Subcutaneous therapy for portal hypertension: PHIN-214, a partial vasopressin receptor 1A agonist.

Cirrhosis is a liver disease that leads to increased intrahepatic resistance, portal hypertension (PH), and splanchnic hyperemia resulting in ascites, variceal bleeding, and hepatorenal syndrome. Terlipressin, a prodrug that converts to a short half-life vasopressin receptor 1 A (V1a) full agonist [8-Lys]-Vasopressin (LVP), is an intravenous treatment for PH complications, but hyponatremia and ischemic side effects require close monitoring. We developed PHIN-214 which converts into PHIN-156, a more biologically stable V1a partial agonist. PHIN-214 enables once-daily subcutaneous administration without causing ischemia or tissue necrosis and has a 10-fold higher therapeutic index than terlipressin in healthy rats. As V1a partial agonists, PHIN-214 and PHIN-156 exhibited maximum activities of 28 % and 42 % of Arginine vasopressin (AVP), respectively. The potency of PHIN-156 and LVP relative to AVP is comparable for V1a (5.20 and 1.65 nM, respectively) and V1b (102 and 115 nM, respectively) receptors. However, the EC50 of PHIN-156 to the V2 receptor was 26-fold higher than that of LVP, indicating reduced potential for dilutional hyponatremia via V2 agonism compared to terlipressin/LVP. No significant off-target binding to 87 toxicologically relevant receptors were observed when evaluated in vitro at 10 µM concentration. In bile duct ligated rats with PH, subcutaneous PHIN-214 reduced portal pressure by 13.4 % ± 3.4 in 4 h. These collective findings suggest that PHIN-214 could be a novel pharmacological treatment for patients with PH, potentially administered outside of hospital settings, providing a safe and convenient alternative for managing PH and its complications.

Pharmacokinetics of cannabidiol-/cannabidiolic acid-rich hemp oil in juvenile cynomolgus macaques (Macaca fascicularis).

Introduction: Cannabinoids are increasingly popular in human and veterinary medicine and have been studied as an alternative treatment for a wide range of disorders. The goal of this study was to perform a pharmacokinetic analysis of oral cannabidiol (CBD)-/cannabidiolic acid (CBDA)-rich hemp oil (CBD/ArHO) in juvenile cynomolgus macaques (Macaca fascicularis). Methods: After a 2 mg/kg CBD/ArHO pilot study, 4 and 8 mg/kg direct-to-mouth CBD/ArHO were administered (n = 4 per dose) once daily for 14 days and blood was collected at 0-, 0.5-, 1-, 2-, 4-, 8-, 12-, and 24-h, and on Days 7 and 14, to quantify serum cannabinoid concentrations by high-performance liquid chromatography-tandem mass spectrometry. Serum biochemistries and complete blood counts were performed on Days 0, 1, and 14. Results: The maximum mean serum concentration (Cmax) of CBDA was 28.6-36.2 times that of CBD at 4 and 8 mg/kg. At 8 mg/kg, the Cmax of CBD was 1.4 times higher (p = 0.0721), and CBDA was significantly 1.8 times higher (p = 0.0361), than at 4 mg/kg. The maximum mean serum concentration of ∆9-tetrahydrocannabinol (THC) was 4.80 ng/mL at 8 mg/kg. Changes in serum biochemistries and complete blood counts over time were not clinically significant. Discussion: Given the low serum CBD concentrations, the doses and frequency used in this study may be insufficient for a therapeutic effect of CBD in particular; therefore, clinical studies are needed to determine the therapeutic dose of CBD and CBDA for macaques, which may differ based on the disorder targeted.

Excited State Proton Transfers in Hybrid Compound Based on Indoline Spiropyran of the Coumarin Type and Azomethinocoumarin in the Presence of Metal Ions.

Spectral-luminescence properties of a hybrid compound containing a coumarin-type spiropyran and an azomethinocoumarin fragment in toluene-acetonitrile solution in the presence of Li+, Ca2+, Zn2+ and Mg2+ ions are reported. Two excited state proton transfers can occur in the hybrid compound-the transfer of a proton from the OH group of the 7-hydroxy coumarin tautomer to the N atom of the C=N bond of the azomethine fragment leading to green ESIPT fluorescence with a maximum at 540 nm and from the OH group of the 7-hydroxy coumarin tautomer to the carbonyl group of the pyrone chromophore, which leads to the formation of the 2-hydroxyl-tautomer T of coumarin with blue fluorescence with a maximum at 475 nm. Dependence of these excited state proton transfers on the metal nature and irradiation with an external UV source is discussed.

Blockade of a Laminin-411-Notch Axis with CRISPR/Cas9 or a Nanobioconjugate Inhibits Glioblastoma Growth through Tumor-Microenvironment Cross-talk.

There is an unmet need for the treatment of glioblastoma multiforme (GBM). The extracellular matrix, including laminins, in the tumor microenvironment is important for tumor invasion and progression. In a panel of 226 patient brain glioma samples, we found a clinical correlation between the expression of tumor vascular laminin-411 (α4ß1γ1) with higher tumor grade and with expression of cancer stem cell (CSC) markers, including Notch pathway members, CD133, Nestin, and c-Myc. Laminin-411 overexpression also correlated with higher recurrence rate and shorter survival of GBM patients. We also showed that depletion of laminin-411 α4 and ß1 chains with CRISPR/Cas9 in human GBM cells led to reduced growth of resultant intracranial tumors in mice and significantly increased survival of host animals compared with mice with untreated cells. Inhibition of laminin-411 suppressed Notch pathway in normal and malignant human brain cell types. A nanobioconjugate potentially suitable for clinical use and capable of crossing blood-brain barrier was designed to block laminin-411 expression. Nanobioconjugate treatment of mice carrying intracranial GBM significantly increased animal survival and inhibited multiple CSC markers, including the Notch axis. This study describes an efficient strategy for GBM treatment via targeting a critical component of the tumor microenvironment largely independent of heterogeneous genetic mutations in glioblastoma.Significance: Laminin-411 expression in the glioma microenvironment correlates with Notch and other cancer stem cell markers and can be targeted by a novel, clinically translatable nanobioconjugate to inhibit glioma growth.

A Novel l-Asparaginase with low l-Glutaminase Coactivity Is Highly Efficacious against Both T- and B-cell Acute Lymphoblastic Leukemias In Vivo.

Acute lymphoblastic leukemia (ALL) is the most common type of pediatric cancer, although about 4 of every 10 cases occur in adults. The enzyme drug l-asparaginase serves as a cornerstone of ALL therapy and exploits the asparagine dependency of ALL cells. In addition to hydrolyzing the amino acid l-asparagine, all FDA-approved l-asparaginases also have significant l-glutaminase coactivity. Since several reports suggest that l-glutamine depletion correlates with many of the side effects of these drugs, enzyme variants with reduced l-glutaminase coactivity might be clinically beneficial if their antileukemic activity would be preserved. Here we show that novel low l-glutaminase variants developed on the backbone of the FDA-approved Erwinia chrysanthemi l-asparaginase were highly efficacious against both T- and B-cell ALL, while displaying reduced acute toxicity features. These results support the development of a new generation of safer l-asparaginases without l-glutaminase activity for the treatment of human ALL.Significance: A new l-asparaginase-based therapy is less toxic compared with FDA-approved high l-glutaminase enzymes Cancer Res; 78(6); 1549-60. ©2018 AACR.

Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation.

The FoxA family of pioneer transcription factors regulates hepatitis B virus (HBV) transcription, and hence viral replication. Hepatocyte-specific FoxA-deficiency in the HBV transgenic mouse model of chronic infection prevents the transcription of the viral DNA genome as a result of the failure of the developmentally controlled conversion of 5-methylcytosine residues to cytosine during postnatal hepatic maturation. These observations suggest that pioneer transcription factors such as FoxA, which mark genes for expression at subsequent developmental steps in the cellular differentiation program, mediate their effects by reversing the DNA methylation status of their target genes to permit their ensuing expression when the appropriate tissue-specific transcription factor combinations arise during development. Furthermore, as the FoxA-deficient HBV transgenic mice are viable, the specific developmental timing, abundance and isoform type of pioneer factor expression must permit all essential liver gene expression to occur at a level sufficient to support adequate liver function. This implies that pioneer transcription factors can recognize and mark their target genes in distinct developmental manners dependent upon, at least in part, the concentration and affinity of FoxA for its binding sites within enhancer and promoter regulatory sequence elements. This selective marking of cellular genes for expression by the FoxA pioneer factor compared to HBV may offer the opportunity for the specific silencing of HBV gene expression and hence the resolution of chronic HBV infections which are responsible for approximately one million deaths worldwide annually due to liver cirrhosis and hepatocellular carcinoma.

Protected graft copolymer-formulated fibroblast growth factors mitigate the lethality of partial body irradiation injury.

We evaluated the mitigating effects of fibroblast growth factor 4 and 7 (FGF4 and FGF7, respectively) in comparison with long acting protected graft copolymer (PGC)-formulated FGF4 and 7 (PF4 and PF7, respectively) administered to C57BL/6J mice a day after exposure to LD50/30 (15.7 Gy) partial body irradiation (PBI) which targeted the gastrointestinal (GI) system. The PGC that we developed increased the bioavailability of FGF4 and FGF7 by 5- and 250-fold compared to without PGC, respectively, and also sustained a 24 hr presence in the blood after a single subcutaneous administration. The dose levels tested for mitigating effects on radiation injury were 3 mg/kg for the PF4 and PF7 and 1.5 mg each for their combination (PF4/7). Amifostine administered prior to PBI was used as a positive control. The PF4, PF7, or PF4/7 mitigated the radiation lethality in mice. The mitigating effect of PF4 and PF7 was similar to the positive control and PF7 was better than other mitigators tested. The plasma citrulline levels and hematology parameters were early markers of recovery and survival. GI permeability function appeared to be a late or full recovery indicator. The villus length and crypt number correlated with plasma citrulline level, indicating that it can act as a surrogate marker for these histology evaluations. The IL-18 concentrations in jejunum as early as day 4 and TPO levels in colon on day 10 following PBI showed statistically significant changes in irradiated versus non-irradiated mice which makes them potential biomarkers of radiation exposure. Other colon and jejunum cytokine levels are potentially useful but require larger numbers of samples than in the present study before their full utility can be realized.

Implantable chemotherapy-loaded silk protein materials for neuroblastoma treatment.

Neuroblastoma is the most common extracranial childhood solid tumor. Treatment of high risk tumors require intense multicycle chemotherapies, resulting in short- and long-term toxicities. Here, we present treatment of an orthotopic neuroblastoma mouse model, with silk fibroin materials loaded with vincristine, doxorubicin or the combination as a intratumoral, sustained release system. The materials, loaded with vincristine with or without doxorubicin, significantly decreased neuroblastoma tumor growth compared to materials loaded without drug or doxorubicin only as well as intravenous (IV) drug treatment. The intratumoral drug concentration was significantly higher with intratumoral delivery versus IV. Furthermore, intratumor delivery decreased the maximum plasma concentration compared to IV delivery, reducing systemic exposure and possibly reduing long-term side effects of chemotherapy exposure. Histopathologically, tumors with remission periods >25 days before recurrence transformed from a "small-round-blue cell" (SBRC) to predominantly "large cell" neuroblastoma (LCN) histopathology, a more aggressive tumor subtype with unfavorable clinical outcomes. These results show that intratumoral chemotherapy delivery may be a treatment strategy for pediatric neuroblastoma, potentially translatable to other focal tumors types. Furthermore, this treatment modality allows for a clinically relevant mouse model of tumor transformation that may be used for studying the phenotypical tumor recurrence and developing more effective treatment strategies for recurrent tumors.

Predictive observation-based endpoint criteria for mice receiving total body irradiation.

Total body irradiation of mice is a commonly used research technique; however, humane endpoints have not been clearly identified. This situation has led to the inconsistent use of various endpoints, including death. To address this issue, we refined a cageside observation-based scoring system specifically for mice receiving total body irradiated. Male and female C57BL/6 mice (age, 8 wk) received 1 of 3 doses of radiation from 1 of 2 different radiation sources and were observed for progression of clinical signs. All mice were scored individually by using cageside observations of their body posture (score, 0 to 3), eye appearance (0 to 3), and activity level (0 to 3). Retrospective analysis of the observation score data indicated that death could be predicted accurately with total scores of 7 or greater, and observation scores were consistent between observers. This scoring system can be used to increase the consistent use of endpoint criteria in total body murine irradiation studies and ultimately to improve animal welfare.

Role for platelet glycoprotein Ib-IX and effects of its inhibition in endotoxemia-induced thrombosis, thrombocytopenia, and mortality.

OBJECTIVE: Poor prognosis of sepsis is associated with bacterial lipopolysaccharide (LPS)-induced intravascular inflammation, microvascular thrombosis, thrombocytopenia, and disseminated intravascular coagulation. Platelets are critical for thrombosis, and there has been increasing evidence of the importance of platelets in endotoxemia. The platelet adhesion receptor, the glycoprotein Ib-IX complex (GPIb-IX), mediates platelet adhesion to inflammatory vascular endothelium and exposed subendothelium. Thus, we have investigated the role of GPIb-IX in LPS-induced platelet adhesion, thrombosis, and thrombocytopenia. APPROACH AND RESULTS: LPS-induced mortality is significantly decreased in mice expressing a functionally deficient mutant of GPIbα. Furthermore, we have developed a micellar peptide inhibitor, MPαC (C13H27CONH-SIRYSGHpSL), which selectively inhibits the von Willebrand factor -binding function of GPIb-IX and GPIb-IX-mediated platelet adhesion under flow without affecting GPIb-IX-independent platelet activation. MPαC inhibits platelet adhesion to LPS-stimulated endothelial cells in vitro and alleviates LPS-induced thrombosis in glomeruli in mice. Importantly, MPαC reduces mortality in LPS-challenged mice, suggesting a protective effect of this inhibitor during endotoxemia. Interestingly, MPαC, but not the integrin antagonist, Integrilin, alleviated LPS-induced thrombocytopenia. CONCLUSIONS: These data indicate an important role for the platelet adhesion receptor GPIb-IX in LPS-induced thrombosis and thrombocytopenia, and suggest the potential of targeting GPIb as an antiplatelet strategy in managing endotoxemia.

Omeprazole and PGC-formulated heparin binding epidermal growth factor normalizes fasting blood glucose and suppresses insulitis in multiple low dose streptozotocin diabetes model.

PURPOSE: Our objective was to develop novel nanocarriers (protected graft copolymer, PGC) that improve the stability of heparin binding EGF (HBEGF) and gastrin and then to use PGC-formulated HBEGF (PGC-HBEGF) and Omeprazole (+/- PGC-gastrin) for normalizing fasting blood glucose (FBG) and improving islet function in diabetic mice. METHODS: HBEGF, PGC-HBEGF, Omeprazole, Omeprazole + PGC-HBEGF, Omeprazole + PGC-gastrin + PGC-HBEGF and epidermal growth factor (EGF) + gastrin were tested in multiple low dose streptozotocin diabetic mice. RESULTS: Omeprazole + PGC-HBEGF normalized FBG and is better than EGF + gastrin at improving islet function and decreasing insulitis. Groups treated with Omeprazole, Omeprazole + PGC-HBEGF, or EGF + gastrin have significantly improved islet function versus saline control. All animals that received PGC-HBEGF had significantly reduced islet insulitis versus saline control. Non-FBG was lower for Omeprazole + PGC-gastrin + PGC-HBEGF but Omeprazole + PGC-HBEGF alone showed better FBG and glucose tolerance. CONCLUSIONS: Omeprazole + PGC-HBEGF provides a sustained exposure to both EGFRA and gastrin, improves islet function, and decreases insulitis in multiple low dose streptozotocin diabetic mice. Although HBEGF or EGF elevates non-FBG, it facilitates a reduction of insulitis and, in the presence of Omeprazole, provides normalization of FBG at the end of treatment. The study demonstrates Omeprazole and PGC-HBEGF is a viable treatment for diabetes.

Enhanced oral bioavailability of the hydrophobic chemopreventive agent (SR13668) in beagle dogs.

Potency and activity of SR13668 in cancer prevention have been proven in several in vitro and in vivo cancer models. However, the compound is highly hydrophobic and its limited oral bioavailability has hindered its clinical translation. In this study, we encapsulated SR13668 into polymeric nanoparticles to increase compound aqueous solubility and therefore bioavailability. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles (100-200 nm) encapsulating SR13668 with narrow size distribution and high drug loading were generated by a continuous and scalable process of flash nanoprecipitation integrated with spray dry. A single gavage dose of SR13668-PLGA nanoparticles at 2.8 mg/kg was administered in eight beagle dogs. Drug levels in animal whole blood and plasma were measured over 24 hours. Enhanced bioavailability of SR13668 using nanoparticles compared with formulations of Labrasol® and neat drug in 0.5% methylcellulose is reported. This is the first attempt to study pharmacokinetics of SR13668 in large animals with orally administrated nanoparticle suspension.

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.

Combination treatment with bortezomib and thiostrepton is effective against tumor formation in mouse models of DEN/PB-induced liver carcinogenesis.

Nanoparticle-encapsulated thiazole antibiotic, thiostrepton, has been shown to be an effective agent for inhibiting tumor growth in solid tumor models through the inhibition of proteasomal activity by the induction of apoptosis in cancer cells. Here, we show the efficacy of thiostrepton-micelles in inhibiting tumor growth in a DEN/PB-induced liver cancer model. We also demonstrate an enhanced anticancer effect of the combination treatment of thiostrepton with bortezomib, another proteasome inhibitor in this liver cancer model.

Enhanced oral bioavailability of a cancer preventive agent (SR13668) by employing polymeric nanoparticles with high drug loading.

SR13668 [2,10-Dicarbethoxy-6-methoxy-5,7-dihydro-indolo-(2,3-b)carbazole] has been proven effective in cancer prevention, but the limited bioavailability has hindered its clinical translation. In this study, we have developed a continuous, scalable process to form stable poly(lactic-co-glycolic acid) nanoparticles encapsulating SR13668, based on understanding of the competitive kinetics of nanoprecipitation and spray drying. The optimized formulation achieved high drug loading (33.3 wt %) and small particles (150 nm) with narrow size distribution. The prepared nanoparticle suspensions through flash nanoprecipitation were spray dried to achieve long-term stability and to conveniently adjust the nanoparticle concentration before use. In vitro release of SR13668 from the nanosuspensions was measured in a solution with separated organic and aqueous phases to overcome the limit of SR13668 low water solubility. Higher oral bioavailability of SR13668 by employing polymeric nanoparticles compared with the Labrasol® formulation was demonstrated in a mouse model.

Effects of bacterial and presystemic nitroreductase metabolism of 2-chloro-5-nitro-N-phenylbenzamide on its mutagenicity and bioavailability.

2-Chloro-5-nitro-N-phenylbenzamide (GW9662), a potent irreversible PPAR-γ antagonist, has shown promise as a cancer chemopreventive agent and is undergoing preclinical evaluations. Studies were initiated to assess its bacterial mutagenicity and pharmacokinetic profile in two animal species prior to subchronic oral toxicity evaluations and the results are reported here. GW9662 was mutagenic in both TA98 and TA100 bacterial strains with and without metabolic activation but was negative in the nitroreductase-deficient strains (TA98NR and TA100NR) also with and without metabolic activation, indicating that GW9662 mutagenicity is dependent on nitroreduction. The mutagenic activity was predominantly via a base-substitution mechanism. Following oral dosing in rats and dogs, the parent compound, GW9662, was virtually absent from plasma samples, but there was chromatographic evidence for the presence of metabolites in the plasma as a result of oral dosing. Metabolite identification studies showed that an amine metabolite ACPB (5-amino-2-chloro-N-phenylbenzamide), a product of nitro reduction, was the predominant species exhibiting large and persistent plasma levels. Thus systemic circulation of GW9662 has been attained largely in the form of its reduced metabolite, probably a product of gut bacterial metabolism. GW9662 was detectable in plasma of rats and dogs after intravenous dose albeit at low concentrations. Pharmacokinetic analysis following intravenous dosing in rats showed a rapid clearance and an extensive tissue distribution which could have accounted for the very low plasma levels. Of note, the amine metabolite was absent following intravenous dosing in both rats and dogs, confirming it being a product of presystemic metabolism. The potential utility of GW9662 as a chemopreventive agent, especially as an Estrogen Receptor-α (ER-α) inducer in an otherwise ER-α negative breast tissue, is of great interest. However, the results shown here suggest that additional animal toxicological and bioavailability studies are required to establish a role of GW9662 as a chemopreventive agent.

Protected graft copolymer (PGC) basal formulation of insulin as potentially safer alternative to Lantus® (insulin-glargine): a streptozotocin-induced, diabetic Sprague Dawley rats study.

PURPOSE: To develop a long-acting formulation of native human insulin with a similar pharmacodynamics (PD) profile as the insulin analogue insulin glargine (Lantus®, Sanofi-Aventis) with the expectation of retaining native human insulin's superior safety profile as insulin glargine is able to activate the insulin-like growth factor 1 (IGF-1) receptor and is linked to a number of malignancies at a higher rate than regular human insulin. METHODS: Development of protected graft copolymer (PGC) excipients that bind native human insulin non-covalently and testing blood glucose control obtained with these formulations in streptozotocin-induced diabetic Sprague Dawley rats compared to equally dosed insulin glargine. RESULTS: PGC-formulations of native human insulin are able to control blood glucose to the same extent and for the same amount of time after s.c. injection as the insulin analogue insulin glargine. No biochemical changes were made to the insulin that would change receptor binding and activation with their possible negative effects on the safety of the insulin. CONCLUSION: Formulation with the PGC excipient offers a viable alternative to biochemically changing insulin or other receptor binding peptides to improve PD properties.

Assessment of oral toxicity and safety of pentamethylchromanol (PMCol), a potential chemopreventative agent, in rats and dogs.

2,2,5,7,8-Pentamethyl-6-chromanol (PMCol) was administered by gavage in rats for 28 days at dose levels of 0, 100, 500, and 2000mg/kg/day. PMCol administration induced decreases in body weight gains and food consumption, hepatotoxicity (increased TBILI, ALB, ALT, TP; increased relative liver weights; increased T4 and TSH), nephrotoxicity (increased BUN and BUN/CREAT, histopathology lesions), effect on lipid metabolism (increased CHOL), anemia, increase in WBC counts (total and differential), coagulation (FBGN upward arrow and PT downward arrow) and hyperkeratosis of the nonglandular stomach in the 2000mg/kg/day dose group (in one or both sexes). In the 500mg/kg/day dose group, toxicity was seen to a lesser extent. In the 100mg/kg/day dose group, only increased CHOL (females) was observed. To assess the toxicity of PMCol in male dogs it was administered orally by capsule administration for 28 days at dose levels of 0, 50, 200 and 800mg/kg/day (four male dogs/dose group). PMCol treatment at 800mg/kg/day resulted in pronounced toxicity to the male dogs. Target organs of toxicity were liver and thymus. Treatment at 200mg/kg/day resulted in toxicity consistent with slight adverse effect on the liver only. The results of the safety pharmacology study indicate that doses of 0, 50, 200 and 800mg/kg administered orally did not have an effect on the QT interval, blood pressures and body temperatures following dosing over a 24-h recording period. Under the conditions of this study, the no-observed-adverse effect level (NOAEL) for daily oral administration of PMCol by gavage for 28 days to male rats was 100mg/kg/day and 50mg/kg in male dogs. In female rats, the NOAEL was not established due to statistically significant and biologically meaningful increases in CHOL level seen in the 100mg/kg/day dose group. The results of these studies indicated that administration of PMCol at higher dose levels resulted in severe toxicity in dogs and moderate toxicity in rats, however, administration at lower levels is considered to be less likely to result in toxicity following 28 days of exposure. Sex-related differences were seen in rats. Male rats appeared to have greater sensitivity to nephrotoxicity, while female animals had a greater incidence of hepatoxicity and changes in hematological parameters evaluated, especially at a dose of 500mg/kg/day, which correlated to the higher plasma drug levels in female rats. It appeared that dogs were generally more sensitive than rats to oral administration of PMCol. Further examination of the potential toxic effects of PMCol in longer term studies is required prior to understanding the full risks of PMCol administration as a chemopreventative agent.

Evaluation of hydrogen ion concentrations in prostates from rats and dogs using fluorescent confocal microscopy.

The knowledge of intracellular spatial distribution of pH in prostates in animal models reflective of human prostate may have implications for drug development upon pH dependent drug delivery and activity. Freshly dissected prostate tissues (in vitro) or the entire prostate gland (in vivo) were loaded with fluorescent dyes and viewed using confocal microscopy. Images were initially taken in tissues perfused with RPMI-1640 medium. Calibration in situ was performed with high potassium buffers of known pH containing nigericin. Acetoxymethyl ester carboxy-SNARF-1 was visible in epithelial cells (but not stroma) in rat and dog prostates. The pH of lysosomes in prostate epithelial cells was 5.2 as determined by fluorescence of Lyso Sensor Green DND-189. A method of in situ confirmation of tissue viability was developed by a secondary loading and visualization of the BCECF fluorescent dye. Besides the direct measurement of the pH in rat and dog tissues (pH approximately 7.0), a method of pH measurement in prostate tissue (rather than in cell culture) was developed.

Plasma pharmacokinetics, oral bioavailability, and interspecies scaling of the DNA methyltransferase inhibitor, zebularine.

PURPOSE: Zebularine is a DNA methyltransferase inhibitor proposed for clinical evaluation. EXPERIMENTAL DESIGN: We developed a liquid chromatography/mass spectrometry assay and did i.v. and oral studies in mice, rats, and rhesus monkeys. RESULTS: In mice, plasma zebularine concentrations declined with terminal half-lives (t(1/2)) of 40 and 91 minutes after 100 mg/kg i.v. and 1,000 mg/kg given orally, respectively. Zebularine plasma concentration versus time curves (area under the curve) after 100 mg/kg i.v. and 1,000 mg/kg given orally were 7,323 and 4,935 mug/mL min, respectively, corresponding to a total body clearance (CL(tb)) of 13.65 mL/min/kg, apparent total body clearance (CL(app)) of 203 mL/min/kg, and oral bioavailability of 6.7%. In rats, plasma zebularine concentrations declined with t(1/2) of 363, 110, and 126 minutes after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally, respectively. Zebularine areas under the curve after 50 mg/kg i.v., 250 mg/kg given orally, and 500 mg/kg given orally were 12,526, 1,969, and 7,612 mug/mL min, respectively, corresponding to a CL(tb) of 3.99 mL/min/kg for 50 mg/kg i.v. and CL(app) of 127 and 66 mL/min/kg for 250 and 500 mg/kg given orally, respectively. Bioavailabilities of 3.1% and 6.1% were calculated for the 250 and 500 mg/kg oral doses, respectively. In monkeys, zebularine t(1/2) was 70 and 150 minutes, CL(tb) was 3.55 and 10.85 mL/min/kg after i.v. administration, and CL(app) was 886 and 39,572 mL/min/kg after oral administration of 500 and 1,000 mg/kg, respectively. Zebularine oral bioavailability was <1% in monkeys. Interspecies scaling produced the following relationship: CL(tb) = 6.46(weight(0.9)). CONCLUSIONS: Zebularine has limited oral bioavailability. Interspecies scaling projects a CL(tb) of 296 mL/min in humans.