HDAC1 ( IC50 = 10 nM ); HDAC3 ( IC50 = 20 nM ); HDAC2; HDAC7; HDAC11; Autophagy; Mitophagy

In comparison to control, the administration of Vorinostat (~100 mg/kg/day) significantly inhibits the growth of CWR22 human prostate xenografts in nude mice, resulting in tumor reductions of 78%, 97%, and 97% at doses of 25 mg/kg/day, 50 mg/kg/day, and 100 mg/kg/day, respectively. Vorinostat causes CWR22 cells to express prostate-specific antigen mRNA and accumulate acetylated core histones, which raises serum prostate-specific antigen levels above those estimated by tumor volume alone. [/2] By increasing histone acetylation in the brain and bridging the blood-brain barrier, oral administration of Vorinostat (0.67g/L) significantly ameliorates the motor impairment in the R6/2 mice model of Huntington's disease.[5]

The Jurkat cell lysate is treated with ice for an hour before being centrifuged at 12,000 g for ten minutes at 4 °C to remove any remaining material. 30 μL of 50% protein G-Sepharose slurry is added to supernatants and left for an hour at 4 °C to preclearase them. Using either the homologous or heterologous immunizing peptide, beads are pelleted by centrifugation, and the supernatants are then incubated for 1 hour at 4 °C with 10 μg of IgG fraction from anti-HDAC1 or HDAC3 polyclonal antisera (preincubated for 2 hours at room temperature). Rabbits are used to raise both antisera against the carboxylterminal peptide of HDAC1 and HDAC3, using synthetic peptides coupled to keyhole limpet hemocyanin). Half an hour is spent at 4°C with 30 μL of a 50% protein G-Sepharose slurry added. After centrifuging the immune complexes, 1 mL of lysis buffer is used to wash them three times. A ³H-acetylated peptide that corresponds to amino acids 1 through 24 of histone H4 is used in the HDAC assay, and beads are resuspended in 200 μL of HDAC buffer (20 mM Tris-HCl, pH 8.0/150 mM NaCl/10% glycerol). By using scintillation counting, released [³H]acetic acid is measured. Vorinostat at varying concentrations is preincubated with the immunoprecipitated complexes for 30 minutes at 4 °C in order to conduct inhibitions studies.

RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) is used to prepare cell lysates, and the Bio-Rad DC Protein Assay is used to measure the protein concentration. Protein lysates are moved to nitrocellulose membrane after being separated via SDS-PAGE. The subsequent dilutions and antibodies are utilized: mouse anti-p21WAF1 (0.5 μg/mL), rabbit anti-HDAC1 (1 μg/mL), rabbit anti-HDAC2 (1 μg/mL), rabbit anti-HDAC3 (9 μg/mL), and rabbit anti-HDAC7 (3 μg/mL). The swine anti-rabbit and rabbit anti-mouse HRP-coupled antibodies were used as secondary antibodies, with a final concentration of 1 μg/mL. All primary antibodies are incubated at 4°C for an entire night before being washed and secondary antibodies are incubated at room temperature for two hours. The enhanced chemiluminescence assay allows for the visualization of particular protein bands. In order to exhibit uniform loading of protein samples, β-tubulin is probed on every western blot.

Absorption, Distribution and Excretion
In vitro studies using human liver microsomes indicate negligible biotransformation by cytochromes P450 (CYP). Vorinostat is eliminated predominantly through metabolism with less than 1% of the dose recovered as unchanged drug in urine, indicating that renal excretion does not play a role in the elimination of vorinostat. However, renal excretion does not play a role in the elimination of vorinostat.
The pharmacokinetics of vorinostat were evaluated in 23 patients with relapsed or refractory advanced cancer. After oral administration of a single 400-mg dose of vorinostat with a high-fat meal, the mean +/- standard deviation area under the curve (AUC) and peak serum concentration (Cmax) and the median (range) time to maximum concentration (Tmax) were 5.5+/-1.8 uM.hr, 1.2+/-0.62 uM and 4 (2-10) hours, respectively.
In the fasted state, oral administration of a single 400-mg dose of vorinostat resulted in a mean AUC and Cmax and median Tmax of 4.2+/-1.9 uM.hr and 1.2+/-0.35 uM and 1.5 (0.5-10) hours, respectively. Therefore, oral administration of vorinostat with a high-fat meal resulted in an increase (33%) in the extent of absorption and a modest decrease in the rate of absorption (Tmax delayed 2.5 hours) compared to the fasted state. However, these small effects are not expected to be clinically meaningful. In clinical trials of patients with CTCL, vorinostat was taken with food.
At steady state in the fed-state, oral administration of multiple 400-mg doses of vorinostat resulted in a mean AUC and Cmax and a median Tmax of 6.0+/-2.0 uM.hr, 1.2+/-0.53 uM and 4 (0.5-14) hours, respectively.
Vorinostat is approximately 71% bound to human plasma proteins over the range of concentrations of 0.5 to 50 ug/mL.
For more Absorption, Distribution and Excretion (Complete) data for Vorinostat (9 total), please visit the HSDB record page.

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Metabolism / Metabolites
The major pathways of vorinostat metabolism involve glucuronidation and hydrolysis followed by β-oxidation. Human serum levels of two metabolites, O-glucuronide of vorinostat and 4-anilino-4-oxobutanoic acid were measured. Both metabolites are pharmacologically inactive. Compared to vorinostat, the mean steady state serum exposures in humans of the O-glucuronide of vorinostat and 4-anilino-4-oxobutanoic acid were 4-fold and 13-fold higher, respectively. In vitro studies using human liver microsomes indicate negligible biotransformation by cytochromes P450 (CYP).
Vorinostat is extensively metabolized to inactive metabolites, principally by glucuronidation and hydrolysis followed by beta-oxidation. The drug is not metabolized by cytochrome P-450 (CYP) isoenzymes.
The major pathways of vorinostat metabolism involve glucuronidation and hydrolysis followed by beta-oxidation. Human serum levels of two metabolites, O-glucuronide of vorinostat and 4-anilino-4-oxobutanoic acid were measured. Both metabolites are pharmacologically inactive. Compared to vorinostat, the mean steady state serum exposures in humans of the O-glucuronide of vorinostat and 4-anilino-4-oxobutanoic acid were 4-fold and 13-fold higher, respectively.
The mean urinary recovery of two pharmacologically inactive metabolites at steady state was 16+/-5.8% of vorinostat dose as the O glucuronide of vorinostat, and 36+/-8.6% of vorinostat dose as 4-anilino-4-oxobutanoic acid. Total urinary recovery of vorinostat and these two metabolites averaged 52+/-13.3% of vorinostat dose.

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Biological Half-Life
2 hours
... Patients (n = 23) received single doses of 400 mg vorinostat on day 1 (fasted) and day 5 (fed) with 48 hours of pharmacokinetic sampling on both days. Patients received 400 mg vorinostat once daily on days 7 to 28. On day 28, vorinostat was given (fed) with pharmacokinetic sampling for 24 hours after dose. The apparent t(1/2) of vorinostat was short (approximately 1.5 hours). ...
The mean terminal half-life was /approximately/ 2.0 hours for both vorinostat and the O-glucuronide metabolite, while that of the 4-anilino-4-oxobutanoic acid metabolite was 11 hours.

Hepatotoxicity
In clinical trials of vorinostat in patients with CTCL, the rates of serum enzyme elevations during therapy were rarely mentioned and only occasional episodes of mild elevations were recorded. Minor elevations in serum ALT levels occurred in 15% to 45% of patients, but values above 5 times ULN were rare and there were no reports of hepatitis, jaundice or clinically apparent liver injury among the treated subjects. Vorinostat has had limited clinical use, but there have been no published reports of its association with significant liver injury.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
71%

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Interactions
Vorinostat is not expected to affect the pharmacokinetics of other agents. As vorinostat is not eliminated via the CYP pathways, it is anticipated that vorinostat will not be subject to drug-drug interactions when co-administered with drugs that are known CYP inhibitors or inducers. However, no formal clinical studies have been conducted to evaluate drug interactions with vorinostat.
Potential prolongation of prothrombin time (PT) or international normalized ratio (INR) in patients receiving vorinostat concomitantly with coumarin-derivative antiacoagulants. PT and INR should be carefully monitored.
Potential severe thrombocytopenia and GI bleeding in patients receiving vorinostat concomitantly with other histone deacetylase (HDAC) inhibitors (eg, valproic acid). Platelet count should be monitored every 2 weeks for the first 2 months.
Suberoylanilide hydroxamic acid (SAHA), a histone deacetylase inhibitor, has been shown to inhibit the development of N-methylnitrosourea (NMU)-induced rat mammary tumors when fed in the diet continuously for the duration of the carcinogenic process. The present study was designed to determine whether the inhibitory effects of SAHA occur during the initiation process or at subsequent stages in the carcinogenic process. In addition, animals with established NMU tumors were administered SAHA to determine whether SAHA could inhibit the continued growth of established mammary tumors. It was found that SAHA fed at 900 ppm in the diet inhibited tumor yields when administered from 14 days prior to NMU administration to termination (-14 to +130) and from +14 and +28 days to termination. However, SAHA had no effect on tumor yields when administered from -14 to +14 or from -14 to +50 days and then returned to the control diets for the remainder of the experimental period (130 days). These results indicate that the inhibitory effects of SAHA are not exerted at the initiation phase of NMU-induced mammary tumorigenesis and appear, instead, to inhibit the subsequent stages in tumor development. Of most interest was the ability of SAHA to inhibit the growth of established mammary tumors. Administration of SAHA in the diet at 900 ppm resulted in significant inhibition of established tumor growth. Thirty-two percent of SAHA-treated tumors exhibited partial regression compared to 12% of controls, growth was stabilized in 24% of treated tumors compared to 12% of controls while 11% exhibited complete regression compared to 0% of controls. Collectively, SAHA-treated tumors exhibited a 7-fold reduction in growth compared to untreated tumors over the test period. ...

[1]. Proc Natl Acad Sci U S A . 1998 Mar 17;95(6):3003-7.

[2]. Cancer Res . 2000 Sep 15;60(18):5165-70.

[3]. Cancer Res . 2001 Dec 1;61(23):8492-7.

[4]. Proc Natl Acad Sci U S A . 2003 Feb 18;100(4):2041-6.

[5]. Proc Natl Acad Sci U S A . 2004 Jan 13;101(2):540-5.

[6]. Clin Cancer Res . 2004 Jun 1;10(11):3839-52.

[7]. Clin Cancer Res . 2008 Sep 1;14(17):5385-99.

[9]. Blood . 2008 Aug 1;112(3):793-804.

Therapeutic Uses
Antineoplastic Agents; Histone Deacetylase Inhibitors
Vorinostat is indicated for the treatment of cutaneous manifestations in patients with cutaneous T-cell lymphoma who have progressive, persistent or recurrent disease on or following two systemic therapies. /Included in US product label/

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Drug Warnings
Risk of pulmonary embolism and deep-vein thrombosis. Clinicians should be alert to signs and symptoms of such effects, especially in patients with a prior history of thromboembolic events.
Risk of dose-related thrombocytopenia and anemia. Dosage should be adjusted or therapy discontinued if thrombocytopenia or anemia occurs.
Risk of nausea, vomiting, and diarrhea; antiemetic and/or antidiarrheal agents may be required. To prevent dehydration, fluid and electrolyte replacement should be administered. Preexisting nausea, vomiting, and diarrhea should be adequately controlled before initiating therapy.
Risk of hyperglycemia. Serum glucose concentrations should be monitored, especially in patients with known or possible diabetes mellitus. Diet and/or antidiabetic therapy should be adjusted, if needed.
For more Drug Warnings (Complete) data for Vorinostat (23 total), please visit the HSDB record page.

C14H20N2O3

264.3

264.147

C, 63.62; H, 7.63; N, 10.60; O, 18.16

149647-78-9

5311

White to off-white solid powder

1.2±0.1 g/cm3

161-162°C

1.567

0.86

3

3

8

19

276

0

O=C(C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])O[H])=O)N([H])C1C([H])=C([H])C([H])=C([H])C=1[H]

WAEXFXRVDQXREF-UHFFFAOYSA-N

InChI=1S/C14H20N2O3/c17-13(15-12-8-4-3-5-9-12)10-6-1-2-7-11-14(18)16-19/h3-5,8-9,19H,1-2,6-7,10-11H2,(H,15,17)(H,16,18)

N'-hydroxy-N-phenyloctanediamide

MK0683; SAHA; M344; CCRIS 8456; CCRIS8456; CCRIS-8456; HSDB 7930; Vorinostat; suberoylanilide hydroxamic acid; MK-0683; MK 0683; MK0683; M344; HSDB 7930; suberoylanilide hydroxamic acid; Zolinza; N-hydroxy-N'-phenyloctanediamide; N1-hydroxy-N8-phenyloctanediamide; Suberanilohydroxamic acid; Trade name: Zolinza

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.46 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (9.46 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 4: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 5: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 6: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 7: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 8: ≥ 2.08 mg/mL (7.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 9: 2% DMSO+30% PEG 300+ddH2O: 5mg/mL

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Solubility in Formulation 10: 3.33 mg/mL (12.60 mM) in 20% HP-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)