Supplementary Methods from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Fig. S2 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Table S1 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity <i>in vitro</i> and <i>in vivo</i>

Supplementary Fig. S2 from KD5170, a novel mercaptoketone-based histone deacetylase inhibitor that exhibits broad spectrum antitumor activity in vitro and in vivo

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).

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.

Abstract INTRODUCTION: We have previously shown robust therapeutic efficacy of miRNAs in preclinical murine models of glioblastoma and were one of the first groups to deliver therapeutic miRNAs intravenously. However a major hurdle to clinical translation is a scalable formulation that affords protection against circulatory RNAses. Nanoparticles can encapsulate and protect the miRNA from degradation and enhance delivery into the immune cell compartment facilitating antitumor effects, in part through the reversal of tumor-mediate immune suppression and increased expression of effector cytokines - thus, overcoming the need for direct tumor delivery of the therapeutic agent. METHODS: FDA acceptable lipid nanoparticles were devised to enhance delivery of miRNA into the peripheral blood mononuclear cells (PBMCs) and verified by in vivo compartmental pharmacokinetic analysis and functional immune monitoring. Nanoparticle test articles contain an active immune modulatory agent - miR-124, which inhibits the signal transducer and activator of transcript 3 (STAT3) pathway. The lead candidate was designated LUNAR-301, and further refinements included unlocking the nucleic acids (LUNAR-302) to enhance efficacy. Nanoparticle formulations were tested in multiple murine models of malignancy including established intracerebral gliomas. RESULTS: In non-tumor bearing mice dosed with intravenous LUNAR-301, miR-124 was delivered to the peripheral blood mononuclear cells (PBMCs) with no clinical signs of toxicity or organ damage on histopathologic exam. In an intracerebral GL261 model, lower pSTAT3 expression was observed in mice treated with LUNAR-301 compared to both empty nanoparticle treated mice or untreated mice, p = 0.0081 and p = 0.0001 respectively. Similarly, lower Foxp3 expression was observed in the LUNAR-301 treated mice, p = 0.0057 and p = 0.0223 respectively. Median survival time for mice treated with LUNAR-301 exceeded 70 days, compared to only 32.5 days for mice treated with the previous gold-standard, miR-124 + lipofectamine. The cure rate difference between LUNAR-301 (9 out of 15 mice) and LUNAR-302 (2 out of 10 mice) was 40% (P = 0.0576); the difference in cure rates between LUNAR-301 and miR-124 + lipofectamine (4 out of 16 mice) was 35% (P = 0.0532). In a subcutaneous murine model of melanoma, tumor growth rate per day without treatment was 44% (i.e., tumor volume was expected to increase 44% cumulatively on a daily basis), while it was reduced to 26.1% in the LUNAR-301-treated group (P = 0.007), and to 16.2% in the LUNAR-302-treated group (P<0.001). CONCLUSIONS: Nanoparticle delivery of miR-124 has a favorable safety and efficacy profile to justify implementation in client-owned canines or human clinical trials for the treatment of gliomas. Citation Format: Nasser K. Yaghi, Jun Wei, Ling-Yuan Kong, Yuuri Hashimoto, Edjah K. Nduom, Neal Huang, Xiaoyang Ling, Shouhao Zhou, Jonathan M. Levine, Virginia R. Fajt, Kiyoshi Tachikawa, Padmanabh Chivukula, David C. Webb, Joseph E. Payne, Amy B. Heimberger. An optimized therapeutic nanoparticle delivery platform of miRNA in preclinical murine models of malignancy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4291. doi:10.1158/1538-7445.AM2015-4291

INTRODUCTION: Melanoma patients that develop brain metastasis have a median survival of 6 months. Radiation, a standard of care for local control of melanoma, has been reported to have immune stimulatory capacities. Therefore, we investigated the combinatorial effect of inhibitors of the signal transducer and activator of transcript 3 (STAT3) pathway, a potent mediator of immune suppression, and whole-brain radiation in a murine brain metastasis model. METHODS: C57BL/6 mice were intracerebrally implanted with B16 melanoma, irradiated with 7.5 Gy, and treated with an advanced lipid nanoparticle system that contained miR-124 (LNP-124) that inhibits the STAT3 pathway, or WP1066, a small molecule inhibitor of STAT3. Mice surviving 130+ days after tumor implantation were subcutaneously rechallenged with tumor. RESULTS: Monotherapy (LNP-124, WP1066, or radiation alone) demonstrated longer survival relative to untreated control mice; combination therapies further extended survival compared to single treatment agents. Median survival for mice receiving radiation combined with LNP124 or WP1066 was 65 days and 45.5 days, respectively, compared to 15 days for untreated mice and 19 - 40 days for monotherapy. While no untreated mice (control) survived over 20 days, the survival rate at day 130 for mice receiving combination therapy was almost 40%. Flow cytometry analysis showed higher IL-2 production in T cells from tumor-draining lymph nodes in mice treated with radiation plus STAT3 inhibitor. SUMMARY: These results indicate the combination of STAT3 inhibition and radiation enhances the therapeutic effect against established murine brain metastasis. The implications for this combination on antitumor immunity, immunologic memory and protection from recurrence will also be discussed.

La presente invention concerne des composes de formule I : R 1 et R 2 etant independamment choisis dans un groupe constitue d'un groupe alkyle en C 10 a C 18 , alcenyle en C 12 a C 18 , et oleyle ; R 3 et R 4 etant independamment choisis dans un groupe constitue d'un alkyle en C 1 a C 6 , et un alcanol en C 2 a C 6 ; X etant choisi dans un groupe constitue de -CH 2 -, -S-, et -O- ou absent ; Y etant choisi parmi -(CH 2 ) n , -S(CH 2 ) n , -O(CH 2 ) n -, thiophene, -SO 2 (CH 2 ) n -, et ester, ou n = 1 a 4 ; ou a = 1 a 4 ; ou b = 1 a 4 ; ou c = 1 a 4 ; et ou Z est un contre-ion ; et des composes constitues de la structure (molecule de ciblage) m -lieur-(molecule de ciblage) n , la molecule de ciblage etant un retinoide ou une vitamine liposoluble ayant un recepteur specifique sur la cellule cible ; m et n etant independamment 0, 1, 2 ou 3 ; et le lieur comprenant une molecule de polyethyleneglycol (PEG) ou similaire a PEG, ainsi que des compositions et des formulations pharmaceutiques comprenant l'un ou les deux de ces composes qui sont utiles pour l'administration d'agents therapeutiques ; et des procedes d'utilisation de ces compositions et formulations pharmaceutiques.

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.