Potent Uncompetitive Inhibitors of Nicotinamide N-Methyltransferase (NNMT) as In Vivo Chemical Probes.

NNMT uses SAM as a cofactor to catalyze the methylation of nicotinamide, producing 1-methylnicotinamide. Recent studies have shown that NNMT upregulation in cancer-associated fibroblasts (CAFs) is required to maintain the CAF phenotype in high-grade serous carcinoma. These observations suggest that NNMT should be evaluated as a therapeutic target, especially in cancer. Although several small-molecule inhibitors of NNMT have been identified, there remains a need for highly potent and selective inhibitors with excellent in vivo activity and ADME properties that can be used as reliable chemical probes. We have identified azaindoline carboxamide 38 as a selective and potent NNMT inhibitor with favorable PK/PD and safety profiles as well as excellent oral bioavailability and pharmaceutical properties. Our mechanistic studies indicate that 38 binds uncompetitively with SAM but competitively with nicotinamide consistent with its binding in the nicotinamide binding site and likely forming a positive interaction with SAM.

Identification of genes for normalization of quantitative real-time PCR data in ovarian tissues.

Increased attention has been paid to the determination of the potential biomarker and therapeutic target for ovarian cancer in recent years. However, the normalization of quantitative real-time PCR is important to obtain accurate gene expression data. We investigated the stability of 20 reference genes in ovarian tissues under different conditions to determine the most adequate for this application. The study characterized the expression of 20 possible reference genes among 52 ovarian tissue samples involving the normal, non-malignant, and primary ovarian carcinomas. One-way analysis of variance (ANOVA) method was used to compare the candidate gene changes brought about by the disease progression. The stability and suitability of the genes with no statistic difference were further validated employing geNorm and NormFinder softwares. Results showed that the expression levels of the 20 reference genes varied, while the RPL4, RPLP0, HSPCB, TPT1, RPL13A, 18S rRNA, PPIA, TBP, and GUSB kept statistic stability despite different ovarian tissue conditions. RPL4, RPLP0, and HSPCB were demonstrated as the most stable reference genes and the combination of the RPLP0 and RPL4 should be recommended as a much more reliable normalization strategy.

Applicability of free drug hypothesis to drugs with good membrane permeability that are not efflux transporter substrates: A microdialysis study in rats.

In clinical pharmacology, the free drug hypothesis has been widely applied in the interpretation of the relationship between pharmacokinetics and pharmacodynamics (PK/PD). The free drug hypothesis assumes that the unbound drug concentration in blood is the same as that in the site of action at steady state. The objective of this study is to demonstrate whether the free drug hypothesis is universally applicable for all drugs. The unbound concentrations of the 18 compounds in blood and in brain interstitial fluids (ISF) at steady state following constant intravenous infusion were simultaneously monitored up to 6 hours via in vivo microdialysis technique. Based on the permeability and efflux ratio (ER), the test compounds can be divided into two classes. Class I includes the compounds with good membrane permeability that are not substrates of efflux transporters (eg, P-gp, BCRP, and MRPs), whereas Class II includes the compounds that are substrates of efflux transporters. The steady-state unbound drug concentrations in blood, brain, and CSF are quantitatively very similar for Class I compounds, whereas the steady-state unbound concentrations in the brain and CSF are significantly lower than those in blood for Class II compounds. These results strongly suggest that the free drug hypothesis is not universal for all drugs but is only applicable for drugs with good permeability that are not substrates of efflux transporters.

Heparanase DNA vaccine delivered by electroporation induces humoral immunity and cytoimmunity in animal models.

Electroporation (EP)-assisted DNA vaccination has been proven effective as an approach to the treatment of cancer. Although heparanase (HPA) is a potential target for patients with advanced tumor diseases, the efficacy of immunotherapeutic strategies targeting HPA has never been evaluated. In this study, humoral immunity was elicited using genetic vaccinations between C57BL/6J mice and Macaca fascicularis. The immunized serum neutralized HPA activity and attenuated the invasion of B16 cells in vitro. In addition, T lymphocytes from the splenic cells of the immunized mice induced HPA-specific cytotoxic lymphocytes (CTLs), which verified cytoimmunity. Prophylactic vaccination significantly suppressed tumor growth and metastasis in vivo and prolonged the survival rate in tumor-bearing murine models. In addition, RT-PCR and Western blot analyses of the primary tumors indicated less proliferation and angiogenesis and more apoptosis in the HPA-immunization immunotherapy groups. Simultaneously, the levels of IL-2, IL-4, and IFN-γ were not significantly greater in the HPA-immunized group than in PBS controls. Thus, we conclude that the combination of an anti-HPA antibody and a CTL response in HPA-immunization gene therapy is enough to attenuate tumor growth and metastasis. This is the first time that a DNA vaccine targeting HPA immunization assisted by EP has induced humoral immunity and cytoimmunity in vivo. This provides a basis for the continued development of DNA vaccines targeting HPA and the use of such vaccines in clinical settings.