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Parenteral chemotherapy is usually administered intravenously, although patient preference and health economics suggest the subcutaneous (sc) route could be an attractive alternative. However, due to the low aqueous solubility of hydrophobic drugs and injection volume limitations, the total amount of drug that can be administered in a single sc injection is frequently insufficient. We have developed hyaluronidase coated nanoparticles (NPs) that efficiently encapsulate such drugs, thus addressing both issues and allowing sufficient amounts of hydrophobic drug to be administered and absorbed effectively. CUDC-101, a poorly water-soluble multitargeted anticancer drug that simultaneously inhibits the receptor tyrosine kinases (RTKs) EGFR and HER2, as well as histone deacetylase (HDAC), was encapsulated in polymeric Molecular Envelope Technology (MET) NPs. The role of polymer chemistry, formulation parameters, and coating with hyaluronidase (HYD) on MET-CUDC-101 NP formulations was examined and optimized to yield high drug loading and colloidal stability, and, after freeze-drying, stable storage at room temperature for up to 90 days. The pharmacokinetic studies in healthy rats showed that plasma AUC 0-24h after sc administration correlates tightly with formulation physical chemistry, specifically in vitro colloidal stability. Compared to uncoated NPs, the HYD-coating doubled the drug plasma exposure. In a murine A431 xenograft model, the coated HYD-MET-CUDC-101 NPs at a dose equivalent to 90 mg kg -1 CUDC-101 increased the survival time from 15 days (control animals treated with hyaluronidase alone) to 43 days. Polymer MET nanoparticles coated with hyaluronidase enabled the subcutaneous delivery of a hydrophobic drug with favorable therapeutic outcomes.

Chidamide is a new subtype-selective histone deacetylase inhibitor (HDACi), which has been approved for the treatment of recurrent or refractory peripheral T-cell lymphoma (PTCL) in China. However, there are few studies about the application of chidamide in PTCL with central nervous system (CNS) involvement. It is essential to investigate the penetration of chidamide in the blood brain barrier (BBB). LC-MS methods were established firstly to determine the concentration of chidamide in rat plasma and CSF. Then five rats were anaesthetized and the plasma and CSF samples were collected at the time of 5, 15, 30, 60, 120, 180, 240, 360 and 480 min after being administered 1 mg/kg chidamide by intravenous injection, respectively. All samples were analyzed with the established LC-MS method by using the precursor/product transitions (m/z) of 391.1/265.1 for chidamide and 441.1/138.2 for internal standard (IS). The PK parameters were calculated after both of the concentrations of chidamide in plasma and CSF were determined. The penetration ratio of chidamide in BBB ranged from 0.19% to 0.67%. Result indicated chidamide could pass through the BBB, enter into the CNS and have the potential to be utilized in PTCL with CNS involvement., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: This was an open label, phase I (3 + 3 design), multi-centre study evaluating panobinostat in pediatric patients with refractory solid tumors., Methods: Primary endpoints were to establish MTD, define and describe associated toxicities, including dose limiting toxicities (DLT) and to characterize its pharmacokinetics (PK). Secondary endpoints included assessing the anti-tumour activity of panobinostat, and its biologic activity, by measuring acetylation of histones in peripheral blood mononuclear cells., Results: Nine patients were enrolled and treated with intravenous panobinostat at a dosing level of 15 mg/m 2 which was tolerated. Six were evaluable for adverse events. Two (33%) patients experienced Grade 3-4 thrombocytopenia, 1 (17%) experienced Grade 3 anemia, and 2 (33%) experienced Grade 3 neutropenia. Grade 4 drug related pain occurred in 2 (33%) of the patients studied. Two (33%) patients experienced a Grade 2 QTcF change (0.478 ± 0.006 ms). One cardiac DLT (T wave changes) was reported. PK values for 15 mg/m 2 (n = 9) dosing were: T max 0.8 h, C max 235.2 ng/mL, AUC 0-t 346.8 h ng/mL and t 1/2 7.3 h. Panobinostat significantly induced acetylation of histone H3 and H4 at all time points measured when compared to pre-treatment samples (p < 0.05). Pooled quantitative Western blot data confirmed that panobinostat significantly induced acetylation of histone H4 at 6 h (p < 0.01), 24 h (p < 0.01) and 28-70 h (p < 0.01) post dose., Conclusion: A significant biological effect of panobinostat, measured by acetylation status of histone H3 and H4, was achieved at a dose of 15 mg/m 2 . PK data and drug tolerability at 15 mg/m 2 was similar to that previously published.

Purpose: Belinostat is a second-generation histone deacetylase inhibitor (HDI) predominantly metabolized by UGT1A1-mediated glucuronidation. Two common polymorphisms (UGT1A1*28 and UGT1A1*60) were previously associated with impaired drug clearance and thrombocytopenia risk, likely from increased drug exposure. This latter phenomenon has been observed with other HDIs such as abexinostat, panobinostat, romidepsin, and vorinostat. It was the intention of this brief report to expand a population pharmacokinetic (PPK) model to include a pharmacodynamic (PD) model describing the change in platelet levels in patients with cancer administered belinostat as a 48-h continuous intravenous infusion, along with cisplatin and etoposide., Methods: The PPK/PD model developed here introduced an additional rate constant to a commonly used mechanistic myelosuppression model to better describe the maturation of megakaryocytes into platelets before degradation and a feedback mechanism. The model employed a proportional error model to describe the observed circulating platelet data., Results: Several covariates were explored, including sex, body weight, UGT1A1 genotype status, liver, and kidney function, but none significantly improved the model. Platelet levels rebounded to baseline within 21 days, before the next cycle of therapy. Simulations predicted that higher belinostat drug exposure does cause lower thrombocyte nadirs compared to lower belinostat levels. However, platelet levels rebound by the start of the next belinostat cycle., Conclusions: This model suggests a q3week schedule allows for sufficient platelet recovery before the next belinostat infusion is optimal.

Alzheimer's disease (AD) is a dreadful neurodegenerative disease that leads to severe impairment of cognitive function, leading to a drastic decline in the quality of life. The primary pathological features of AD include senile plaques (SPs) and intracellular neurofibrillary tangles (NFTs), comprising aggregated amyloid β (Aβ) and hyperphosphorylated tau protein, respectively, in the hippocampus of AD patients. Histone deacetylase 6 (HDAC6) is a key enzyme in this neurodegenerative disease, in particular, as it relates to tau hyperphosphorylation. This study aimed to investigate the protective effects and mechanism of the novel HDAC6 inhibitor, MPT0G211, using an AD model. Our results indicated that MPT0G211 significantly reduced tau phosphorylation and aggregation, the processes highly correlated with the formation of NFTs. This HDAC6 inhibitory activity resulted in an increase in acetylated Hsp90, which decreased Hsp90 and HDAC6 binding, causing ubiquitination of phosphorylated tau proteins. In addition, a significant increase of phospho-glycogen synthase kinase-3β (phospho-GSK3β) on Ser9 (the inactive form) through Akt phosphorylation was associated with the inhibition of phospho-tau Ser396 in response to MPT0G211 treatment. In AD in vivo models, MPT0G211 appeared to ameliorate learning and memory impairment in animals. Furthermore, MPT0G211 treatment reduced the amount of phosphorylated tau in the hippocampal CA1 region. In summary, MPT0G211 treatment appears to be a promising strategy for improving the AD phenotypes, including tau hyperphosphorylation and aggregation, neurodegeneration, and learning and memory impairment, making it a valuable agent for further investigation.

Aim/hypothesis: Combination therapy targeting the major actors involved in the immune-mediated destruction of pancreatic beta cells appears to be an indispensable approach to treat type 1 diabetes effectively. We hypothesised that the combination of an orally active pan-histone deacetylase inhibitor (HDACi: givinostat) with subtherapeutic doses of CD3 antibodies may provide ideal synergy to treat ongoing autoimmunity., Methods: NOD mice transgenic for the human CD3ε (also known as CD3E) chain (NOD-huCD3ε) were treated for recent-onset diabetes with oral givinostat, subtherapeutic doses of humanised CD3 antibodies (otelixizumab, 50 μg/day, 5 days, i.v.) or a combination of both drugs. Disease remission, metabolic profiles and autoreactive T cell responses were analysed in treated mice., Results: We demonstrated that givinostat synergised with otelixizumab to induce durable remission of diabetes in 80% of recently diabetic NOD-huCD3ε mice. Remission was obtained in only 47% of mice treated with otelixizumab alone. Oral givinostat monotherapy did not reverse established diabetes but reduced the in situ production of inflammatory cytokines (IL-1β, IL-6, TNF-α). Importantly, the otelixizumab + givinostat combination strongly improved the metabolic status of NOD-huCD3ε mice; the mice recovered the capacity to appropriately produce insulin, control hyperglycaemia and sustain glucose tolerance. Finally, diabetes remission induced by the combination therapy was associated with a significant reduction of insulitis and autoantigen-specific CD8 + T cell responses., Conclusions/interpretation: HDACi and low-dose CD3 antibodies synergised to abrogate in situ inflammation and thereby improved pancreatic beta cell survival and metabolic function leading to long-lasting diabetes remission. These results support the therapeutic potential of protocols combining these two drugs, both in clinical development, to restore self-tolerance and insulin independence in type 1 diabetes.

A sensitive and rapid LC-MS/MS method was developed and validated for the simultaneous quantitation of four HDAC inhibitors, namely belinostat (BST), panobinostat (PST), rocilinostat (RST) and vorinostat (VST), in mouse plasma as per regulatory guidelines. The analytes and internal standard were extracted from 50 μL mouse plasma by protein precipitation, followed by chromatographic separation using an Atlantis C 18 column with an isocratic mobile phase comprising 0.1% formic acid-acetonitrile (25:75, v/v) at a flow rate of 0.5 mL/min within 2.5 min. Detection and quantitation were done by multiple reaction monitoring on a triple quadrupole mass spectrometer following the transitions: m/z 319 → 93, 350 → 158, 434 → 274 and 265 → 232 for BST, PST, RST and VST, respectively, in the positive ionization mode. The calibration curves were linear from 2.92 to 2921 ng/mL for BST and PST and from 1.01 to 1008 ng/mL for RST and VST with r 2  ≥ 0.99 for all of the analytes. The intra- and inter-batch accuracy and precision (CV) across quality controls varied from 85.5 to 112% and from 2.30 to 12.5, respectively, for all of the analytes. Analytes were found to be stable under different stability conditions. The method was applied to an i.v. pharmacokinetic study in mice., (Copyright © 2016 John Wiley & Sons, Ltd.)

Purpose This phase I trial evaluated epigenetic modulation of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor by using a histone deacetylase abexinostat in combination with pazopanib to enhance response and reverse resistance. Patients and Methods Pazopanib was administered once a day on days 1 to 28 and abexinostat was administered orally twice a day on days 1 to 5, 8 to 12, and 15 to 19 (schedule A) or on days 1 to 4, 8 to 11, and 15 to 18 (schedule B). Dose escalation (3 + 3 design) in all solid tumors was followed by dose expansion in renal cell carcinoma (RCC). Results Fifty-one patients with RCC (N = 22) were enrolled, including 30 (59%) with one or more lines of prior VEGF-targeting therapy. Five dose-limiting toxicities, including fatigue (n = 2), thrombocytopenia (n = 2), and elevated AST/ALT (n = 1), were observed with schedule A; one dose-limiting toxicity was observed (elevated AST/ALT) was observed with schedule B. Grade ≥ 3 related adverse events included fatigue (16%), thrombocytopenia (16%), and neutropenia (10%). The recommended phase II dose was established as abexinostat 45 mg/m 2 twice a day administered per schedule B plus pazopanib 800 mg/d. Objective response rate was 21% overall and 27% in the RCC subset. Median duration of response was 9.1 months (1.2 to > 49 months). Eight patients (16%) had durable control of disease for > 12 months. Durable tumor regressions were observed in seven (70%) of 10 patients with pazopanib-refractory disease, including one patients with RCC with ongoing response > 3.5 years. Peripheral blood histone acetylation and HDAC2 gene expression were associated with durable response to treatment. Conclusion Abexinostat is well tolerated in combination with pazopanib, allowing prolonged exposure and promising durable responses in pazopanib- and other VEGF inhibitor-refractory tumors, which supports epigenetically mediated reversal of treatment resistance.

Histone deacetylase inhibitors (HDAC inhibitors) are used to treat malignancies such as cutaneous T cell lymphoma and peripheral T cell lymphoma. Only four drugs are approved by the US Food and Drug Administration, namely vorinostat, romidepsin, panobinostat and belinostat, while chidamide has been approved in China. There are a number of bioanalytical methods reported for the measurement of HDAC inhibitors in clinical (human plasma and serum) and preclinical (mouse plasma, rat plasma, urine and tissue homogenates, etc.) studies. This review covers various HDAC inhibitors such as vorinostat, romidepsin, panobinostat, belinostat and chidamide. In addition to providing a comprehensive review of the available methods for the above mentioned HDAC inhibitors, it also provides case studies with perspectives for chosen drugs. Based on the review, it is concluded that the published methodologies using either HPLC or LC-MS/MS are well suited for the quantification of HDAC inhibitors in various biological fluids to delineate pharmacokinetic data., (Copyright © 2016 John Wiley & Sons, Ltd.)

Novel selective histone deacetylase 6 (HDAC6) inhibitors using the quinazoline as the cap were designed, synthesized, and evaluated for HDAC enzymatic assays. N-Hydroxy-4-(2-methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)phenoxy)butanamide, 23bb, was the most potent selective inhibitor for HDAC6 with an IC50 of 17 nM and showed 25-fold and 200-fold selectivity relative to HDAC1 and HDAC8, respectively. In vitro, 23bb presented low nanomolar antiproliferative effects against panel of cancer cell lines. Western blot analysis further confirmed that 23bb increased acetylation level of α-tubulin in vitro. 23bb has a good pharmacokinetic profile with oral bioavailability of 47.0% in rats. In in vivo efficacy evaluations of colorectal HCT116, acute myelocytic leukemia MV4-11, and B cell lymphoma Romas xenografts, 23bb more effectively inhibited the tumor growth than SAHA even at a 4-fold reduced dose or ACY-1215 at the same dose. Our results indicated that 23bb is a potent oral anticancer candidate for selective HDAC6 inhibitor and deserves further investigation.

Histone deacetylase inhibitors (HDACi) are approved for treating rare cancers and are of interest as potential therapies for neurodegenerative disorders. We evaluated a triple combination formulation (TCF) comprising the pan-HDACi vorinostat, the caging agent 2-hydroxypropyl-β-cyclodextrin (HPBCD), and polyethylene glycol (PEG) for treating a mouse model (the Npc1(nmf164) mouse) of Niemann-Pick type C (NPC) disease, a difficult-to-treat cerebellar disorder. Vorinostat alone showed activity in cultured primary cells derived from Npc1(nmf164) mice but did not improve animal survival. However, low-dose, once-weekly intraperitoneal injections of the TCF containing vorinostat increased histone acetylation in the mouse brain, preserved neurites and Purkinje cells, delayed symptoms of neurodegeneration, and extended mouse life span from 4 to almost 9 months. We demonstrate that the TCF boosted the ability of HDACi to cross the blood-brain barrier and was not toxic even when used long term. Further, the TCF enabled dose reduction, which has been a major challenge in HDACi therapy. TCF simultaneously treats neurodegenerative and systemic symptoms of Niemann-Pick type C disease in a mouse model., (Copyright © 2016, American Association for the Advancement of Science.)

Chidamide (epidaza), a new oral isotype-selective histone deacetylase inhibitor (HDACi), which is just approved in China for the treatment of recurrent or refractory peripheral T-cell lymphoma (PTCL) in December 2014, is the first listed benzamide class of HDACi in the world, and is currently undergoing global clinical trials for solid tumor treatments. Here, we report a sensitive, rapid and robust HPLC-MS/MS method for determination of chidamide in human plasma. Plasma sample was subjected to a simple acetonitrile protein precipitation containing MS-275 used as an internal standard (IS). Chromatography was performed on a Hypersil GOLD C18 analytical column, using a gradient methanol/water mobile phase containing 0.1% formic acid. A tandem mass spectrometer equipped with electrospray ionization source was used as detector and operated in the positive-ion mode. Selected reaction monitoring (SRM) using the precursor/ product transitions (m/z) of 391.1/265.1 for chidamide and 377.1/359.2 for IS were used for quantification, respectively. Good linearity was obtained in the range of 1-1000ng/mL. The method gave R.S.D.% values for precision always lower than 13.8% and R.E.% values for accuracy between -3.7 and 9.1%. In addition, the specificity, recovery, stability and matrix effect were satisfactory too. The method is now being successfully applied to plasma samples as part of an ongoing chidamide phase Ib clinical trial in patients with solid tumors, and had demonstrated consistent AUClast and t1/2 results with the published phase I pharmacokinetic data, which was also analyzed by this method, thus further confirming the reproducibility and accuracy during its clinical application. Considering the excellent performance of this method, it will continue being utilized for future clinical developments of chidamide and for routine monitoring of plasma exposure of chidamide during its clinical therapy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

IKH12 is a novel histone deacetylase 6 selective inhibitor. A rapid and sensitive liquid chromatography tandem mass spectrometry method was developed and validated for the quantification of IKH12 in rat plasma and tissue with kendine 91 as internal standard (IS). The samples were prepared by liquid-liquid extraction with tert-butyl methyl ether. The chromatographic separation was accomplished by using a Zorbax Extend C18 4.6 × 150 mm, 5 µm column, with a mobile phase consisting of methanol and 0.1% formic acid (75:25 v/v). Multiple reaction monitoring, using electrospray ionization in positive ion mode, was employed to quantitatively detect IKH12 and IS. The monitored transitions were set at m/z 418 → 252 and 444 → 169 for IKH12 and kendine 91, respectively. The calibration curve was linear over the concentration range 2-1000 ng mL(-1) . The intra- and inter-assay precision and accuracy of the quality controls and the limit of quantification were satisfactory in all cases (according to European Medicines Agency guidelines). Stability studies showed that plasma samples were stable in the chromatography rack for 24 h and at -80°C for 2 months and also after three freeze-thaw cycles. This method was successfully applied to a pharmacokinetic study of IKH12 in rat., (Copyright © 2015 John Wiley & Sons, Ltd.)

Purpose: This study assessed the pharmacokinetics and safety of oral panobinostat and its metabolite BJB432 in patients with advanced solid tumors and normal to severely impaired renal function., Methods: Patients with varying degrees of renal impairment, defined by their 24-h baseline urine creatinine clearance (as normal, mild, moderate or severe), received a single oral dose of 30 mg panobinostat. Serial plasma samples were collected pre-dose and up to 96-h post-dose. Serial urine samples were collected for 24-h post-dose. Following the serial PK sampling, patients received 30 mg oral panobinostat thrice weekly for as long as the patient had benefit. Pharmacokinetic parameters were derived using non-compartmental analysis., Results: Thirty-seven patients were enrolled, and median age was 64 (range 40-81) years. Eleven patients had normal renal function; 10, 10, and 6 patients had mild, moderate, and severe renal impairment, respectively. Geometric means of AUC(0-∞) in the normal, mild, moderate, and severe groups were 224.5, 144.3, 223.1, and 131.7 ng h/mL, respectively. Geometric mean ratio of BJB432 to parent drug plasma AUC(0-∞) was 0.64 in the normal group and increased to 0.81, 1.13, and 1.20 in the mild, moderate, and severe groups, respectively. The fraction excreted as unchanged panobinostat was small (<2 %), with a large variability. The renal clearance of panobinostat and tolerability was similar across all four groups., Conclusion: Systemic exposure to panobinostat did not increase with severity of renal impairment, and the drug was tolerated equally; thus, patients with renal impairment do not require starting dose adjustments.

Mocetinostat (MGCD0103) is a benzamide histone deacetylase inhibitor undergoing clinical trials for treatment of various cancers including Hodgkin's lymphoma, follicular lymphoma and acute myelogenous leukemia. A sensitive and selective liquid chromatography mass spectrometry method for determination of MGCD0103 in rat plasma was developed. After addition of midazolam as internal standard (IS), protein precipitation by acetonitrile was used as sample preparation. Chromatographic separation was achieved on a C18 (2.1 mm×50 mm, 3.5 µm) column with acetonitrile-0.1% formic acid in water as mobile phase with gradient elution. An electrospray ionization source was applied and operated in positive ion mode; selective ion monitoring (SIM) mode was used for quantification using target fragment ions m/z 397 for MGCD0103 and m/z 326 for the IS. Calibration plots were linear over the range of 5-5000 ng/mL for MGCD0103 in rat plasma. Mean recoveries of MGCD0103 in rat plasma were in the range of 89.7-96.1%. RSD of intra-day and inter-day precision were both<11%. The accuracy of the method ranged from 96.5% to 109.7%. The matrix effects for MGCD0103 were between 94.5% and 97.4%. The method was successfully applied to pharmacokinetic study of MGCD0103 after oral (15 mg/kg) and intravenous (3 mg/kg) administration in rats. The bioavailability of MGCD0103 was 29.3% in rats.

Vorinostat (suberoylanilide hydroxamic acid) is the first approved histone deacetylase (HDAC) inhibitor for the treatment of cutaneous T-cell lymphoma after progressive disease following two systemic therapies. Intracellular access of vorinostat is essential to exert its epigenetic effects. Therefore, we studied the relationship between vorinostat extracellular (plasma) and intracellular (peripheral blood mononuclear cells, PBMCs) concentration and assessed its concentration-effect relationship by HDAC activity testing. Assays were developed and validated for the low nanomolar quantification of vorinostat and two inactive metabolites in human plasma and PBMCs. For the vorinostat extraction from plasma and PBMCs solid-phase extraction and liquid-liquid extraction methods were applied. Extraction recoveries ranged from 88.6% to 114.4% for all analytes and extraction methods. Extracts were chromatographed on a Phenomenex Luna column isocratically (plasma) or by gradient (PBMCs) consisting of acidic ammonium acetate, acetonitrile, and methanol. The analytes were quantified using deuterated internal standards and positive electrospray tandem mass spectrometry (multiple reaction monitoring) with lower limits of quantification of 11.0 ng/mL (plasma) and 0.1 ng/3 × 10(6) cells (PBMCs). The calibrated ranges were linear for vorinostat in plasma 11.0-1100 (11,000) ng/mL (metabolites) and PBMCs 0.1-10.0 ng/3 × 10(6) cells with correlation coefficients >0.99, an overall accuracy varying between -6.7% and +3.8% in plasma, -8.1% and -1.5% in PBMCs, and an overall precision ranging from 3.2% to 6.1% in plasma and 0.8% to 4.0% in PBMCs (SD batch-to-batch). The application to blood samples from healthy volunteers incubated with vorinostat revealed accumulation of vorinostat in PBMCs, effective intracellular HDAC inhibition at therapeutic vorinostat concentrations and a direct vorinostat concentration dependency to HDAC inhibition., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: A phase I study to assess the maximum-tolerated dose (MTD), dose-limiting toxicity (DLT), pharmacokinetics (PK) and antitumor activity of vorinostat in combination with bortezomib in patients with advanced solid tumors., Methods: Patients received vorinostat orally once daily on days 1-14 and bortezomib intravenously on days 1, 4, 8 and 11 of a 21-day cycle. Starting dose (level 1) was vorinostat (400 mg) and bortezomib (0.7 mg/m(2)). Bortezomib dosing was increased using a standard phase I dose-escalation schema. PKs were evaluated during cycle 1., Results: Twenty-three patients received 57 cycles of treatment on four dose levels ranging from bortezomib 0.7 mg/m(2) to 1.5 mg/m(2). The MTD was established at vorinostat 400 mg daily and bortezomib 1.3 mg/m(2). DLTs consisted of grade 3 fatigue in three patients (1 mg/m(2),1.3 mg/m(2) and 1.5 mg/m(2)) and grade 3 hyponatremia in one patient (1.5 mg/m(2)). The most common grade 1/2 toxicities included nausea (60.9%), fatigue (34.8%), diaphoresis (34.8%), anorexia (30.4%) and constipation (26.1%). Objective partial responses were observed in one patient with NSCLC and in one patient with treatment-refractory soft tissue sarcoma. Bortezomib did not affect the PKs of vorinostat; however, the Cmax and AUC of the acid metabolite were significantly increased on day 2 compared with day 1., Conclusions: This combination was generally well-tolerated at doses that achieved clinical benefit. The MTD was established at vorinostat 400 mg daily × 14 days and bortezomib 1.3 mg/m(2) on days 1, 4, 8 and 11 of a 21-day cycle.

The fatty acids, n-butyric acid (BA), 4-phenylbutyric acid (PBA) and valproic acid (VPA, 2-propylpentanoic acid) have been used for many years in the treatment of a variety of CNS and peripheral organ diseases including cancer. New information that these drugs alter epigenetic processes through their inhibition of histone deacetylases (HDACs) has renewed interest in their biodistribution and pharmacokinetics and the relationship of these properties to their therapeutic and side effect profiles. In order to determine the pharmacokinetics and biodistribution of these drugs in primates, we synthesized their carbon-11 labeled analogues and performed dynamic positron emission tomography (PET) in six female baboons over 90 min. The carbon-11 labeled carboxylic acids were prepared by using (11)CO2 and the appropriate Grignard reagents. [(11)C]BA was metabolized rapidly (only 20% of the total carbon-11 in plasma was parent compound at 5 min post injection) whereas for VPA and PBA 98% and 85% of the radioactivity were the unmetabolized compound at 30 min after their administration respectively. The brain uptake of all three carboxylic acids was very low (<0.006%ID/cc, BA>VPA>PBA), which is consistent with the need for very high doses for therapeutic efficacy. Most of the radioactivity was excreted through the kidneys and accumulated in the bladder. However, the organ biodistribution between the drugs differed. [(11)C]BA showed relatively high uptake in spleen and pancreas whereas [(11)C]PBA showed high uptake in liver and heart. Notably, [(11)C]VPA showed exceptionally high heart uptake possibly due to its involvement in lipid metabolism. The unique biodistribution of each of these drugs may be of relevance in understanding their therapeutic and side effect profile including their teratogenic effects., (© 2013.)

A pediatric study has established a maximum tolerated dose (MTD) for temsirolimus (Tem) of more than 150 mg/m intravenously/week. A phase I trial was conducted to establish the MTD for Tem in combination with valproic acid (VPA) in children and adolescents with refractory solid tumors. The secondary aims included expression of mammalian target of rapamycin (mTOR) markers on archival tumor tissue; Tem pharmacokinetics; assessment of histone acetylation (HA); and tumor response. Patients were treated with VPA (5 mg/kg orally three times daily) with a target serum level of 75-100 mcg/ml. Tem was started at an initial dose of 60 mg/m/week. Pharmacokinetics and HA measurements were performed during weeks 1 and 5. Two of the first three patients experienced dose-limiting toxicity (grade 3 mucositis). Tem at 35 mg/m/week was found to be tolerable. Peak Tem concentrations were higher in all patients compared with those in previously published reports of single agent Tem. Increases in HA are correlated with VPA levels. All tumor samples expressed mTORC1 and mTORC2. An objective response was observed in one patient (melanoma), whereas transient stable disease was observed in four other patients (spinal cord ependymoma, alveolar soft part sarcoma, medullary thyroid carcinoma, and hepatocellular carcinoma). The MTD of Tem when administered with VPA is considerably lower than when used as a single agent, with mucositis the major dose-limiting toxicity. The combination merits further study and may have activity in melanoma. Attention to drug-drug interactions will be important in future multiagent trials including Tem.

Nonspecific histone deacetylase (HDAC) inhibition has been shown to facilitate the extinction of drug-seeking behavior in a manner resistant to reinstatement. A key open question is which specific HDAC is involved in the extinction of drug-seeking behavior. Using the selective HDAC3 inhibitor RGFP966, we investigated the role of HDAC3 in extinction and found that systemic treatment with RGFP966 facilitates extinction in mice in a manner resistant to reinstatement. We also investigated whether the facilitated extinction is related to the enhancement of extinction consolidation during extinction learning or to negative effects on performance or reconsolidation. These are key distinctions with regard to any compound being used to modulate extinction, because a more rapid decrease in a defined behavior is interpreted as facilitated extinction. Using an innovative combination of behavioral paradigms, we found that a single treatment of RGFP966 enhances extinction of a previously established cocaine-conditioned place preference, while simultaneously enhancing long-term object-location memory within subjects. During extinction consolidation, HDAC3 inhibition promotes a distinct pattern of histone acetylation linked to gene expression within the infralimbic cortex, hippocampus, and nucleus accumbens. Thus, the facilitated extinction of drug-seeking cannot be explained by adverse effects on performance. These results demonstrate that HDAC3 inhibition enhances the memory processes involved in extinction of drug-seeking behavior.

Background: Control of epigenetic changes using histone deacetylase inhibitors (HDACi) is thought to be a promising target in therapy of gastrointestinal (GI) cancer. In this study, we evaluated the safety, pharmacokinetics, and efficacy of two dosing regimens of vorinostat, an oral HDACi, in patients with GI tumors., Methods: Patients received either vorinostat 300 mg bid for 3 consecutive days followed by 4 rest days per cycle (n = 10) or vorinostat 400 mg qd for 21 consecutive days per cycle (n = 6). Pharmacokinetic parameters were assessed for the first treatment cycle. Efficacy was determined through evaluation of tumors and assessment of treatment response., Results: The median treatment duration of 300 mg bid was 52.0 days and of 400 mg qd was 51.5 days. The most common drug-related adverse events were anorexia, nausea, fatigue, and hyperglycemia. Two patients taking 400 mg qd had dose-limiting toxicities (DLTs) of thrombocytopenia. No patients taking 300 mg bid experienced DLT. Five patients taking 300 mg bid and 2 patients taking 400 mg qd maintained stable disease for >8 weeks, with the maximum duration of 245 days. Mean drug exposure (±SD) was generally higher with 400 mg qd (area under the curve [AUC(0-∞)] of 7.75 ± 2.79 μM h on Day 1 post-dose) compared with 300 mg bid (AUC(0-∞) of 3.94 ± 1.56 μM h on Day 1 post-dose)., Conclusions: Vorinostat 300 mg bid for 3 consecutive days followed by 4 days of rest was better tolerated in patients with GI cancer than a higher once daily dose. Additionally, there were patients in both groups who achieved stable disease, most maintaining it for longer than 8 weeks, suggesting vorinostat as a possible active agent in the treatment of GI cancer.

Concentrations of acetyl-coenzyme A and nicotinamide adenine dinucleotide (NAD(+)) affect histone acetylation and thereby couple cellular metabolic status and transcriptional regulation. We report that the ketone body d-β-hydroxybutyrate (βOHB) is an endogenous and specific inhibitor of class I histone deacetylases (HDACs). Administration of exogenous βOHB, or fasting or calorie restriction, two conditions associated with increased βOHB abundance, all increased global histone acetylation in mouse tissues. Inhibition of HDAC by βOHB was correlated with global changes in transcription, including that of the genes encoding oxidative stress resistance factors FOXO3A and MT2. Treatment of cells with βOHB increased histone acetylation at the Foxo3a and Mt2 promoters, and both genes were activated by selective depletion of HDAC1 and HDAC2. Consistent with increased FOXO3A and MT2 activity, treatment of mice with βOHB conferred substantial protection against oxidative stress.

Long-term memory formation is known to be critically dependent upon de novo gene expression in the brain. As a consequence, pharmacological enhancement of the transcriptional processes mediating long-term memory formation provides a potential therapeutic strategy for cognitive disorders involving aberrant neuroplasticity. Here we focus on the identification and characterization of small molecule inhibitors of histone deacetylases (HDACs) as enhancers of CREB (cAMP response element-binding protein)-regulated transcription and modulators of chromatin-mediated neuroplasticity. Using a CREB reporter gene cell line, we screened a library of small molecules structurally related to known HDAC inhibitors leading to the identification of a probe we termed crebinostat that produced robust activation of CREB-mediated transcription. Further characterization of crebinostat revealed its potent inhibition of the deacetylase activity of recombinant class I HDACs 1, 2, 3, and class IIb HDAC6, with weaker inhibition of the class I HDAC8 and no significant inhibition of the class IIa HDACs 4, 5, 7, and 9. In cultured mouse primary neurons, crebinostat potently induced acetylation of both histone H3 and histone H4 as well as enhanced the expression of the CREB target gene Egr1 (early growth response 1). Using a hippocampus-dependent, contextual fear conditioning paradigm, mice systemically administered crebinostat for a ten day time period exhibited enhanced memory. To gain insight into the molecular mechanisms of memory enhancement by HDAC inhibitors, whole genome transcriptome profiling of cultured mouse primary neurons treated with crebinostat, combined with bioinformatic analyses of CREB-target genes, was performed revealing a highly connected protein-protein interaction network reflecting modules of genes important to synaptic structure and plasticity. Consistent with these findings, crebinostat treatment increased the density of synapsin-1 punctae along dendrites in cultured neurons. Finally, crebinostat treatment of cultured mouse primary neurons was found to upregulate Bdnf (brain-derived neurotrophic factor) and Grn (granulin) and downregulate Mapt (tau) gene expression-genes implicated in aging-related cognitive decline and cognitive disorders. Taken together, these results demonstrate that crebinostat provides a novel probe to modulate chromatin-mediated neuroplasticity and further suggests that pharmacological optimization of selective of HDAC inhibitors may provide an effective therapeutic approach for human cognitive disorders. This article is part of a Special Issue entitled 'Cognitive Enhancers'., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Purpose: Combining proteasome and histone deacetylase (HDAC) inhibition has been seen to provide synergistic anti-tumor activity, with complementary effects on a number of signaling pathways. The novel bi-cyclic structure of marizomib with its unique proteasome inhibition, toxicology and efficacy profiles, suggested utility in combining it with an HDAC inhibitor such as vorinostat. Thus, in this study in vitro studies assessed the potential utility of combining marizomib and vorinostat, followed by a clinical trial with the objectives of assessing the recommended phase 2 dose (RP2D), pharmacokinetics (PK), pharmacodynamics (PD), safety and preliminary anti-tumor activity of the combination in patients., Experimental Design: Combinations of marizomib and vorinostat were assessed in vitro. Subsequently, in a Phase 1 clinical trial patients with melanoma, pancreatic carcinoma or Non-small Cell Lung Cancer (NSCLC) were given escalating doses of weekly marizomib in combination with vorinostat 300 mg daily for 16 days in 28 day cycles. In addition to standard safety studies, proteasome inhibition and pharmacokinetics were assayed., Results: Marked synergy of marizomib and vorinostat was seen in tumor cell lines derived from patients with NSCLC, melanoma and pancreatic carcinoma. In the clinical trial, 22 patients were enrolled. Increased toxicity was not seen with the combination. Co-administration did not appear to affect the PK or PD of either drug in comparison to historical data. Although no responses were demonstrated using RECIST criteria, 61% of evaluable patients demonstrated stable disease with 39% having decreases in tumor measurements., Conclusions: Treatment of multiple tumor cell lines with marizomib and vorinostat resulted in a highly synergistic antitumor activity. The combination of full dose marizomib with vorinostat is tolerable in patients with safety findings consistent with either drug alone.

A rapid and sensitive liquid chromatography/tandem mass spectrometry (LC-MS/MS) method has been developed and fully validated to determine HS270, a new histone deacetylase (HDAC) inhibitor, in rat plasma using SAHA as the internal standard (IS). After a single step liquid-liquid extraction with acetoacetate, analytes were subjected to LC-MS/MS analysis using positive electro-spray ionization (ESI(+)) under selected reaction monitoring mode (SRM). The chromatographic separation was achieved on a Hypurity C(18) column (50 mm × 2.1 mm, i.d., 5 μm). The MS/MS detection was conducted by monitoring the fragmentation of m/z 392.3→100.1 for HS270, m/z 265.1→232.1 for IS. The method had a chromatographic running time of 2.5 min and linear calibration curves over the concentrations of 0.5-1000 ng/mL. The recovery of the method was 70.8-82.5% and the lower limit of quantification (LLOQ) was 0.5 ng/mL. The intra- and inter-batch precisions were less than 15% for all quality control samples at concentrations of 1.0, 100.0, and 750.0 ng/mL. The validated LC-MS/MS method has successfully applied to a HS270 pharmacokinetic study after oral doses of 25, 50, 100, 200 mg/kg, and i.v. dose of 5 mg/kg to rats., (Copyright © 2011. Published by Elsevier B.V.)

Purpose: The objectives of this study were to develop physiologically based models for the pharmacokinetics (PK) and organ distribution of apicidin in rats and mice and to predict human PK in blood and organs., Methods: The PK of apicidin was characterized in rats and mice after i.v. bolus injection, and distribution to various tissues was determined in rats following i.v. infusions at steady state. The developed models were prospectively validated within rat and within mouse and by scaling from rat to mouse using data after multiple i.v. injections. Human PK was predicted by the physiologically based modeling using intrinsic clearance data for humans from in vitro experiments., Results: The Cl(s) predicted for human (9.8 ml/min/kg) was lower than those found in mice (116.9 ml/min/kg) and rats (61.6 ml/min/kg), and the V(ss) predicted for human (1.9 l/kg) was less than in mice (2.0 l/kg) and rats (2.5 l/kg). Consequently, the predicted t (1/2) was longer in human (2.3 h) than in mice and rats (0.4 and 0.9 h, respectively). The highest concentrations of apicidin were predicted in liver followed by adipose tissue, kidney, lung, spleen, heart, arterial blood, venous blood, small intestine, stomach, muscle, testis, and brain., Conclusions: The developed models adequately described the PK of apicidin in rats and mice and were applied to predict human PK. These models may be useful in predicting human blood and tissue concentrations of apicidin under different exposure conditions.

Purpose: To evaluate pharmacological properties of H6CAHA, an adamantyl-hydroxamate histone deacetylase inhibitor, and to investigate its effect on prostate cancer cells following exposure to γ-radiation in vitro and in vivo., Methods and Materials: H6CAHA was assessed for in vitro solubility, lipophilicity and growth inhibition, and in vivo plasma pharmacokinetics. The effect of H6CAHA on radiation clonogenic survival and DNA damage repair was evaluated in human prostate cancer (PC3, DU145, LNCaP) and nonmalignant control epithelial (RWPE1 and 267B1) cell lines. The effect of this agent on the growth of prostate cancer xenografts was also assessed in mice., Results: H6CAHA demonstrated good solubility and permeability profiles and preferentially inhibited the growth of prostate cancer cells over nonmalignant cells. Plasma pharmacokinetics revealed that the area under the curve of H6CAHA was 8.08 ± 0.91 μM × h, and its half-life was 11.17 ± 0.87 h. Radiation clonogenic assays revealed that H6CAHA decreased the survival of prostate cancer cells at the dose that exerted limited effect on normal cells. Concomitantly, delayed DNA damage repair following combination treatment was evident in cancer cells, indicated by the prolonged appearance of γH2AX and Rad51 foci and suppression of DNA damage repair genes (ATM, BRCA1, and BRCA2). Combined modality of H6CAHA (daily intraperitoneal injections for 10 days) with γ-radiation (10 × 2 Gy) completely blocked the growth of PC3 tumor xenografts (p < 0.001) over 60 days., Conclusion: These results support the potential therapeutic value of H6CAHA in combination with radiation and support the rationale for further clinical investigation., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Decitabine and azacitidine, two DNA methyltransferase (DNMT) inhibitors, are the current standard of treatment for myelodysplastic syndrome (MDS). Histone deacetylase (HDAC) inhibitors are also being tested against MDS. Both drug classes synergize in their gene reactivating and anticancer activities. The combination of hydralazine and valproate (Transkrip®), a DNMT and HDAC inhibitor, respectively), has been developed as epigenetic therapy under the drug repositioning concept. To evaluate the clinical efficacy and safety of hydralazine and valproate against MDS, an open phase-II study for previously treated patients with MDS was conducted. The hydralazine dose was given according with the acetylator phenotype, and valproate was dosed at 30 mg/kg/day. Response was graded with International Working Group criteria. Toxicity was evaluated by the Common Toxemia Criteria-National Cancer Institute version 3 scale. From November 2007 to January 2010, 12 patients were included. Median age±SD was 53±19.78 years (range, 23-79 years); median time from diagnosis to inclusion in the study was 7.9 months (range 2.6-36.1 months). Median of previous treatment was 2 (range, 1-6). Refractory cytopenia with multilineage dysplasia was diagnosed in ten cases, and refractory anemia with excess of blasts in two. Overall response was documented in six (50%) of 12 cases, including one CR, one PR, and four hematological improvements of the erythroid series. Two patients (16.6%) progressed to acute myeloid leukemia. Hemoglobin increased from 7.4 to 10.3 g/dL (in 13 weeks), neutrophils, from 1.1 to 2.0 (in 3 weeks), and platelets, from 66×10(9) to 72×10(9)/L (in 2 weeks). Transfusional requirements decreased from 2.3 to 0 U bi-monthly for red blood cells and from 0.5 to 0 U bi-monthly for platelets in responding patients. Main toxicities were mild, including somnolence and nausea. Preliminary results of this phase-II study suggest that the combination of hydralazine and valproate is a promising non-toxic and effective therapy for MDS.

The pharmacokinetics and metabolism of KBH-A40, a novel δ-lactam-based histone deacetylase inhibitor, were characterized in male Sprague-Dawley rats. KBH-A40 exhibited a high clearance (12.0 ± 2.8 l h⁻¹kg⁻¹), a large volume of distribution at steady state, V(ss) (3.9 ± 1.5 l kg⁻¹), and a short half-life, t₁/₂ (2.0 ± 0.3 h). KBH-A40 was rapidly converted to its metabolite, KBH-A40 carboxylate, after intravenous (2 and 20 mg kg⁻¹ and oral (10 mg kg⁻¹) administration; the carboxylate metabolite remained at elevated concentrations in the plasma for more than 8 h. Glucuronide conjugate of KBH-A40 was identified qualitatively by using liquid chromatography tandem mass spectrometry in rat plasma. KBH-A40 was rapidly absorbed (t(max) = 0.4 h) after oral dose, consistent with its permeability in Caco-2 cells. Its oral bioavailability was low (14.2-14.8%). An apparent "double peak" phenomenon was observed for both KBH-A40 and KBH-A40 carboxylate after oral administration. KBH-A40 was degraded rapidly by glucuronidation, but not by cytochrome P450-mediated oxidation, in rat liver microsomes. These results suggest that the rapid metabolism of KBH-A40 could be a major reason for its poor pharmacokinetics. Therefore, this work provides valuable structural information to improve pharmacokinetic properties of KBH-A40, a lead compound.

Background: This study assessed the population pharmacokinetics and metabolic conversion of a novel histone deacetylase (HDAC) inhibitor, SD-2007, into its active metabolite, apicidin, in rats., Methods: SD-2007 was given to rats by intravenous injection (4 mg/kg) and oral administration (40 mg/kg). Serum concentrations of SD-2007 and apicidin were determined by LC-MS/MS. All concentrations were analyzed using a population pharmacokinetic model with 9 compartments in S-ADAPT., Results: The area under the curve for apicidin was 96 ± 16 mg·h/ml after 4 mg/kg administered intravenously and 2,455 ± 1,211 mg·h/ml after 40 mg/kg given orally. The population pharmacokinetic model described all profiles well. After oral administration of SD-2007, the median absolute bioavailability of SD-2007 was 6.67% (range 3.83-9.89) and the median apparent bioavailability was 22.3% (range 15.7-35.8) for apicidin, whereas only a median of 8.85% (range 7.57-9.34) of an intravenous SD-2007 dose was converted to apicidin., Conclusions: Oral SD-2007 displayed a substantial presystemic metabolism to active apicidin. The high serum concentrations of apicidin after oral administration of SD-2007 may cause significant HDAC inhibition., (Copyright © 2011 S. Karger AG, Basel.)

Oral sodium phenylbutyrate (SPB) is currently under investigation as a histone deacetylation (HDAC) inhibitor in Huntington disease (HD). Ongoing studies indicate that symptoms related to HD genetic abnormalities decrease with SPB therapy. In a recently reported safety and tolerability study of SPB in HD, we analyzed overall chromatographic patterns from a method that employs gradient liquid chromatography with series electrochemical array, ultraviolet (UV), and fluorescence (LCECA/UV/F) for measuring SPB and its metabolite phenylacetate (PA). We found that plasma and urine from SPB-treated patients yielded individual-specific patterns of approximately 20 metabolites that may provide a means for the selection of subjects for extended trials of SPB. The structural identification of these metabolites is of critical importance because their characterization will facilitate understanding the mechanisms of drug action and possible side effects. We have now developed an iterative process with LCECA, parallel LCECA/LCMS, and high-performance tandem MS for metabolite characterization. Here we report the details of this method and its use for identification of 10 plasma and urinary metabolites in treated subjects, including indole species in urine that are not themselves metabolites of SPB. Thus, this approach contributes to understanding metabolic pathways that differ among HD patients being treated with SPB., (Copyright 2010 Elsevier Inc. All rights reserved.)

Structurally diverse histone deacetylase inhibitors (HDACI) have emerged as chemotherapeutic agents. Here, we report the first mercaptoacetamide HDACIs (coded 6MAQH and 5MABMA) for use in treatment against prostate cancer cells in vitro and in vivo and correlate their plasma pharmacokinetics and tissue-pharmacodynamics with tumor sensitivity. HDACIs were assessed for in vitro microsomal stability and growth inhibition against prostate cancer and nonmalignant cells. Antitumor activity was determined following i.p. administration of 6MAQH and 5MABMA (0.5 and 5 mg/Kg) using mice bearing PC3 tumor xenografts (n = 10). The plasma pharmacokinetics of 6MAQH and 5MABMA and their effects on the acetylation of histone H4 in tissues were determined in athymic mice. Both HDACIs significantly inhibited the growth of cancer cells while exerting limited effect on nonmalignant cells. They exhibited stability in human, dog, and rat microsomes [t(1/2 (min)) = 83, 72, and 66 for 6MAQH and 68, 43, and 70 for 5MABMA, respectively]. Both HDACIs (0.5 mg/Kg) led to tumor regression (P < 0.01), which was sustained for at least 60 days. In vivo data show favorable plasma pharmacokinetics with the area under the curve of 4.97 +/- 0.6 micromol/L x h for 6MAQH and 4.23 +/- 0.43 micromol/L x h for 5MABMA. The clearance rates for 6MAQH and 5MABMA were 4.05 +/- 0.15 and 4.87 +/- 0.2 L/h, whereas the half-lives were 2.2 +/- 0.33 and 1.98 +/- 0.21 h, respectively. Both HDACIs markedly enhanced the acetylation of histone H4 within 30 minutes in tissues, including the brain, liver, and spleen. Taken together, the results provide a rationale for further investigation of these mercaptoacetamide HDACIs as potent anticancer agents.