Differential effects of two phosphodiesterase 4 inhibitors against lipopolysaccharide-induced neuroinflammation in mice.

BACKGROUND: Several phosphodiesterase 4 (PDE4) inhibitors have emerged as potential therapeutics for central nervous system (CNS) diseases. This study investigated the pharmacological effects of two selective PDE4 inhibitors, roflumilast and zatolmilast, against lipopolysaccharide-induced neuroinflammation. RESULTS: In BV-2 cells, the PDE4 inhibitor roflumilast reduced the production of nitric oxide and tumor necrosis factor-α (TNF-α) by inhibiting NF-κB phosphorylation. Moreover, mice administered roflumilast had significantly reduced TNF-α, interleukin-1ß (IL-1ß), and IL-6 levels in plasma and brain tissues. By contrast, zatolmilast, a PDE4D inhibitor, showed no anti-neuroinflammatory effects in vitro or in vivo. Next, in vitro and in vivo pharmacokinetic studies of these compounds in the brain were performed. The apparent permeability coefficients of 3 µM roflumilast and zatolmilast were high (> 23 × 10-6 cm/s) and moderate (3.72-7.18 × 10-6 cm/s), respectively, and increased in a concentration-dependent manner in the MDR1-MDCK monolayer. The efflux ratios were < 1.92, suggesting that these compounds are not P-glycoprotein substrates. Following oral administration, both roflumilast and zatolmilast were slowly absorbed and eliminated, with time-to-peak drug concentrations of 2-2.3 h and terminal half-lives of 7-20 h. Assessment of their brain dispositions revealed the unbound brain-to-plasma partition coefficients of roflumilast and zatolmilast to be 0.17 and 0.18, respectively. CONCLUSIONS: These findings suggest that roflumilast, but not zatolmilast, has the potential for use as a therapeutic agent against neuroinflammatory diseases.

Integrative Analyses Reveal the Anticancer Mechanisms and Sensitivity Markers of the Next-Generation Hypomethylating Agent NTX-301.

Epigenetic dysregulation characterized by aberrant DNA hypermethylation is a hallmark of cancer, and it can be targeted by hypomethylating agents (HMAs). Recently, we described the superior therapeutic efficacy of a novel HMA, namely, NTX-301, when used as a monotherapy and in combination with venetoclax in the treatment of acute myeloid leukemia. Following a previous study, we further explored the therapeutic properties of NTX-301 based on experimental investigations and integrative data analyses. Comprehensive sensitivity profiling revealed that NTX-301 primarily exerted anticancer effects against blood cancers and exhibited improved potency against a wide range of solid cancers. Subsequent assays showed that the superior efficacy of NTX-301 depended on its strong effects on cell cycle arrest, apoptosis, and differentiation. Due to its superior efficacy, low doses of NTX-301 achieved sufficiently substantial tumor regression in vivo. Multiomics analyses revealed the mechanisms of action (MoAs) of NTX-301 and linked these MoAs to markers of sensitivity to NTX-301 and to the demethylation activity of NTX-301 with high concordance. In conclusion, our findings provide a rationale for currently ongoing clinical trials of NTX-301 and will help guide the development of novel therapeutic options for cancer patients.

Pharmacokinetics and Tissue Distribution of 13C-Labeled Succinic Acid in Mice.

Succinic acid is widely used as a food additive, and its effects on sepsis, cancer, ataxia, and obesity were recently reported. Dietary drug exposure studies have been conducted to evaluate the in vivo efficacy of succinic acid, but limited pharmacokinetic information is available. Therefore, this study evaluated the pharmacokinetic profiles and tissue distribution of succinic acid following a single intravenous or oral dose. A surrogate analyte, succinic acid-13C4 (13C4SA), was administrated to distinguish endogenous and exogenous succinic acid. The concentration of 13C4SA was determined by a validated analytical method using mass spectrometry. After a 10 mg/kg intravenous dose, non-compartmental pharmacokinetic analysis in plasma illustrated that the clearance, volume of distribution, and terminal half-life of 13C4SA were 4574.5 mL/h/kg, 520.8 mL/kg, and 0.56 h, respectively. Oral 13C4SA was absorbed and distributed quickly (bioavailability, 1.5%) at a dose of 100 mg/kg. In addition, 13C4SA exposure was the highest in the liver, followed by brown adipose tissue, white adipose tissue, and the kidneys. This is the first report on the pharmacokinetics of succinic acid after a single dose in mice, and these results could provide a foundation for selecting dosing regimens for efficacy studies.

Pharmacokinetic Characterization of Supinoxin and Its Physiologically Based Pharmacokinetic Modeling in Rats.

Supinoxin is a novel anticancer drug candidate targeting the Y593 phospho-p68 RNA helicase, by exhibiting antiproliferative activity and/or suppression of tumor growth. This study aimed to characterize the in vitro and in vivo pharmacokinetics of supinoxin and attempt physiologically based pharmacokinetic (PBPK) modeling in rats. Supinoxin has good permeability, comparable to that of metoprolol (high permeability compound) in Caco-2 cells, with negligible net absorptive or secretory transport observed. After an intravenous injection at a dose range of 0.5-5 mg/kg, the terminal half-life (i.e., 2.54-2.80 h), systemic clearance (i.e., 691-865 mL/h/kg), and steady state volume of distribution (i.e., 2040-3500 mL/kg) of supinoxin remained unchanged, suggesting dose-independent (i.e., dose-proportional) pharmacokinetics for the dose ranges studied. After oral administration, supinoxin showed modest absorption with an absolute oral bioavailability of 56.9-57.4%. The fecal recovery following intravenous and oral administration was 16.5% and 46.8%, respectively, whereas the urinary recoveries in both administration routes were negligible. Supinoxin was mainly eliminated via NADPH-dependent phase I metabolism (i.e., 58.5% of total clearance), while UDPGA-dependent phase II metabolism appeared negligible in the rat liver microsome. Supinoxin was most abundantly distributed in the adipose tissue, gut, and liver among the nine major tissues studied (i.e., the brain, liver, kidneys, heart, lungs, spleen, gut, muscles, and adipose tissue), and the tissue exposure profiles of supinoxin were well predicted with physiologically based pharmacokinetics.

Discovery of a Novel Chemical Scaffold Against Mutant Isocitrate Dehydrogenase 1 (IDH1).

BACKGROUND: Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are frequently found in various cancer types. IDH1 mutants produce 2-hydroxyglutarate (2-HG), an oncometabolite, from alpha-ketoglutarate (α-KG). This 2-HG plays a key role in tumorigenesis via inhibition of α-KG dependent enzymes. For this reason, IDH1 mutant could be an ideal target for the treatment of cancer. MATERIALS AND METHODS: To find a new IDH1 inhibitor, 8,364 compounds were obtained from Korea Chemical Bank. Using high-throughput screening (HTS) of a chemical library, we unveiled a compound that could inhibit the IDH1 mutant. RESULTS: According to the enzyme assay, our compound (KRC-09) effectively inhibited the activity of IDH1 R132H mutant. In addition, KRC-09 decreased the concentration of intracellular 2-HG in the U-87 MG cell line harboring IDH1 R132H. CONCLUSION: In this article, we present a novel chemical scaffold that suppresses the activity of an IDH1 mutant.

Design and characterization of cereblon-mediated androgen receptor proteolysis-targeting chimeras.

Proteolysis-targeting chimera (PROTAC)-mediated protein degradation is a rapidly emerging therapeutic intervention that induces the degradation of targeted proteins. Herein, we report the design and biological evaluation of a series of androgen receptor (AR) PROTAC degraders for the treatment of metastatic castration-resistant prostate cancer. Predominantly, instead of thalidomide, we utilized the TD-106 scaffold, a novel cereblon (CRBN) binder that was identified in our previous study. Our results suggest that the linker position in the TD-106 CRBN binder is critical for the efficiency of AR degradation. The compounds attached to the 6-position of TD-106 promoted better degradation of AR than those at the 5- and 7-positions. Among the synthesized AR PROTACs, the representative degrader 33c (TD-802) effectively induced AR protein degradation, with a degradation concentration 50% of 12.5 nM and a maximum degradation of 93% in LNCaP prostate cancer cells. Additionally, most AR PROTAC degraders, including TD-802, displayed good liver microsomal stability and in vivo pharmacokinetic properties. Finally, we showed that TD-802 effectively inhibited tumor growth in an in vivo xenograft study.

αVß3-Targeted Delivery of Camptothecin-Encapsulated Carbon Nanotube-Cyclic RGD in 2D and 3D Cancer Cell Culture.

Integrin αvß3 is widely expressed in various types of human cancer lines and plays a key role in angiogenesis for tumor growth and metastasis. Delivery of therapeutics to αvß3-expressing tumors can thus be a promising approach for treating cancer. For targeted delivery of anticancer therapeutics to αvß3-expressing tumor cells, cyclic arginylglycylaspartic acid (RGD) peptide was covalently conjugated to the surface of carboxylic acid-functionalized carbon nanotubes (fCNTs), and the topoisomerase I inhibitor camptothecin (CPT) was encapsulated in the fCNTs (CPT@fCNT-RGD). CPT@fCNT-RGD was successfully delivered to αvß3-expressing A375 cells, and compared with nontargeted CPT@fCNT, it provided 3.78- and 3.02-fold increases in the anticancer effect in 2D and 3D culture. Analysis of apoptosis-related gene expression shows that the expression levels of Bax, cleaved caspase-3, and nuclear factor kappa-light-chain-enhancer of activated B cells were significantly increased in A375 cells incubated with CPT@fCNT-RGD compared with those incubated with CPT@fCNT. These results suggest that cyclic RGD-conjugated CNTs encapsulating an anticancer therapeutic can be a promising platform for treating cancer.

Encapsulation and Enhanced Delivery of Topoisomerase I Inhibitors in Functionalized Carbon Nanotubes.

The topoisomerase I inhibitors SN-38 and camptothecin (CPT) have shown potent anticancer activity, but water insolubility and metabolic instability limits their clinical application. Utilizing carbon nanotubes as a protective shell for water-insoluble SN-38 and CPT while maintaining compatibility with aqueous media via a carboxylic acid-functionalized surface can thus be a strategy to overcome this limitation. Through hydrophobic-hydrophobic interactions, SN-38 and CPT were successfully encapsulated in carboxylic acid functionalized single-walled carbon nanotubes and dispersed in water. The resulting cell proliferation inhibition and drug distribution profile inside the cells suggest that these drug-encapsulated carbon nanotubes can serve as a promising delivery strategy for water-insoluble anticancer drugs.

Pharmacokinetics of tafamidis, a transthyretin amyloidosis drug, in rats.

1. We characterized the pharmacokinetics of tafamidis, a novel drug to treat transthyretin-related amyloidosis, in rats after intravenous and oral administration at doses of 0.3-3 mg/kg. In vitro Caco-2 cell permeability and liver microsomal stability, as well as in vivo tissue distribution and plasma protein binding were also examined. 2. After intravenous injection, systemic clearance (CL), volumes of distribution at steady state (Vss) and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 6.41-7.03 mL/h/kg, 270-354 mL/kg and 39.5-46.9 h, respectively. Following oral administration, absolute bioavailability was 99.7-104% and was independent of doses from 0.3 to 3 mg/kg. In the urine and faeces, 4.36% and 48.9% of tafamidis, respectively, were recovered. 3. Tafamidis was distributed primarily in the liver and not in the brain, kidney, testis, heart, spleen, lung, gut, muscle, or adipose tissue. Further, tafamidis was very stable in rat liver microsomes, and its plasma protein binding was 99.9%. 4. In conclusion, tafamidis showed dose-independent pharmacokinetics with intravenous and oral doses of 0.3-3 mg/kg. Tafamidis undergoes minimal first-pass metabolism, distributes mostly in the liver and plasma, and appears to be eliminated primarily via biliary excretion.

Synthesis and Molecular Modeling Studies of N'-Hydroxyindazolecarboximidamides as Novel Indoleamine 2,3-Dioxygenase 1 (IDO1) Inhibitors.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme that is highly overexpressed in various cancer cells and antigen-presenting cells. It has emerged as an attractive therapeutic target for cancer immunotherapy, which has prompted high interest in the development of small-molecule inhibitors. To discover novel IDO1 inhibitors, we designed and synthesized a series of N'-hydroxyindazolecarboximidamides. Among the compounds synthesized, compound 8a inhibited both tryptophan depletion and kynurenine production through the IDO1 enzyme. Molecular docking studies revealed that 8a binds to IDO1 with the same binding mode as the analog, epacadostat (INCB24360). Here, we report the synthesis and biological evaluation of these hydroxyindazolecarboximidamides and present the molecular docking study of 8a with IDO1.

Replacing the terminal piperidine in ceritinib with aliphatic amines confers activities against crizotinib-resistant mutants including G1202R.

The piperidine fragment in ceritinib was replaced with diverse aliphatic amines to improve inherent resistance issues of ceritinib. While most of the prepared compounds exhibit as similar in vitro activities as ceritinib, compound 10 shows encouraging activities against wild-type ALK as well as crizotinib-resistant mutants including extremely resistant G1202R mutant with an IC50 of 1.8 nM. Furthermore, pharmacokinetic profiles of 10 is apparently better than that of ceritinib. In murine xenograft studies, compound 10 turns out to be as active as ceritinib, suggesting that further optimization of 10 may lead to clinical candidates overcoming ALK mutant issues.

Minor modifications to ceritinib enhance anti-tumor activity in EML4-ALK positive cancer.

Ceritinib, an ALK inhibitor, was hurriedly approved by the US FDA last year, and demonstrates impressive results in EML4-ALK positive patients. To get a superior ALK inhibitor, we synthesized several ceritinib derivatives with minor modifications to the phenylpiperidine moiety. Biochemical and cellular assays demonstrated the improved activity of KRCA-386 over that of ceritinib. KRCA-386 has superior inhibitory activity against ALK mutants commonly found in crizotinib-resistant patients. Particularly, KRCA-386 has considerably greater activity than ceritinib against the G1202R mutant, one of the most challenging mutations to overcome. The cell cycle analysis indicates that ALK inhibitors induce G1/S arrest, resulting in apoptosis. The in vivo xenograft data also demonstrate that KRCA-386 is significantly better than ceritinib. KRCA-386 dosed at 25 mpk caused 105% tumor growth inhibition (TGI) compared to 72% TGI with ceritinib dosed at 25 mpk. (n = 8, P = 0.010) The kinase profiling assay revealed that several kinases, which are known to be critical for tumor growth, are inhibited by KRCA-386, but not by ceritinib. We anticipate that this characteristic of KRCA-386 enhances its in vivo efficacy. In addition, KRCA-386 shows excellent blood brain barrier penetration compared to ceritinib. These results suggest that KRCA-386 could be useful for crizotinib-resistant patients with brain metastases.

Novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety against anaplastic lymphoma kinase (ALK): Synthesis, in vitro, ex vivo, and in vivo efficacy studies.

A series of novel 2,4-diaminopyrimidines bearing tetrahydronaphthalenyl moiety were synthesized and evaluated for their anti-anaplastic lymphoma kinase (ALK) activities using enzymatic and cell-based assays. Among the compounds synthesized, compound 17b showed promising pharmacological results in in vitro, ex vivo, and pharmacokinetic studies. An in vivo efficacy study with compound 17b demonstrated highly potent inhibitory activity in H3122 tumor xenograft model mice. A series of kinase assays showed that compound 17b inhibited various kinases including FAK, ACK1, FGFR, RSK1, IGF-1R, among others, thus demonstrating its potential for synergistic anti-tumor activity and development as a multi-targeted non-small cell lung cancer (NSCLC) therapy.

Discovery of novel tetrahydroisoquinoline-containing pyrimidines as ALK inhibitors.

Exploration of the two-position side chain of pyrimidine in LDK378 with tetrahydroisoquinolines (THIQs) led to discovery of 8 and 17 as highly potent ALK inhibitors. THIQs 8 and 17 showed encouraging in vitro and in vivo xenograft efficacies, comparable with those of LDK378. Although THIQ analogs (8a-o and 17a-i) prepared were not as active as their parent compounds, both 8 and 17 have significant inhibitory activities against various ALK mutant enzymes including G1202R, indicating that this series of compounds could be further optimized as useful ALK inhibitors overcoming the resistance issues found from crizotinib and LDK378.

Synthesis and evaluation of novel 2,4-diaminopyrimidines bearing bicyclic aminobenzazepines for anaplastic lymphoma kinase (ALK) inhibitor.

A series of novel 2,4-diaminopyrimidine compounds bearing bicyclic aminobenzazepine were synthesized and evaluated for their anti-ALK activities. The activities of these compounds were confirmed in both enzyme- and cell-based ALK assays. Amongst compounds synthesized, KRCA-0445 showed very promising results in pharmacokinetic study and in vivo efficacy study with H3122 xenograft mouse model.

Pharmacokinetics of enzalutamide, an anti-prostate cancer drug, in rats.

We characterized the pharmacokinetics of enzalutamide, a novel anti-prostate cancer drug, in rats after intravenous and oral administration in the dose range 0.5-5 mg/kg. Tissue distribution, liver microsomal stability, and plasma protein binding were also examined. After intravenous injection, systemic clearance, volumes of distribution at steady state (Vss), and half-life (T½) remained unaltered as a function of dose, with values in the ranges of 80.4-86.3 mL/h/kg, 1020-1250 mL/kg, and 9.13-10.6 h, respectively. Following oral administration, absolute oral bioavailability was 89.7 % and not dose-dependent. The recoveries of enzalutamide in urine and feces were 0.0620 and 2.04 %, respectively. Enzalutamide was distributed primarily in 10 tissues (brain, liver, kidneys, testis, heart, spleen, lungs, gut, muscle, and adipose) and tissue-to-plasma ratios of enzalutamide ranged from 0.406 (brain) to 10.2 (adipose tissue). Further, enzalutamide was stable in rat liver microsomes, and its plasma protein binding was 94.7 %. In conclusion, enzalutamide showed dose-independent pharmacokinetics at intravenous and oral doses of 0.5-5 mg/kg. Enzalutamide distributed primarily to 10 tissues and appeared to be eliminated primarily by metabolism.

Design, synthesis, and biological evaluation of stable colchicine binding site tubulin inhibitors as potential anticancer agents.

To block the metabolically labile sites of novel tubulin inhibitors targeting the colchicine binding site based on SMART, ABI, and PAT templates, we have designed, synthesized, and biologically tested three focused sets of new derivatives with modifications at the carbonyl linker, the para-position in the C ring of SMART template, and modification of A ring of the PAT template. Structure-activity relationships of these compounds led to the identification of new benzimidazole and imidazo[4,5-c]pyridine-fused ring templates, represented by compounds 4 and 7, respectively, which showed enhanced antitumor activity and substantially improved the metabolic stability in liver microsomes compared to SMART. MOM group replaced TMP C ring and generated a potent analogue 15, which showed comparable potency to the parent SMART compound. Further modification of PAT template yielded another potent analogue 33 with 5-indolyl substituent at A ring.

Selective androgen receptor modulators (SARMs) negatively regulate triple-negative breast cancer growth and epithelial:mesenchymal stem cell signaling.

INTRODUCTION: The androgen receptor (AR) is the most highly expressed steroid receptor in breast cancer with 75-95% of estrogen receptor (ER)-positive and 40-70% of ER-negative breast cancers expressing AR. Though historically breast cancers were treated with steroidal androgens, their use fell from favor because of their virilizing side effects and the emergence of tamoxifen. Nonsteroidal, tissue selective androgen receptor modulators (SARMs) may provide a novel targeted approach to exploit the therapeutic benefits of androgen therapy in breast cancer. MATERIALS AND METHODS: Since MDA-MB-453 triple-negative breast cancer cells express mutated AR, PTEN, and p53, MDA-MB-231 triple-negative breast cancer cells stably expressing wildtype AR (MDA-MB-231-AR) were used to evaluate the in vitro and in vivo anti-proliferative effects of SARMs. Microarray analysis and epithelial:mesenchymal stem cell (MSC) co-culture signaling studies were performed to understand the mechanisms of action. RESULTS: Dihydrotestosterone and SARMs, but not bicalutamide, inhibited the proliferation of MDA-MB-231-AR. The SARMs reduced the MDA-MB-231-AR tumor growth and tumor weight by greater than 90%, compared to vehicle-treated tumors. SARM treatment inhibited the intratumoral expression of genes and pathways that promote breast cancer development through its actions on the AR. SARM treatment also inhibited the metastasis-promoting paracrine factors, IL6 and MMP13, and subsequent migration and invasion of epithelial:MSC co-cultures. CONCLUSION: 1. AR stimulation inhibits paracrine factors that are important for MSC interactions and breast cancer invasion and metastasis. 2. SARMs may provide promise as novel targeted therapies to treat AR-positive triple-negative breast cancer.

Direct endothelial junction restoration results in significant tumor vascular normalization and metastasis inhibition in mice.

Tumor blood vessels are leaky and immature, which causes inadequate blood supply to tumor tissues resulting in hypoxic microenvironment and promotes metastasis. Here we have explored tumor vessel modulating activity of Sac-1004, a recently developed molecule in our lab, which directly potentiates VE-cadherin-mediated endothelial cell junction. Sac-1004 could enhance vascular junction integrity in tumor vessels and thereby inhibit vascular leakage and enhance vascular perfusion. Improved perfusion enabled Sac-1004 to have synergistic anti-tumor effect on cisplatin-mediated apoptosis of tumor cells. Interestingly, characteristics of normalized blood vessels namely reduced hypoxia, improved pericyte coverage and decreased basement membrane thickness were readily observed in tumors treated with Sac-1004. Remarkably, Sac-1004 was also able to inhibit lung and lymph node metastasis in MMTV and B16BL6 tumor models. This was in correlation with a reduction in epithelial-to-mesenchymal transition of tumor cells with considerable diminution in expression of related transcription factors. Moreover, cancer stem cell population dropped substantially in Sac-1004 treated tumor tissues. Taken together, our results showed that direct restoration of vascular junction could be a significant strategy to induce normalization of tumor blood vessels and reduce metastasis.

Discovery of 4-Aryl-2-benzoyl-imidazoles as tubulin polymerization inhibitor with potent antiproliferative properties.

A series of 4-aryl-2-benzoyl-imidazoles were designed and synthesized based on our previously reported 2-aryl-4-benzoyl-imidazole (ABI) derivatives. The new structures reversed the aryl group and the benzoyl group of previous ABI structures and were named as reverse ABI (RABI) analogues. RABIs were evaluated for biological activity against eight cancer cell lines including multidrug-resistant cancer cell lines. In vitro assays indicated that several RABI compounds had excellent antiproliferative properties, with IC50 values in the low nanomolar range. The average IC50 of the most active compound 12a is 14 nM. In addition, the mechanism of action of these new analogues was investigated by cell cycle analysis, tubulin polymerization assay, competitive mass spectrometry binding assay, and molecular docking studies. These studies confirmed that these new RABI analogues maintain their mechanisms of action by disrupting tubulin polymerization, similar to their parental ABI analogues.