Property-Driven Design and Development of Lipids for Efficient Delivery of siRNA.

Ionizable cationic lipids are critical components involved in nanoparticle formulations, which are utilized in delivery platforms for RNA therapeutics. While general criteria regarding lipophilicity and measured pKa in formulation are understood to have impacts on utility in vivo, greater granularity with respect to the impacts of the structure on calculated and measured physicochemical parameters and the subsequent performance of those ionizable cationic lipids in in vivo studies would be beneficial. Herein, we describe structural alterations made within a lipid class exemplified by 4, which allow us to tune calculated and measured physicochemical parameters for improved performance, resulting in substantial improvements versus the state of the art at the outset of these studies, resulting in good in vivo activity within a range of measured basicity (pKa = 6.0-6.6) and lipophilicity (cLogD = 10-14).

Immune modulatory nanoparticle therapeutics for intracerebral glioma.

Background: Previously we showed therapeutic efficacy of unprotected miR-124 in preclinical murine models of glioblastoma, including in heterogeneous genetically engineered murine models by exploiting the immune system and thereby negating the need for direct tumor delivery. Although these data were promising, to implement clinical trials, we required a scalable formulation that afforded protection against circulatory RNases. Methods: We devised lipid nanoparticles that encapsulate and protect the miRs from degradation and provide enhanced delivery into the immune cell compartment and tested in vivo antitumor effects. Results: Treatment with nanoparticle-encapsulated miR-124, LUNAR-301, demonstrated a median survival exceeding 70 days, with an associated reversal of tumor-mediated immunosuppression and induction of immune memory. In both canine and murine models, the safety profile of LUNAR-301 was favorable. Conclusions: For the first time, we show that nanoparticles can direct a therapeutic response by targeting intracellular immune pathways. Although shown in the context of gliomas, this therapeutic approach would be applicable to other malignancies.

Pharmacological characterization of KLYP961, a dual inhibitor of inducible and neuronal nitric-oxide synthases.

Nitric oxide (NO) derived from neuronal nitric-oxide synthase (nNOS) and inducible nitric-oxide synthase (iNOS) plays a key role in various pain and inflammatory states. KLYP961 (4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one) inhibits the dimerization, and hence the enzymatic activity of human, primate, and murine iNOS and nNOS (IC(50) values 50-400 nM), with marked selectivity against endothelial nitric-oxide synthase (IC(50) >15,000 nM). It has ideal drug like-properties, including excellent rodent and primate pharmacokinetics coupled with a minimal off-target activity profile. In mice, KLYP961 attenuated endotoxin-evoked increases in plasma nitrates, a surrogate marker of iNOS activity in vivo, in a sustained manner (ED(50) 1 mg/kg p.o.). KLYP961 attenuated pain behaviors in a mouse formalin model (ED(50) 13 mg/kg p.o.), cold allodynia in the chronic constriction injury model (ED(50) 25 mg/kg p.o.), or tactile allodynia in the spinal nerve ligation model (ED(50) 30 mg/kg p.o.) with similar efficacy, but superior potency relative to gabapentin, pregabalin, or duloxetine. Unlike morphine, the antiallodynic activity of KLYP961 did not diminish upon repeated dosing. KLYP961 also attenuated carrageenin-induced edema and inflammatory hyperalgesia and writhing response elicited by phenylbenzoquinone with efficacy and potency similar to those of celecoxib. In contrast to gabapentin, KLYP961 did not impair motor coordination at doses as high as 1000 mg/kg p.o. KLYP961 also attenuated capsaicin-induced thermal allodynia in rhesus primates in a dose-related manner with a minimal effective dose (≤ 10 mg/kg p.o.) and a greater potency than gabapentin. In summary, KLYP961 represents an ideal tool with which to probe the physiological role of NO derived from iNOS and nNOS in human pain and inflammatory states.

Discovery of dual inducible/neuronal nitric oxide synthase (iNOS/nNOS) inhibitor development candidate 4-((2-cyclobutyl-1H-imidazo[4,5-b]pyrazin-1-yl)methyl)-7,8-difluoroquinolin-2(1H)-one (KD7332) part 2: identification of a novel, potent, and selective series of benzimidazole-quinolinone iNOS/nNOS dimerization inhibitors that are orally active in pain models.

Three isoforms of nitric oxide synthase (NOS), dimeric enzymes that catalyze the formation of nitric oxide (NO) from arginine, have been identified. Inappropriate or excessive NO produced by iNOS and/or nNOS is associated with inflammatory and neuropathic pain. Previously, we described the identification of a series of amide-quinolinone iNOS dimerization inhibitors that although potent, suffered from high clearance and limited exposure in vivo. By conformationally restricting the amide of this progenitor series, we describe the identification of a novel series of benzimidazole-quinolinone dual iNOS/nNOS inhibitors with low clearance and sustained exposure in vivo. Compounds were triaged utilizing an LPS challenge assay coupled with mouse and rhesus pharmacokinetics and led to the identification of 4,7-imidazopyrazine 42 as the lead compound. 42 (KD7332) (J. Med. Chem. 2009, 52, 3047 - 3062) was confirmed as an iNOS dimerization inhibitor and was efficacious in the mouse formalin model of nociception and Chung model of neuropathic pain, without showing tolerance after repeat dosing. Further 42 did not affect motor coordination up to doses of 1000 mg/kg, demonstrating a wide therapeutic margin.

KLYP956 is a non-imidazole-based orally active inhibitor of nitric-oxide synthase dimerization.

Nitric-oxide synthases (NOS) generate nitric oxide (NO) through the oxidation of l-arginine. Inappropriate or excessive production of NO by NOS is associated with the pathophysiology of various disease states. Efforts to treat these disorders by developing arginine mimetic, substrate-competitive NOS inhibitors as drugs have met with little success. Small-molecule-mediated inhibition of NOS dimerization represents an intriguing alternative to substrate-competitive inhibition. An ultra-high-throughput cell-based screen of 880,000 small molecules identified a novel quinolinone with inducible NOS (iNOS) inhibitory activity. Exploratory chemistry based on this initial screening hit resulted in the synthesis of KLYP956, which inhibits iNOS at low nanomolar concentrations. The iNOS inhibitory potency of KLYP956 is insensitive to changes in concentrations of the substrate arginine, or the cofactor tetrahydrobiopterin. Mechanistic analysis suggests that KLYP956 binds the oxygenase domain in the vicinity of the active site heme and inhibits iNOS and neuronal NOS (nNOS) by preventing the formation of enzymatically active dimers. Oral administration of KLYP956 [N-(3-chlorophenyl)-N-((8-fluoro-2-oxo-1,2-dihydroquinolin-4-yl)methyl)-4-methylthiazole-5-carboxamide] inhibits iNOS activity in a murine model of endotoxemia and blocks pain behaviors in a formalin model of nociception. KLYP956 thus represents the first nonimidazole-based inhibitor of iNOS and nNOS dimerization and provides a novel pharmaceutical alternative to previously described substrate competitive inhibitors.

Discovery of inducible nitric oxide synthase (iNOS) inhibitor development candidate KD7332, part 1: Identification of a novel, potent, and selective series of quinolinone iNOS dimerization inhibitors that are orally active in rodent pain models.

There are three isoforms of dimeric nitric oxide synthases (NOS) that convert arginine to citrulline and nitric oxide. Inducible NOS is implicated in numerous inflammatory diseases and, more recently, in neuropathic pain states. The majority of existing NOS inhibitors are either based on the structure of arginine or are substrate competitive. We describe the identification from an ultra high-throughput screen of a novel series of quinolinone small molecule, nonarginine iNOS dimerization inhibitors. SAR studies on the screening hit, coupled with an in vivo lipopolysaccharide (LPS) challenge assay measuring plasma nitrates and drug levels, rapidly led to the identification of compounds 12 and 42--potent inhibitors of the human and mouse iNOS enzyme that were highly selective over endothelial NOS (eNOS). Following oral dosing, compounds 12 and 42 gave a statistical reduction in pain behaviors in the mouse formalin model, while 12 also statistically reduced neuropathic pain behaviors in the chronic constriction injury (Bennett) model.

Identification of KD5170: a novel mercaptoketone-based histone deacetylase inhibitor.

We report the identification of KD5170, a potent mercaptoketone-based Class I and II-histone deacetylase inhibitor that demonstrates broad spectrum cytotoxic activity against a range of human tumor-derived cell lines. KD5170 exhibits robust and sustained histone H3 hyperacetylation in HCT-116 xenograft tumors following single oral or i.v. dose and inhibition of tumor growth following chronic dosing.

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor, exerts antimyeloma effects by DNA damage and mitochondrial signaling.

Histone deacetylase inhibitors have emerged as promising anticancer drugs. Using an unbiased ultrahigh throughput screening system, a novel mercaptoketone-based histone deacetylase inhibitor series was identified that was optimized to the lead compound, KD5170. KD5170 inhibited the proliferation of myeloma cell lines and the viability of CD138(+) primary myeloma cells by induction of apoptosis, accompanied by an increase of acetylation of histones and activation of caspase-3, caspase-8, and caspase-9. Treatment with KD5170 caused a loss of mitochondrial membrane potential resulting in release of apoptogenic factors such as cytochrome c, Smac, and apoptosis-inducing factor. Furthermore, KD5170 induced oxidative stress and oxidative DNA damage in myeloma cells as evidenced by the up-regulation of heme oxygenase-1 and H2A.X phosphorylation. Combination of KD5170 with proteasome inhibitor bortezomib or tumor necrosis factor-related apoptosis-inducing ligand synergistically enhanced the antimyeloma activity. We further found that resistance of myeloma cells to KD5170 was associated with activation of the extracellular signal-regulated kinase/mitogen-activated protein kinase pathway under treatment with KD5170. Pretreatment with the mitogen-activated protein kinase inhibitor U0126 restored sensitivity to KD5170, suggesting that the combination of KD5170 with U0126 could overcome drug resistance. Growth of myeloma tumor xenografts in KD5170-treated nude mice was significantly inhibited and survival was prolonged. Histone acetylation was increased in spleen and tumor tissues of animals treated with KD5170. Our data indicate that KD5170 has potent antimyeloma activity in vitro and in vivo, which is mediated by DNA damage and mitochondrial signaling and subsequent induction of apoptosis.

KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity in vitro and in vivo.

Histone deacetylase (HDAC) inhibitors have garnered significant attention as cancer drugs. These therapeutic agents have recently been clinically validated with the market approval of vorinostat (SAHA, Zolinza) for treatment of cutaneous T-cell lymphoma. Like vorinostat, most of the small-molecule HDAC inhibitors in clinical development are hydroxamic acids, whose inhibitory activity stems from their ability to coordinate the catalytic Zn2+ in the active site of HDACs. We sought to identify novel, nonhydroxamate-based HDAC inhibitors with potentially distinct pharmaceutical properties via an ultra-high throughput small molecule biochemical screen against the HDAC activity in a HeLa cell nuclear extract. An alpha-mercaptoketone series was identified and chemically optimized. The lead compound, KD5170, exhibits HDAC inhibitory activity with an IC50 of 0.045 micromol/L in the screening biochemical assay and an EC50 of 0.025 micromol/L in HeLa cell-based assays that monitor histone H3 acetylation. KD5170 also exhibits broad spectrum classes I and II HDAC inhibition in assays using purified recombinant human isoforms. KD5170 shows significant antiproliferative activity against a variety of human tumor cell lines, including the NCI-60 panel. Significant tumor growth inhibition was observed after p.o. dosing in human HCT-116 (colorectal cancer), NCI-H460 (non-small cell lung carcinoma), and PC-3 (prostate cancer) s.c. xenografts in nude mice. In addition, a significant increase in antitumor activity and time to end-point occurred when KD5170 was combined with docetaxel in xenografts of the PC-3 prostate cancer cell line. The biological and pharmaceutical profile of KD5170 supports its continued preclinical and clinical development as a broad spectrum anticancer agent.

Novel approach to pro-drugs of lactones: water soluble imidate and ortho-ester derivatives of a furanone-based COX-2 selective inhibitor.

Interest in water soluble COX-2 inhibitors that can be administered intravenously led to the development of novel pro-drugs of a furanone based COX-2 inhibitor 2. Transforming the lactone moiety of the furanone to an imidate or an ortho-ester with a hydrophilic, endogenous appendage resulted in water soluble pro-drugs that converted to the parent drug in vivo.