Oxycodone withdrawal induces HDAC1/HDAC2-dependent transcriptional maladaptations in the reward pathway in a mouse model of peripheral nerve injury.

The development of physical dependence and addiction disorders due to misuse of opioid analgesics is a major concern with pain therapeutics. We developed a mouse model of oxycodone exposure and subsequent withdrawal in the presence or absence of chronic neuropathic pain. Oxycodone withdrawal alone triggered robust gene expression adaptations in the nucleus accumbens, medial prefrontal cortex and ventral tegmental area, with numerous genes and pathways selectively affected by oxycodone withdrawal in mice with peripheral nerve injury. Pathway analysis predicted that histone deacetylase (HDAC) 1 is a top upstream regulator in opioid withdrawal in nucleus accumbens and medial prefrontal cortex. The novel HDAC1/HDAC2 inhibitor, Regenacy Brain Class I HDAC Inhibitor (RBC1HI), attenuated behavioral manifestations of oxycodone withdrawal, especially in mice with neuropathic pain. These findings suggest that inhibition of HDAC1/HDAC2 may provide an avenue for patients with chronic pain who are dependent on opioids to transition to non-opioid analgesics.

Safety, Tolerability, and Pharmacokinetics of Single Ascending Doses of ELX-02, a Potential Treatment for Genetic Disorders Caused by Nonsense Mutations, in Healthy Volunteers.

ELX-02 is an investigational synthetic eukaryotic ribosome-selective glycoside optimized as a translational read-through molecule that induces read through of nonsense mutations, resulting in normally localized full-length functional proteins. ELX-02 is being developed as a therapy for genetic diseases caused by nonsense mutations. Two phase 1a, randomized, double-blind placebo-controlled, single-ascending-dose studies were conducted in healthy human subjects to evaluate the safety and pharmacokinetics of ELX-02. Single subcutaneously injected doses of ELX-02 between 0.3 mg/kg and 7.5 mg/kg showed an acceptable safety profile without severe or serious drug-related adverse events, including a lack of renal and ototoxicity events. Injection of ELX-02 resulted in a rapid time to peak concentration and elimination phase, with complete elimination from the vascular compartment within 10 hours. ELX-02 area under the concentration-time curve to infinity showed dose-exposure linearity (24-fold increase for a 25-fold dose increase), and the maximum concentration showed dose proportionality (17-fold increase for a 25-fold increase). The mean apparent volume of distribution was dose dependent, suggesting an increased distribution and tissue uptake of ELX-02 at higher doses. The primary route of excretion was in the urine, with the majority of the compound excreted unchanged. These results support the evaluation of the safety, pharmacokinetics, and efficacy of repeat dosing in future studies.

HDAC6 inhibition effectively reverses chemotherapy-induced peripheral neuropathy.

Chemotherapy-induced peripheral neuropathy is one of the most common dose-limiting side effects of cancer treatment. Currently, there is no Food and Drug Administration-approved treatment available. Histone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase whose function includes regulation of α-tubulin-dependent intracellular mitochondrial transport. Here, we examined the effect of HDAC6 inhibition on established cisplatin-induced peripheral neuropathy. We used a novel HDAC6 inhibitor ACY-1083, which shows 260-fold selectivity towards HDAC6 vs other HDACs. Our results show that HDAC6 inhibition prevented cisplatin-induced mechanical allodynia, and also completely reversed already existing cisplatin-induced mechanical allodynia, spontaneous pain, and numbness. These findings were confirmed using the established HDAC6 inhibitor ACY-1215 (Ricolinostat), which is currently in clinical trials for cancer treatment. Mechanistically, treatment with the HDAC6 inhibitor increased α-tubulin acetylation in the peripheral nerve. In addition, HDAC6 inhibition restored the cisplatin-induced reduction in mitochondrial bioenergetics and mitochondrial content in the tibial nerve, indicating increased mitochondrial transport. At a later time point, dorsal root ganglion mitochondrial bioenergetics also improved. HDAC6 inhibition restored the loss of intraepidermal nerve fiber density in cisplatin-treated mice. Our results demonstrate that pharmacological inhibition of HDAC6 completely reverses all the hallmarks of established cisplatin-induced peripheral neuropathy by normalization of mitochondrial function in dorsal root ganglia and nerve, and restoration of intraepidermal innervation. These results are especially promising because one of the HDAC6 inhibitors tested here is currently in clinical trials as an add-on cancer therapy, highlighting the potential for a fast clinical translation of our findings.

Treatment with a selective histone deacetylase 6 inhibitor decreases lupus nephritis in NZB/W mice.

To date, there are 18 histone deacetylase (HDAC) enzymes, divided into four classes, which alter protein function by removing acetyl groups from lysine residues. Prior studies report that non-selective HDAC inhibitors decrease disease in lupus mouse models. Concern for adverse side effects of non-selective HDAC inhibition supports investigation of selective-HDAC inhibition. We hypothesized that a selective HDAC-6 inhibitor (HDAC6i) will alleviate disease in a mouse model of lupus by increasing acetylation of alpha-tubulin. Intraperitoneal injections of the selective HDAC6i ACY-1083 (0.3 mg/kg, 1 mg/kg, or 3 mg/kg), vehicle control, or dexamethasone were administered to 21-week-old, female NZB/W mice, 5 days a week, for 13 weeks. Disease progression was evaluated by proteinuria, serum levels of anti-dsDNA antibody, cytokines and immunoglobulins, and post mortem evaluation of nephritis and T cell populations in the spleen. HDAC6i treatment decreased proteinuria, glomerular histopathology, IgG, and C3 scores when compared to vehicle-treated mice. Within glomeruli of HDAC6i-treated mice, there was increased acetylation of alpha-tubulin and decreased NF-κB. Additionally, HDAC6i decreased serum IL-12/IL-23 and Th17 cells in the spleen. Taken together, these results suggest HDAC-6 inhibition may decrease lupus nephritis in NZB/W mice via mechanisms involving acetylation of alpha-tubulin and decreased NF-κB in glomeruli as well as inhibition of Th17 cells.

Selective Inhibitors of Histone Deacetylases 1 and 2 Synergize with Azacitidine in Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous group of hematopoietic stem cell disorders characterized by defects in myeloid differentiation and increased proliferation of neoplastic hematopoietic precursor cells. Outcomes for patients with AML remain poor, highlighting the need for novel treatment options. Aberrant epigenetic regulation plays an important role in the pathogenesis of AML, and inhibitors of DNA methyltransferase or histone deacetylase (HDAC) enzymes have exhibited activity in preclinical AML models. Combination studies with HDAC inhibitors plus DNA methyltransferase inhibitors have potential beneficial clinical activity in AML, however the toxicity profiles of non-selective HDAC inhibitors in the combination setting limit their clinical utility. In this work, we describe the preclinical development of selective inhibitors of HDAC1 and HDAC2, which are hypothesized to have improved safety profiles, for combination therapy in AML. We demonstrate that selective inhibition of HDAC1 and HDAC2 is sufficient to achieve efficacy both as a single agent and in combination with azacitidine in preclinical models of AML, including established AML cell lines, primary leukemia cells from AML patient bone marrow samples and in vivo xenograft models of human AML. Gene expression profiling of AML cells treated with either an HDAC1/2 inhibitor, azacitidine, or the combination of both have identified a list of genes involved in transcription and cell cycle regulation as potential mediators of the combinatorial effects of HDAC1/2 inhibition with azacitidine. Together, these findings support the clinical evaluation of selective HDAC1/2 inhibitors in combination with azacitidine in AML patients.

Selective HDAC inhibition by ACY-241 enhances the activity of paclitaxel in solid tumor models.

ACY-241 is a novel, orally available and selective histone deacetylase (HDAC) 6 inhibitor in Phase 1b clinical development in multiple myeloma (NCT 02400242). Like the structurally related drug ACY-1215 (ricolinostat), ACY-241 has the potential for a substantially reduced side effect profile versus current nonselective HDAC inhibitor drug candidates due to reduced potency against Class I HDACs while retaining the potential for anticancer effectiveness. We now show that combination treatment of xenograft models with paclitaxel and either ricolinostat or ACY-241 significantly suppresses solid tumor growth. In cell lines from multiple solid tumor lineages, combination treatment with ACY-241 and paclitaxel enhanced inhibition of proliferation and increased cell death relative to either single agent alone. Combination treatment with ACY-241 and paclitaxel also resulted in more frequent occurrence of mitotic cells with abnormal multipolar spindles and aberrant mitoses, consistent with the observed increase of aneuploid cells. At the molecular level, multipolar mitotic spindle formation was observed to be NuMA-dependent and γ-tubulin independent, suggesting that treatment-induced multipolar spindle formation does not depend on centrosomal amplification. The significantly enhanced efficacy of ACY-241 plus paclitaxel observed here, in addition to the anticipated superior safety profile of a selective HDAC6 inhibitor versus pan-HDAC inhibitors, provides a strong rationale for clinical development of this combination in patients with advanced solid tumors.

Chemical Inhibition of Histone Deacetylases 1 and 2 Induces Fetal Hemoglobin through Activation of GATA2.

Therapeutic intervention aimed at reactivation of fetal hemoglobin protein (HbF) is a promising approach for ameliorating sickle cell disease (SCD) and ß-thalassemia. Previous studies showed genetic knockdown of histone deacetylase (HDAC) 1 or 2 is sufficient to induce HbF. Here we show that ACY-957, a selective chemical inhibitor of HDAC1 and 2 (HDAC1/2), elicits a dose and time dependent induction of γ-globin mRNA (HBG) and HbF in cultured primary cells derived from healthy individuals and sickle cell patients. Gene expression profiling of erythroid progenitors treated with ACY-957 identified global changes in gene expression that were significantly enriched in genes previously shown to be affected by HDAC1 or 2 knockdown. These genes included GATA2, which was induced greater than 3-fold. Lentiviral overexpression of GATA2 in primary erythroid progenitors increased HBG, and reduced adult ß-globin mRNA (HBB). Furthermore, knockdown of GATA2 attenuated HBG induction by ACY-957. Chromatin immunoprecipitation and sequencing (ChIP-Seq) of primary erythroid progenitors demonstrated that HDAC1 and 2 occupancy was highly correlated throughout the GATA2 locus and that HDAC1/2 inhibition led to elevated histone acetylation at well-known GATA2 autoregulatory regions. The GATA2 protein itself also showed increased binding at these regions in response to ACY-957 treatment. These data show that chemical inhibition of HDAC1/2 induces HBG and suggest that this effect is mediated, at least in part, by histone acetylation-induced activation of the GATA2 gene.

Antidepressant-like properties of novel HDAC6-selective inhibitors with improved brain bioavailability.

HDAC inhibitors have been reported to produce antidepressant and pro-cognitive effects in animal models, however, poor brain bioavailability or lack of isoform selectivity of current probes has limited our understanding of their mode of action. We report the characterization of novel pyrimidine hydroxyl amide small molecule inhibitors of HDAC6, brain bioavailable upon systemic administration. We show that two compounds in this family, ACY-738 and ACY-775, inhibit HDAC6 with low nanomolar potency and a selectivity of 60- to 1500-fold over class I HDACs. In contrast to tubastatin A, a reference HDAC6 inhibitor with similar potency and peripheral activity, but more limited brain bioavailability, ACY-738 and ACY-775 induce dramatic increases in α-tubulin acetylation in brain and stimulate mouse exploratory behaviors in novel, but not familiar environments. Interestingly, despite a lack of detectable effect on histone acetylation, we show that ACY-738 and ACY-775 share the antidepressant-like properties of other HDAC inhibitors, such as SAHA and MS-275, in the tail suspension test and social defeat paradigm. These effects of ACY-738 and ACY-775 are directly attributable to the inhibition of HDAC6 expressed centrally, as they are fully abrogated in mice with a neural-specific loss of function of HDAC6. Furthermore, administered in combination, a behaviorally inactive dose of ACY-738 markedly potentiates the anti-immobility activity of a subactive dose of the selective serotonin reuptake inhibitor citalopram. Our results validate new isoform-selective probes for in vivo pharmacological studies of HDAC6 in the CNS and reinforce the viability of this HDAC isoform as a potential target for antidepressant development.

Preclinical activity, pharmacodynamic, and pharmacokinetic properties of a selective HDAC6 inhibitor, ACY-1215, in combination with bortezomib in multiple myeloma.

Histone deacetylase (HDAC) enzymatic activity has been linked to the transcription of DNA in cancers including multiple myeloma (MM). Therefore, HDAC inhibitors used alone and in combination are being actively studied as novel therapies in MM. In the present study, we investigated the preclinical activity of ACY-1215, an HDAC6-selective inhibitor, alone and in combination with bortezomib in MM. Low doses of ACY-1215 combined with bortezomib triggered synergistic anti-MM activity, resulting in protracted endoplasmic reticulum stress and apoptosis via activation of caspase-3, caspase-8, and caspase-9 and poly (ADP) ribosome polymerase. In vivo, the anti-MM activity of ACY-1215 in combination with bortezomib was confirmed using 2 different xenograft SCID mouse models: human MM injected subcutaneously (the plasmacytoma model) and luciferase-expressing human MM injected intravenously (the disseminated MM model). Tumor growth was significantly delayed and overall survival was significantly prolonged in animals treated with the combination therapy. Pharmacokinetic data showed peak plasma levels of ACY-1215 at 4 hours after treatment coincident with an increase in acetylated α-tubulin, a marker of HDAC6 inhibition, by immunohistochemistry and Western blot analysis. These studies provide preclinical rationale for acetylated α-tubulin use as a pharmacodynamic biomarker in future clinical trials.

Rifalazil and derivative compounds show potent efficacy in a mouse model of H. pylori colonization.

The rifamycin rifalazil (RFZ), and derivatives (NCEs) were efficacious in a mouse model of Helicobacter pylori colonization. Select NCEs were more active in vitro and showed greater efficacy than RFZ. A systemic component contributes to efficacy.

Rifalazil and other benzoxazinorifamycins in the treatment of chlamydia-based persistent infections.

Rifalazil is a benzoxazinorifamycin which inhibits bacterial DNA-dependent RNA polymerase. The benzoxazine ring endows benzoxazinorifamycins with unique physical and chemical characteristics which favor the use of rifalazil and derivatives in treating diseases caused by the obligate intracellular pathogens of the genus chlamydia. Minimal inhibitory concentrations of benzoxazinorifamycins against chlamydia are in the pg/mL range. These compounds have potential as monotherapeutic agents to treat chlamydia-associated disease because they retain activity against chlamydia strains resistant to currently approved rifamycins such as rifampin. A pivotal clinical trial with rifalazil has been initiated for the treatment of peripheral arterial disease. The rationale for this innovative use of rifalazil, including the association of C. pneumoniae in atherosclerotic plaque formation, as well as rifalazil's potency and efficacy against chlamydia in both preclinical and clinical studies, is discussed. Other benzoxazino derivatives may have utility as stand-alone topical antibacterials or combination antibacterials to treat serious Gram-positive infections. None of the benzoxazinorifamycins examined to date induce the cytochrome P450 3A4 enzyme. This is in contrast to currently approved rifamycins which are strong inducers of P450 enzymes, resulting in drug-drug interactions that limit the clinical utility of this drug class.

Efficacy of novel rifamycin derivatives against rifamycin-sensitive and -resistant Staphylococcus aureus isolates in murine models of infection.

Novel rifamycins (new chemical entities [NCEs]) having MICs of 0.002 to 0.03 microg/ml against Staphylococcus aureus and retaining some activity against rifampin-resistant mutants were tested for in vivo efficacy against susceptible and rifampin-resistant strains of S. aureus. Rifalazil and rifampin had a 50% effective dose (ED50) of 0.06 mg/kg of body weight when administered as a single intravenous (i.v.) dose in a murine septicemia model against a susceptible strain of S. aureus. The majority of NCEs showed efficacy at a lower i.v. dose (0.003 to 0.06 mg/kg). In addition, half of the NCEs tested for oral efficacy had ED50s in the range of 0.015 to 0.13 mg/kg, i.e., lower or equivalent to the oral ED50s of rifampin and rifalazil. NCEs were also tested in the septicemia model against a rifampin-resistant strain of S. aureus. Twenty-four of 169 NCEs were efficacious when administered as a single oral dose of 80 mg/kg. These NCEs were examined in the murine thigh infection model against a susceptible strain of S. aureus. Several NCEs dosed by intraperitoneal injection at 0.06 mg/kg caused a significant difference in bacterial titer compared with placebo-treated animals. No NCEs showed efficacy in the thigh model against a highly rifampin-resistant strain. However, several NCEs showed an effect when tested against a partially rifampin-resistant strain. The NCEs having a 25-hydroxyl moiety were more effective as a group than their 25-O-acetyl counterparts. These model systems defined candidate NCEs as components of potential combination therapies to treat systemic infections or as monotherapeutic agents for topical applications.

In vitro activity of novel rifamycins against rifamycin-resistant Staphylococcus aureus.

We describe novel rifamycin derivatives (new chemical entities [NCEs]) that retain significant activity against a comprehensive collection of Staphylococcus aureus strains that are resistant to rifamycins. This collection of resistant strains contains 21 of the 26 known single-amino-acid alterations in RpoB, the target of rifamycins. Some NCEs also demonstrated a lower frequency of resistance development than rifampin and rifalazil in S. aureus as measured in a resistance emergence test. When assayed for activity against the strongest rifamycin-resistant mutants, several NCEs had MICs of 2 microg/ml, in contrast to MICs of rifampin and rifalazil, which were 512 microg/ml for the same strains. The properties of these NCEs therefore demonstrate a significant improvement over those of earlier rifamycins, which have been limited primarily to combination therapy due to resistance development, and suggest a potential use of these NCEs for monotherapy in several clinical indications.

Discovery of potent poly(ADP-ribose) polymerase-1 inhibitors from the modification of indeno[1,2-c]isoquinolinone.

Novel indeno[1,2-c]isoquinolinone derivatives were synthesized and evaluated as inhibitors of the nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1). These potent nonmutagenic PARP-1 inhibitors possess an additional five-membered ring between the B and C rings of 6(5H)-phenanthridinone. The most potent PARP-1 inhibitors were obtained from the substitution of the D ring at the C-9 position, in particular sulfonamide and N-acyl analogues (6 and 11). The 9-sulfonamide analogues 11a and 12a exhibited IC(50) values of 1 and 10 nM, respectively.

Activities of rifamycin derivatives against wild-type and rpoB mutants of Chlamydia trachomatis.

Rifalazil, a semisynthetic rifamycin, was shown previously to have exceptional potency against Chlamydia trachomatis (MIC of 0.00025 microg/ml). We therefore tested 250 additional rifamycin derivatives and identified 12 with activities that are eightfold more potent than that of rifalazil. These compounds also showed exceptional activities against rifampin-resistant strains that carry missense mutations in the rpoB gene. The antimicrobial potency and intracellular penetration of these agents suggest their potential in treatment of chlamydial infections.

Preclinical pharmacology of lumiracoxib: a novel selective inhibitor of cyclooxygenase-2.

1. This manuscript presents the preclinical profile of lumiracoxib, a novel cyclooxygenase-2 (COX-2) selective inhibitor. 2. Lumiracoxib inhibited purified COX-1 and COX-2 with K(i) values of 3 and 0.06 microM, respectively. In cellular assays, lumiracoxib had an IC(50) of 0.14 microM in COX-2-expressing dermal fibroblasts, but caused no inhibition of COX-1 at concentrations up to 30 microM (HEK 293 cells transfected with human COX-1). 3. In a human whole blood assay, IC(50) values for lumiracoxib were 0.13 microM for COX-2 and 67 microM for COX-1 (COX-1/COX-2 selectivity ratio 515). 4. Lumiracoxib was rapidly absorbed following oral administration in rats with peak plasma levels being reached between 0.5 and 1 h. 5. Ex vivo, lumiracoxib inhibited COX-1-derived thromboxane B(2) (TxB(2)) generation with an ID(50) of 33 mg kg(-1), whereas COX-2-derived production of prostaglandin E(2) (PGE(2)) in the lipopolysaccharide-stimulated rat air pouch was inhibited with an ID(50) value of 0.24 mg kg(-1). 6. Efficacy of lumiracoxib in rat models of hyperalgesia, oedema, pyresis and arthritis was dose-dependent and similar to diclofenac. However, consistent with its low COX-1 inhibitory activity, lumiracoxib at a dose of 100 mg kg(-1) orally caused no ulcers and was significantly less ulcerogenic than diclofenac (P<0.05). 7. Lumiracoxib is a highly selective COX-2 inhibitor with anti-inflammatory, analgesic and antipyretic activities comparable with diclofenac, the reference NSAID, but with much improved gastrointestinal safety.

Part I: pathogenetic role of peroxynitrite in the development of diabetes and diabetic vascular complications: studies with FP15, a novel potent peroxynitrite decomposition catalyst.

BACKGROUND: Peroxynitrite is a cytotoxic oxidant formed from nitric oxide (NO) and superoxide. Tyrosine nitration, a footprint of peroxynitrite, has been demonstrated in the pancreatic islets as well as in the cardiovascular system of diabetic subjects. Delineation of the pathogenetic role of peroxynitrite in disease conditions requires the use of potent, in vivo active peroxynitrite decomposition catalysts. The aim of the current work was to produce a potent peroxynitrite decomposition catalyst and to test its effects in rodent models of diabetes and its complications. METHODS: FP15 was synthesized and analyzed using standard chemical methods. Diabetes was triggered by the administration of streptozotocin. Tyrosine nitration was measured immunohistochemically. Cardiovascular and vascular measurements were conducted according to standard physiologic methods. RESULTS: FP15, a potent porphyrinic peroxynitrite decomposition catalyst, potently inhibited tyrosine nitration and peroxynitrite-induced cytotoxicity in vitro and in vivo. FP15 treatment (3-10 mg/kg/d) dose dependently and reduced the incidence and severity of diabetes mellitus in rats subjected to multiple low doses of streptozotocin, as well as in nonobese mice developing spontaneous autoimmune diabetes. Furthermore, treatment with FP15 protected against the development of vascular dysfunction (loss of endothelium-dependent relaxations) and the cardiac dysfunction (loss of myocardial contractility) in diabetic mice. FP15 treatment reduced tyrosine nitration in the diabetic pancreatic islets. CONCLUSIONS: The current results demonstrate the importance of endogenous peroxynitrite generation in the pathogenesis of autoimmune diabetes and diabetic cardiovascular complications. Peroxynitrite decomposition catalysts may be of therapeutic utility in diabetes and other pathophysiologic conditions.