelin synthase 1 (SMS1), recent research has focused on the link between SMS1 and apoptosis in different cells. To investigate whether SMS1 is involved in the process of oxidative stress-induced apoptosis in neurons and to explore the possible underlying mechanism, we treated mouse neuroblastoma Neuro-2A (N2a) cells with hydrogen peroxide (H2O2). Incubation with H2O2 significantly upregulated the expression of SMS1, increased the intracellular levels of ceramide and sphingomyelin synthase activity, and induced apoptosis. Moreover, pretreatment of N2a cells with D609, an sphingomyelin synthase inhibitor, or SMS1-silencing RNA (siRNA) further increased ceramide and potentiated H2O2-induced apoptosis which could be reversed by SB203580 (a p38 inhibitor). Thus, our study has shown that SMS1 regulates ceramide levels in N2a cells and plays a potent protective role in this oxidative stress-induced apoptosis partly through the p38 pathway.

3H-1,2-dithiole-3-thione as a novel therapeutic agent for the treatment of experimental autoimmune encephalomyelitis.

3H-1,2-dithiole-3-thione (D3T), the simplest member of the sulfur-containing dithiolethiones, is found in cruciferous vegetables, and has been previously reported to be a potent inducer of antioxidant genes and glutathione biosynthesis by activation of the transcription factor Nrf2. D3T is a cancer chemopreventive agent and possesses anti-inflammatory properties. Although D3T has been shown to protect against neoplasia, the effect of D3T in the autoimmune inflammatory disease multiple sclerosis/experimental autoimmune encephalomyelitis (EAE) is unknown. The present study is the first report of the therapeutic effect of D3T in EAE. Our results show D3T, administered post immunization, not only delays disease onset but also dramatically reduces disease severity in EAE. Strikingly, D3T, administered post disease onset of EAE, effectively prevents disease progression and exacerbation. Mechanistic studies revealed that D3T suppresses dendritic cell activation and cytokine production, inhibits pathogenic Th1 and Th17 differentiation, represses microglia activation and inflammatory cytokine expression, and promotes microglia phase II enzyme induction. In summary, these results indicate that D3T affects both innate and adaptive immune cells, and the protective effect of D3T in EAE might be attributed to its effects on modulating dendritic cell and microglia activation and pathogenic Th1/Th17 cell differentiation.

SMIFH2-mediated mDia formin functional inhibition potentiates chemotherapeutic targeting of human ovarian cancer spheroids.

Due to a lack of effective screening or prevention protocol for epithelial ovarian cancer (EOC), there is a critical unmet need to develop therapeutic interventions for EOC treatment. EOC metastasis is unique. Initial dissemination is not primarily hematogenous, yet is facilitated through shedding of primary tumor cells into the peritoneal fluid and accumulating ascites. Increasingly, isolated patient spheroids point to a clinical role for spheroids in EOC metastasis. EOC spheroids are highly invasive structures that disseminate upon peritoneal mesothelium, and visceral tissues including liver and omentum. Selection for this subset of chemoresistant EOC cells could influence disease progression and/or recurrence. Thus, targeting spheroid integrity/structure may improve the chemotherapeutic responsiveness of EOC. We discovered a critical role for mammalian Diaphanous (mDia)-related formin-2 in maintaining EOC spheroid structure. Both mDia2 and the related mDia1 regulate F-actin networks critical to maintain cell-cell contacts and the integrity of multi-cellular epithelial sheets. We investigated if mDia2 functional inhibition via a small molecule inhibitor SMIFH2 combined with chemotherapeutics, such as taxol and cisplatin, inhibits the viability of EOC monolayers and clinically relevant spheroids. SMIFH2-mediated mDia formin inhibition significantly reduced both ES2 and Skov3 EOC monolayer viability while spheroid viability was minimally impacted only at the highest concentrations. Combining either cisplatin or taxol with SMIFH2 did not significantly enhance the effects of either drug alone in ES2 monolayers, while Skov3 monolayers treated with taxol or cisplatin and SMIFH2 showed significant additive inhibition of viability. ES2 spheroids were highly responsive with clear additive anti-viability effects with dual taxol or cisplatin when combined with SMIFH2 treatments. While combined taxol with SMIFH2 in spheroids showed an additive effect relative to single treatments, Skov3 spheroids showed no additive effects from combined cisplatin and SMIFH2 treatments. Our data indicate that mDia formin inhibition combined with taxol to drive enhanced and/or additive anti-viability effects targeting 3D EOC structures, including ES2 and Skov3 spheroids. Combined mDia formin inhibition with cisplatin may be most effective in EOC spheroids where cisplatin sensitivity is retained at moderate levels, such as ES2 cells.

AP39, A Mitochondrially Targeted Hydrogen Sulfide Donor, Exerts Protective Effects in Renal Epithelial Cells Subjected to Oxidative Stress in Vitro and in Acute Renal Injury in Vivo.

This study evaluated the effects of AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol-5yl) phenoxy)decyl) triphenyl phosphonium bromide], a mitochondrially targeted donor of hydrogen sulfide (H2S) in an in vitro model of hypoxia/oxidative stress injury in NRK-49F rat kidney epithelial cells (NRK cells) and in a rat model of renal ischemia-reperfusion injury. Renal oxidative stress was induced by the addition of glucose oxidase, which generates hydrogen peroxide in the culture medium at a constant rate. Glucose oxidase (GOx)-induced oxidative stress led to mitochondrial dysfunction, decreased intracellular ATP content, and, at higher concentrations, increased intracellular oxidant formation (estimated by the fluorescent probe 2, 7-dichlorofluorescein, DCF) and promoted necrosis (estimated by the measurement of lactate dehydrogenase release into the medium) of the NRK cells in vitro. Pretreatment with AP39 (30-300 nM) exerted a concentration-dependent protective effect against all of the above effects of GOx. Most of the effects of AP39 followed a bell-shaped concentration-response curve; at the highest concentration of GOx tested, AP39 was no longer able to afford cytoprotective effects. Rats subjected to renal ischemia/reperfusion responded with a marked increase (over four-fold over sham control baseline) blood urea nitrogen and creatinine levels in blood, indicative of significant renal damage. This was associated with increased neutrophil infiltration into the kidneys (assessed by the myeloperoxidase assay in kidney homogenates), increased oxidative stress (assessed by the malondialdehyde assay in kidney homogenates), and an increase in plasma levels of IL-12. Pretreatment with AP39 (0.1, 0.2, and 0.3 mg/kg) provided a dose-dependent protection against these pathophysiological alterations; the most pronounced protective effect was observed at the 0.3 mg/kg dose of the H2S donor; nevertheless, AP39 failed to achieve a complete normalization of any of the injury markers measured. The partial protective effects of AP39 correlated with a partial improvement of kidney histological scores and reduced TUNEL staining (an indicator of DNA damage and apoptosis). In summary, the mitochondria-targeted H2S donor AP39 exerted dose-dependent protective effects against renal epithelial cell injury in vitro and renal ischemia-reperfusion injury in vivo. We hypothesize that the beneficial actions of AP39 are related to the reduction of cellular oxidative stress, and subsequent attenuation of various positive feed-forward cycles of inflammatory and oxidative processes.

Oral TNFα Modulation Alters Neutrophil Infiltration, Improves Cognition and Diminishes Tau and Amyloid Pathology in the 3xTgAD Mouse Model.

Cytokines such as TNFα can polarize microglia/macrophages into different neuroinflammatory types. Skewing of the phenotype towards a cytotoxic state is thought to impair phagocytosis and has been described in Alzheimer's Disease (AD). Neuroinflammation can be perpetuated by a cycle of increasing cytokine production and maintenance of a polarized activation state that contributes to AD progression. In this study, 3xTgAD mice, age 6 months, were treated orally with 3 doses of the TNFα modulating compound isoindolin-1,3 dithione (IDT) for 10 months. We demonstrate that IDT is a TNFα modulating compound both in vitro and in vivo. Following long-term IDT administration, mice were assessed for learning & memory and tissue and serum were collected for analysis. Results demonstrate that IDT is safe for long-term treatment and significantly improves learning and memory in the 3xTgAD mouse model. IDT significantly reduced paired helical filament tau and fibrillar amyloid accumulation. Flow cytometry of brain cell populations revealed that IDT increased the infiltrating neutrophil population while reducing TNFα expression in this population. IDT is a safe and effective TNFα and innate immune system modulator. Thus small molecule, orally bioavailable modulators are promising therapeutics for Alzheimer's disease.

Pre-clinical pharmacokinetics and acute toxicological evaluation of a monastrol derivative anticancer candidate LaSOM 65 in rats.

1. The present work investigated the pharmacokinetic and tissue distribution as well as acute toxicity of a new chemical entity (NCE), the anticancer candidate LaSOM 65 in Wistar rats. 2. LaSOM 65 pharmacokinetics was investigated after intravenous (i.v., 1 mg/kg) and oral (p.o., 10 and 30 mg/kg) dosing. Tissue distribution was assessed after i.v. bolus dose. Acute toxicity was evaluated after i.v. (1, 2.5 and 5 mg/kg) and p.o. (50, 100 and 150 mg/kg) administration. 3. Short half-life (1.75 ± 0.71 h), a clearance of 0.85 ± 0.18 L/h/kg and a volume of distribution of 1.76 ± 0.24 L/kg were observed after i.v. dosing. The compound showed good bioavailability and linear pharmacokinetics after oral doses. The NCE distributes consistently in lung and fatty tissues, with penetration ratios of 2.7 and 1.4, respectively. The other tissues investigated presented smaller penetration ratios. Adverse clinical symptoms were observed only after i.v. administration, and regressed 3 h after dosing. Compared with controls, no statistical differences were found for serum analysis, body weight and relative organ weight, indicating no acute toxicological effects. 4. Overall, LaSOM 65 showed good pharmacokinetic characteristics and no signs of acute toxicity, indicating that it is a promising anticancer candidate.

Evidence for the role of phosphatidylcholine-specific phospholipase C in sustained hypoxic pulmonary vasoconstriction.

The aim of the study was to investigate the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in hypoxic pulmonary vasoconstriction (HPV) and elucidate its possible interactions within HPV mechanism. Inhibition of PC-PLC with D609 (30µM) resulted in partial reduction of the transient phase and almost complete abolition of the sustained phase of HPV in isolated rat intrapulmonary arteries (IPAs). Intravenous injection of D609 (5mg/kg) 30min before the onset of hypoxia prevented the development of acute hypoxic pulmonary hypertension (AHPH) in rats. D609 also inhibited pulmonary vasoconstriction induced with a generator of superoxide anions LY83583, but not the one induced with hydrogen peroxide. Protein kinase C (PKC) inhibition with Ro-31-8220 partially diminished the transient phase of hypoxic contraction in IPA while the sustained phase remained unchanged. Phosphocholine, known to be released due to phosphatidylcholine breakdown by PC-PLC, induced sustained contraction in isolated IPA and also transient pulmonary and systemic hypertension if administered intravenously (70mg/kg). We conclude that PC-PLC plays an important role in sustained HPV possibly through the activation of PKC-independent mechanism, which may be coupled with phosphocholine release.

Oral toxicity and pharmacokinetic studies of SHetA2, a new chemopreventive agent, in rats and dogs.

SHetA2 is a heteroarotinoid that has shown selective inhibition of cancer cell growth and an induction of apoptosis without activation of nuclear retinoic acid receptors. In the rat study, SHetA2 was administered in 1% aqueous methylcellulose/0.2% Tween 80 by oral gavage at 0, 100, 500, and 2,000 mg/kg/day for 28 days. The high-dose administration induced decreased activity in male rats, decreased body-weight gains and food consumption, and changes in organ weights. The major metabolite of SHetA2 in rat plasma was monohydroxy SHetA2, which was considerably higher than the parent compound after oral and intravenous administration. Pharmacokinetic analysis showed extremely low (<1%) systemic bioavailability of SHetA2 for all doses tested. The dose of 2,000 mg/kg/day was considered as the lowest observed adverse effect level. The no observed adverse effect level (NOAEL) was 500 mg/kg/day. In the dog study, no toxicity of SHetA2 in 30% aqueous Solutol(®) HS 15 was observed in any tested dose groups (0, 100, 400, and 1,500 mg/kg/day). The major metabolite of SHetA2 in dog plasma was also monohydroxy SHetA2, which was equal to or lower than the parent compound after oral administration. SHetA2 levels in dog plasma were notably higher, when compared to levels in rat plasma. However, exposure was not dose proportional, as exemplified by a lack of proportional increase in maximum concentration or area under the plasma concentration-time curve with increasing dose. The NOAEL was not established and was considered to be above 1,500 mg/kg/day.

MLN0905, a small-molecule plk1 inhibitor, induces antitumor responses in human models of diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common of the non-Hodgkin lymphomas, accounting for up to 30% of all newly diagnosed lymphoma cases. Current treatment options for this disease are effective, but not always curative; therefore, experimental therapies continue to be investigated. We have discovered an experimental, potent, and selective small-molecule inhibitor of PLK1, MLN0905, which inhibits cell proliferation in a broad range of human tumor cells including DLBCL cell lines. In our report, we explored the pharmacokinetic, pharmacodynamic, and antitumor properties of MLN0905 in DLBCL xenograft models grown in mice. These studies indicate that MLN0905 modulates the pharmacodynamic biomarker phosphorylated histone H3 (pHisH3) in tumor tissue. The antitumor activity of MLN0905 was evaluated in three human subcutaneous DLBCL xenograft models, OCI LY-10, OCI LY-19, and PHTX-22L (primary lymphoma). In each model, MLN0905 yielded significant antitumor activity on both a continuous (daily) and intermittent dosing schedule, underscoring dosing flexibility. The antitumor activity of MLN0905 was also evaluated in a disseminated xenograft (OCI LY-19) model to better mimic human DLBCL disease. In the disseminated model, MLN0905 induced a highly significant survival advantage. Finally, MLN0905 was combined with a standard-of-care agent, rituximab, in the disseminated OCI LY-19 xenograft model. Combining rituximab and MLN0905 provided both a synergistic antitumor effect and a synergistic survival advantage. Our findings indicate that PLK1 inhibition leads to pharmacodynamic pHisH3 modulation and significant antitumor activity in multiple DLBCL models. These data strongly suggest evaluating PLK1 inhibitors as DLBCL anticancer agents in the clinic.

Anti-inflammatory and immunomodulatory effects of RDV-8 [C18H22N2O2S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] in a rat model of induced pleurisy and in vitro lymphoproliferation.

RDV-8 [C(18)H(22)N(2)O(2)S (ethyl 1-butyl-6-methyl-2-phenyl-4-thioxo-1,4-dihydropyrimidine-5-carboxylate)] is derived from the 4-thioxopyrimidine, and presents important clinical effects. The present study explored the RDV-8 effects in the proliferation of human peripheral blood mononuclear cells (PBMCs), as well as in a pleurisy-induced rat model. PBMCs were directly plated in four different RDV-8 concentrations (0.0125, 0.025, 0.05 and 0.1 mg/mL). RDV-8 decreased cell proliferation and monocyte chemotactic protein 1 synthesis. The interleukin 1 levels and the cytotoxic effect were not significantly affected by RDV-8 treatment. In the carrageenan-induced pleurisy model, the RDV-8 (3 mg/kg) treatment induced a significant reduction in the exudate volume, in the polymorphonuclear leukocyte migration and in the pleural exudate NO levels. The results indicate that RDV-8 may have an immunomodulatory effect, as well as anti-inflammatory actions suggesting that it could represent a new strategy in the inflammatory response modulation.