Hydralazine-valproate: a repositioned drug combination for the epigenetic therapy of cancer.

Drug Evaluation

1.

Introduction

2.

Overview of the market

3.

Formulation of

Expert Opin. Drug Metab. Toxicol. Downloaded from informahealthcare.com by University of Sydney on 08/29/14 For personal use only.

hydralazine--valproate

Hydralazine--valproate: a repositioned drug combination for the epigenetic therapy of cancer Alfonso Duenãs-Gonzalez†, Jaime Coronel, Lucely Cetina, Aurora Gonza´lez-Fierro, Alma Chavez-Blanco & Lucia Taja-Chayeb †

4.

Clinical efficacy

5.

Safety and tolerability

6.

Postmarketing surveillance

7.

Regulatory affairs

8.

Conclusion

9.

Expert opinion

Instituto de Investigaciones Biomedicas UNAM/Instituto Nacional de Cancerologia, Mexico

Introduction: DNA methylation (DNMTi) and histone deacetylase inhibitors (HDACi) are in development for cancer therapy. So far, four epigenetic drugs are approved for myelodysplastic syndrome (MDS) and cutaneous T-cell lymphoma (CTCL). The combination of hydralazine-valproate (TRANSKRIP) is being repositioned as an oral DNMT and HDAC inhibitor. Areas covered: Brief discussion on the current status of epigenetic drugs and studies published on the preclinical and clinical development of the hydralazine-valproate combination. Expert opinion: Drug repositioning is a strategy for prompt and cost-efficient drug discovery. There is evidence that combining DNMTi with HDACi would be more efficacious than administering each agent on its own. Hydralazinevalproate is safe when used alone or in combination with chemotherapy or chemoradiation. The fact that both drugs are orally administered is another advantage over current epigenetic drugs. This combination is promising but larger studies are needed. Among these, the randomized Phase III trials in advanced and in locally advanced cervical cancer combined with chemotherapy and cisplatin-radiation respectively, would eventually confirm its efficacy. Studies on MDS and CTCL would also eventually prove the efficacy of hydralazine valproate so that in the coming years hydralazine-valproate could have a role in cancer epigenetic therapy. Keywords: cervical cancer, cutaneous T-cell lymphoma, drug repositioning, epigenetics, hydralazine, myelodysplastic syndrome, orphan drug, valproate Expert Opin. Drug Metab. Toxicol. [Early Online]

1.

Introduction

Epigenetics is generally understood to be the study of heritable regulatory changes that do not involve any changes in the DNA sequence. The functional unit of the DNA is the nucleosome, and its status within the nuclear context determines gene expression [1]. Epigenetics, therefore, can be referred as the study of all elements that participate in nucleosome-chromatin regulation as determinants of gene expression. In the 1970s, it was discovered that the addition of a methyl group at the fifth position of the cytosine in a CpG dinucleotide could inactivate the expression of genes [2]. Thus, DNA methylation driven by DNA methyltransferase (DNMT) enzymes gained momentum as the most important epigenetic factor [3]. Afterwards, the regulatory effect of histone acetylases and deacetylases (HDAC) added another level of complexity by regulating the acetylation status at the lysine tails at the histone core of the nucleosomes [4]; in addition, it was discovered that these enzymes were also able to bind and regulate DNMT and other proteins having a DNA methylation binding domain [5,6]. The recognition that histone proteins can undergo in addition 10.1517/17425255.2014.947263 © 2014 Informa UK, Ltd. ISSN 1742-5255, e-ISSN 1744-7607 All rights reserved: reproduction in whole or in part not permitted

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A. Duenãs-Gonzalez et al.

Box 1. Drug summary.


Drug name Phase Indication

Pharmacology description Route of administration Chemical structure

Hydralazine--magnesium valproate Phase II and III Cervical cancer (Mexican Regulatory Authority) Orphan drug designation for myelodysplastic syndromes and cutaneous T-cell lymphoma Inhibitor of DNA methyltransferase and histone deacetylase Oral Magnesium valproate H3C

CH3

Mg2+ OH

O

Hydralazine N N NH H2N

Pivotal trial(s)

[61]

Pharmaprojects -- copyright to Citeline Drug Intelligence (an Informa business). Readers are referred to Pipeline (http://informa-pipeline. citeline.com) and Citeline (http://informa.citeline.com).

potential effectiveness of epigenetic-targeted therapies in hematological malignancies, but successful application of epigenetic drugs in solid tumors remains to be realized. On the other hand, drug discovery and development is an expensive, time-consuming and risky enterprise. In order to accelerate the drug development process with reduced risk of failure and relatively lower costs, pharmaceutical companies have adopted drug repositioning as an alternative. This strategy involves exploration of drugs that have already been approved for the treatment of other diseases and/or whose targets have already been discovered. Various techniques, including data mining, bioinformatics and usage of novel screening platforms, have been used for identification and screening of potential repositioning candidates. However, challenges in clinical trials and intellectual property issues may be encountered during the repositioning process. Nevertheless, such initiatives not only add value to the portfolio of pharmaceutical companies but also provide an opportunity for academia and government laboratories to develop new and innovative uses of existing drugs for cancer and other diseases [15,16]. This review focuses on Alpharma’s (TRANSKRIP), which contains the blood pressure-lowering hydralazine and the antiepileptic magnesium valproate, that are repositioned as DNMTi and HDACi, respectively. This drug is registered in Mexico for the treatment of advanced cervical cancer in combination with chemotherapy, and it gained orphan drug designation by the US FDA for MDS and CTCL. 2.

to acetylation/deacetylation, methylation, phosphorylation and other modifications led to discovery of histone methyltransferases, histone demethylases and their numerous interactions with both components of nucleosomes, histones and DNA itself as well as a number of protein complexes having nucleosome-remodeling activities [7,8]. The discovery of noncoding RNAs as another set of epigenetic players that are themselves regulated by epigenetic machinery elements and the discovery of DNA demethylases [9] -- the ten eleven translocation enzymes -- which convert 5-methylcytosine to 5-hydroxymethylcytosine have resulted in an unanticipated higher complexity of how epigenetics drives the fine-tune regulation of gene expression [10]. The fact that epigenetic traits are dynamic and reversible, and the simple concept prevailing when DNA methylation and histone deacetylation were thought as the main responsible for the transcriptional silencing of tumor suppressor genes in cancer led to the development of DNA methylation and histone deacetylase inhibitors (DNMTi and HDACi), respectively. So far, the US FDA has approved four epigenetic-targeted anticancer drugs. Two DNMTi: [Celgene’s Vidaza (azacitidine) and Eisai’s Dacogen (decitabine)] for myelodysplastic syndrome (MDS) [11,12], and two HDACi: [Merck’s Zolinza (vorinostat) and Celgene’s Istodax (romidepsin)] for cutaneous T-cell lymphoma (CTCL) [13,14]. These initial successes demonstrate the 2

Overview of the market

Among cancer-targeted therapies, epigenetic drugs are highly promising due to the fact that they potentially can be used for all cancers in general as epigenetic alterations (at least DNA methylation and histone deacetylation) are common to both hematological and solid neoplasias. In addition, epigenetic drugs, especially those having an excellent safety profile suitable for long-term administration, can bear potential for cancer chemoprevention [17,18]. Considerable activity and interest are focused on the therapeutic segment of the epigenetic field despite the current market is small (MDS and CTCL). As expected, epigenetic drugs may prove to be useful for treatment of hematological cancers and solid tumors. Currently, over 40 large and emerging companies are active in this field searching for novel non-nucleoside DMNTi, whereas programs for developing HDACi are very competitive ones. In the field of DNMTi, the two nucleoside analogs are myelotoxic and not well suited to be combined with cytotoxic chemotherapy for solid tumors, whereas for HDACi there are certainly a number of agents in late stage of development [19]. Introduction to hydralazine and magnesium valproate 2.1.1 Chemistry 2.1

Hydralazine (C8H8N4), molecular weight 160.175, is also known as 1-hydrazinophthalazine, apresolin, apresoline,

Expert Opin. Drug Metab. Toxicol. (2014) 10(10)


Hydralazine-magnesium valproate

hydralazin, hydrazinophthalazine, hypophthalin, apressin, aprezolin, hidralazin and has long been utilized for management of hypertensive disorders and heart failure; nonetheless, its current use is almost limited to hypertensive disorders during pregnancy [20]. In 1988, it was shown that hydralazine induced selfreactivity in cloned T-cell lines and DNA hypomethylation, and in 2003 its ability to restore expression of tumor suppressor genes silenced by promoter hypermethylation in cancer cell lines and primary tumors was shown (Box 1) [21,22]. Magnesium valproate (C16H30MgO4), molecular weight 310.712, also known as magnesium dipropylacetate, magnesium 2-propylvalerate, magnesium 2-propylpentanoate, is a fatty acid with anticonvulsant properties used in the treatment of epilepsy and bipolar disorder. The mechanisms of its therapeutic actions are not well understood. It may act by increasing GABA levels in the brain or by altering the properties of voltage-dependent sodium channels [23]. In 1991, it was found that valproic acid causes N-terminal tail hyperacetylation of histones H3 and H4 in vitro and in vivo and was proven to inhibit HDAC enzymatic activity directly at a concentration of 0.5 mM [24]. 2.1.2

Pharmacodynamics Hydralazine

2.1.2.1

In silico models have demonstrated that residues Lys162 and Arg 240 within the enzyme active site interact with hydralazine at distances between these residues and hydralazine nitrogen atoms not exceeding 4A . These interactions are energetically stable, supporting that hydralazine may inhibit DNMT [25]. These observations have been further supported by Singh et al., who also found in a validated model of human DNMT1 enzyme that hydralazine has inhibitory activity upon this enzyme [26]. Though it reduces the enzyme activity weakly [27] , this may not be disadvantageous as a strong DNA demethylating agent may induce genomic instability. Other authors have reported that hydralazine decreases DNMT1, DNMT3a and DNMT3b in different model systems [28,29]. This discrepancy with regard to hydralazine’s precise mechanism of action as DNA methylation inhibitor extends to other non-nucleoside DNMTi, which may stem from technical issues [30]. A number of preclinical studies have further confirmed the DNA methylation inhibitory activity of hydralazine upon genes such as APC [31], stanniocalcin-2 [32], WWOX [33], SERCA2a [34], Tead4 [35] and androgen receptor [29]. Hydralazine-induced DNA demethylation reverses doxorubicin resistance in a MCF-7 model that is associated with increased DNMTs, global DNA methylation and upregulation of MDR gene. The results of this study in the MCF-7/Dox model demonstrate that global DNA hypermethylation participates in the development of doxorubicin resistance and that the demethylating agent hydralazine can revert the resistant phenotype [36]. Of further interest, hydralazine reverses gemcitabine resistance and leads to hENT1 and dCK gene reactivation in a DNA promoter methylation-independent manner via

inhibition of G9A histone methyltransferase, which methylate H3K9m2 at these promoters [37]. More recently, it has been reported that hydralazine downregulates a number of proteins from the EGFR signaling pathway in prostate cancer cells [29]. Magnesium valproate Valproic acid inhibits HDAC1 in vitro in a dose-dependent manner, with an inhibitory concentration of 0.4 mM, falling within the therapeutic range in humans. Valproic acid inhibits HDACs other than HDAC1, including HDAC1, -2, -3, -4 and -8 with a 50% inhibition between 0.5 and 2 mM and induces hyperacetylation of H4 and nonhistone proteins such as p53 at concentrations as low as 1 -- 2 mM [38] Gurvich et al. found that valproic acid inhibits class I HDACs (HDACs 1 -- 3) with IC50 values ranging from 0.7 to 1 mM and inhibits class II subclass I HDACs 4, -5 and -7 with IC50 values ranging from 1 to 1.5 mM; on the contrary, it does not inhibit HDAC 6 or -10 (class II subclass II). Interestingly, relative valproic acid-analog potencies to inhibit HDACs correlate with their potencies in inducing leukemia cell-line differentiation, which led the authors to conclude that valproic acid effects on differentiation are most likely due to the inhibition of HDACs [39]. Further, in breast cancer cells, it has been shown that valproic acid depletes several members of structural maintenance of chromatin proteins, SMC-associated proteins, DNMT and heterochromatin proteins, which lead to chromatin decondensation, enhanced DNA sensitivity to nucleases, and increased DNA interaction with intercalating agents. This modulation is not a direct -- but is rather a downstream -- effect of histone acetylation reversible upon drug withdrawal [40]. In a variety of in vitro and in vivo systems, valproic acid has shown potent antitumor effects by modulating multiple pathways, including cell cycle arrest, apoptosis, angiogenesis, metastasis, differentiation and senescence. These effects appear to be cell typespecific, which may also depend on the differentiation level and the underlying genetic alterations [41,24]. 2.1.2.2

2.1.3

Pharmacokinetics and metabolism Hydralazine

2.1.3.1

This drug is well absorbed through the gastrointestinal tract, but systemic bioavailability is low. Because the acetylated compound is inactive, the dose required to produce a systemic effect is higher in fast acetylators. N-acetylation of hydralazine occurs in bowel and/or liver. Hydralazine’s half-life is 1 h, and systemic clearance of the drug is ~ 50 ml/kg/min. Systemic metabolism of hydralazine depends on hydroxylation followed by conjugation with glucoronic acid in liver, which is not dependent on acetylation rate; therefore, half-life does not differ to a great degree between slow and fast acetylators [42]. Hydralazine peak concentration in plasma and the drug’s peak hypotensive effect occurs within 30 -- 120 min of ingestion. Although its half-life in plasma is ~ 1 h, the duration of the hypotensive effect can last as long as 12 h. Hydralazine’s antihypertensive


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effect possesses no clear dose--response effects. The dose varies from 10 mg four times a day (q.i.d.) to 50 mg q.i. d.. After stabilization with multiple daily doses, a twice-daily dose regimen can be effective. Slow acetylators require a lower dose. For heart failure, recommended doses are higher (up to 800 mg daily or more); as a rule, 10 -- 100 mg q.i.d. can be effective. Common side effects include headache, nausea, flushing, low blood pressure, palpitation, tachycardia, dizziness and angina pectoris. Hydralazine causes autoimmune reactions, among which the drug-induced lupus-like syndrome is the most common [43]. Magnesium valproate Valproic acid is rapidly absorbed after oral administration, with peak serum levels occurring ~ 1 -- 4 h after a single oral dose. Valproic acid half-life in serum falls typically within the range of 7 -- 16 h. When the drug is administered with meals, a slight delay in absorption occurs, but this does not affect total absorption. Its distribution throughout the body is rapid, the drug is strongly bound (95%) to human plasma proteins and decreases in the extent of protein binding and variable changes in valproic acid clearance and elimination may result from dosage increases. As an antiepileptic, the therapeutic plasma concentration is believed to range from 50 to 100 µg/ml. Valproic acid is primarily metabolized to the glucoronide conjugate in the liver and only very little unmetabolized parent drug is excreted in urine. Valproic acid and its metabolites are eliminated mainly in urine, with minor amounts appearing in feces [44]. This drug is in general well tolerated by patients. Neurological side effects such as sedation, dizziness and tremor, as well as mild gastrointestinal toxicities, usually take place early during treatment. The most serious adverse events are liver failure and teratogenicity. Fatal hepatotoxicity is rare (~ 1:15,000) and principally occurs in children aged < 2 years treated with multiple drugs. It can induce birth defects such as neural tube closure defects and other malformations when administered during early pregnancy [45]. Pharmacokinetic studies comparing various salts of the valproic acid demonstrate that magnesium valproate and sodium valproate are bioequivalent in terms of bioavailability. Magnesium valproate shows lesser interindividual variability and therefore it offers additional advantages in comparison with sodium valproate. The gastrointestinal absorption of magnesium valproate is slower than that of sodium valproate, whereby plasma levels rise more slowly to similar maximum concentrations. This helps to maintain more constant levels for longer periods [46,47]. 2.1.3.2

3.

Formulation of hydralazine--valproate

Hydralazine--valproate is an extended-release product formulated in two versions depending on the individual acetylating metabolism of the patient. The packaging box of hydralazine--valproate includes both drugs, and each weekly blister 4

has 28 tablets: 7 yellow with hydralazine and 21 white with magnesium valproate. Fast acetylators are prescribed with hydralazine--valproate where the 7 yellow tablets of hydralazine contain 182 mg; and the slow acetylators where the yellow 7 tablets contain 83 mg of hydralazine. The white tablets of magnesium valproate contain 700 mg in either presentation. The pharmacokinetics of orally administered hydralazine was evaluated in 26 healthy volunteers (13 slow and 13 fast acetylators) after a hydralazine single dose of 182 mg formulated in a controlled-release tablet. The acetylation phenotype was determined using sulfamethazine (a single oral dose of 500 mg taken in the morning after an overnight fast). Urine was collected 0 -- 6 h after medication. Acetylated and unchanged sulfamethazine were determined by the Bratton-Marshall method and the ratio of acetylated/ unchanged sulfamethazine determined. Subjects were classified as fast or slow taking a cut-off ratio of 70% [48]. The Cmax and Tmax of hydralazine for fast acetylators were 208.4 SD ± 56.9 ng/ml and 2.8 SD 2.5 h, respectively. The corresponding results for slow acetylators were 470.4 SD 162.8 ng/ml, and 4.4 SD ± 3.1 h. Healthy volunteers with a fast acetylator phenotype had no clinically significant changes in blood pressure and heart rate or any other side effect; however, slow acetylators had transient episodes of headache, tachycardia and faintness. Among 85 cancer patients that received either 182 or 83 mg of hydralazine daily, according to their acetylator status, the mean concentrations of hydralazine in plasma were not statistically different 239.1 and 259.2 ng/ml for fast and slow acetylators, respectively, p = 0.3868. These results demonstrate that the administration of dose-adjusted controlled-release hydralazine according to the acetylation status of cancer patients yields similar levels of hydralazine [48]. More recently, the pharmacokinetic parameters of a single dose of hydralazine in 24 h (one tablet with 83 mg for slow acetylators and one tablet with 182 mg for fast acetylators) and three fixed doses of valproate (one tablet of controlled-release with 700 mg every 8 h) were evaluated in healthy, genetically (acetylator genotype) selected volunteers. Selection was performed based on their NAT2 enzyme activity as deduced from their genotype after genotyping three SNPs that cover 99.9% of the NAT2 gene variants in the Mexican population. The study showed almost identical AUC 0 -- 48 h and Cmax (1410 ± 560 vs 1446 ± 509 ng h/ml and 93.4 ± 16.7 vs 112.5 ± 42.1 ng/ml) in both groups with NAT2 genotypeadjusted doses, whereas the multidose parameters of valproate were not significantly affected neither by the selection of the NAT2 genotype nor by the co-administration of 83 or 182 mg of hydralazine [49]. Altogether, these results indicate that dose adjustments of hydralazine for slow and rapid acetylators are adequate when acetylation status is determined by phenotype with sulfamethazine. One ongoing study is aimed to determine whether genotyping of NAT2 gene polymorphism could replace phenotyping.



4.

Clinical efficacy

Phase I studies 4.1.1 Hydralazine


4.1

Sixteen patients with untreated cervical carcinoma were studied in the window from the diagnosis to the beginning of definitive treatment with chemoradiation. These patients had a macroscopic tumor accessible for punch biopsy. Hydralazine was administered to cohorts of four patients at the following dose levels: i) 50 mg/day; ii) 75 mg/day; iii) 100 mg/day and iv) 150 mg/day for 10 days. Tumor biopsies and peripheral blood samples were taken the day before and at day 11. The genes APC, MGMT, ER, GSTP1, DAPK, RARb, FHIT and p16 were evaluated pre- and post-treatment for DNA promoter methylation and gene expression by methylationspecific PCR (MSP) and RT-PCR, respectively, in each of the tumor samples. Methylation of the imprinted H19 gene and the ‘normally methylated’ sequence clone 1.2 was also analyzed. Global DNA methylation was analyzed by capillary electrophoresis and cytosine extension assay. Toxicity was evaluated using the NCI Common Toxicity Criteria. Hydralazine was well tolerated. Methylation analysis of the biopsies taken before and after 10 days of hydralazine administration was performed in all 16 patients. Methylation results involving the genes analyzed were variable. Overall, 70% (89 out of 128) of the pretreatment samples analyzed (8 genes for each of the 16 pretreatment biopsies) had at least one methylated gene, and all 16 patients had at least one methylated gene. Analysis by individual genes showed the following rates of methylated genes: APC (94%), ER (25%) FHIT (88%), GSTp1 (88%), MGMT (81%), p16 (19%), RARb (62%),and DAPK (100%), irrespective of the dose of hydralazine used, the post-treatment biopsies showed a variable demethylation rate according to the gene, varying from 15% (2/13 samples) for the MGMT gene, to 67% of demethylation for the p16 gene (2 out of 3 samples). There was no correlation between the dose of hydralazine and the demethylation rate, most likely due to the small sample size. Gene expression analysis showed only 12 informative cases, of these 9 (75%) re-expressed the gene. There was neither change in the methylation status of H19 and clone 1.2 nor changes in global DNA methylation. This study concluded that hydralazine at doses between 50 and 150 mg/day are well tolerated and effective to demethylate and reactivate the expression of tumor suppressor genes without significantly affecting global DNA methylation [50]. Magnesium valproate Twelve patients with untreated cervical carcinoma were studied in the window from the diagnosis to the beginning of definitive treatment with chemoradiation. These patients had a macroscopic tumor accessible for punch biopsy and the study consisted in the administration of magnesium valproate in cohorts of four patients at 20, 30 and 40 mg/kg 4.1.2

from day 1 to 5. At day 6, tumor biopsies and blood samples were taken. All patients completed the study medication, and the mean daily dose for all patients was 1890 mg. According to dose level, the mean daily dose for the 20 mg/kg dose level was 1245 mg (range 1000 -- 1400 mg), whereas it was 2000 mg (1800 -- 2100) for the 30 mg/kg and 2425 mg (1800 -- 3300) for the 40 mg/kg dose level. Pharmacodynamic evaluation of histone acetylation and HDAC inhibition was done in both the primary tumor (in 10 patients) and the peripheral blood at day 6 (in 4 patients). A clear increase in tumor total H3 acetylation as evaluated by western blot was observed in 9 out of 10 (90%) patients, whereas for total H4 hyperacetylation was seen in 7 out of 10 (70%) patients. Both histones were hyperacetylated in six (60%) individuals. Further, evaluation of HDAC inhibition was observed in 8 of 10 (80%) patients, whereas patients 3 and 8 (20%) had either no change or a mild increase in deacetylase activity. Unfortunately, the small sample size did not allow for establishing a correlation with H3 and H4 histone acetylation with HDAC inhibition. Nevertheless, two patients with no HDAC inhibition showed histone hyperacetylation. Histone hyperacetylation was observed in peripheral blood of the four patients (100%) that could be evaluated and correlated with hyperacetylation in tumors. These effects were achieved at plasma concentration of valproic acid that ranged from 73.6 -- 170.49 µg/ml, although there was lack of correlation between plasma levels with dose level. Mean values for patients were 94.06 µg/ml at 20 mg/kg, 123.46 µg/ml at 30 mg/kg and 90.93 µg/ml at 40 mg/kg. These data indicate that at the range of doses tested there is a plateau in the molecular response (100, 50 and 50%) for the three tested doses [51]. 4.2

Phase II studies Breast cancer

4.2.1

Eligible patients for the study were 18 years of age and older, ECOG 0 -- 2, and adequate organic function with histologically proven invasive T2-3, N0-2 and M0 (stages IIB--IIIA) breast carcinoma. Patients were treated with a daily dose of a slow-release formulation of hydralazine tablets containing either 182 mg for rapid acetylators or 83 mg for slow acetylators and magnesium valproate tablets of 700 mg at a dose of 30 mg/kg three-times a day. Both hydralazine and magnesium valproate were administered from day -7 until the last day of the fourth chemotherapy cycle, which consisted on doxorubicin 60 mg/m2 and cyclophosphamide 600 mg/m2 at day 1 every 21 days. Sixteen patients were included, and all received four cycles of doxorubicin and cyclophosphamide plus hydralazine and magnesium valproate as planned. In addition, all patients were evaluated for clinical response and toxicity, and 14 for pathological response. There were five (31%) clinical complete responses and eight (50%); partials for an overall response rate of 81% and no one progressed. Among pathological responses, one (6.6%) had complete response; however, in 70% of cases, the residual disease was < 3 cm, 33% of cases had pathological negative lymph


5



nodes and no case had extranodal extension. The pharmacodynamic evaluation showed a statistically significant reduction in the 5mC content from the DNA extracted from peripheral blood cells as well as a reduction in the enzymatic activity of HDAC. These effects were achieved at mean concentrations of valproic acid that varied from 78.5 to 100.3 µg/ml, for an overall mean of 87.5 µg/ml. Microarray analysis was intended to be done in all patients, but adequate samples only sufficed for analysis in three patients (three pretreatment biopsies and only one post-treatment). The number of genes up- or downregulated by at least threefold difference was 1091 and 89, respectively (GEO GSE6304). These data led the authors to conclude that valproic acid and hydralazine exert their proposed molecular effects of HDAC inhibition, DNA demethylation and gene reactivation in primary tumors of patients with breast cancer, and that despite the regimen containing epigenetic drugs with cytotoxics was apparently more myelotoxic it may have increased the efficacy of chemotherapy [52]. Refractory solid tumors This Phase II study was undertaken on the basis that chemotherapy resistance, either innate or acquired, requires expression changes in a large number of genes for its development, being possible that epigenetic-mediated changes could be the driving force responsible for chemotherapy resistance [53]. Thus, it could be expected that DNMTi and HDACi would overcome chemotherapy resistance. Eligible patients were 18 years of age and older who have an ECOG performance status, 0 -- 2; and adequate organ function with histologically proven malignant solid tumors who were receiving their second, third or fourth line of palliative chemotherapy and who showed -- at the second or third chemotherapy course -progressive disease as their maximum response according to Response Evaluation Criteria in Solid Tumors (RECIST) or Gynecologic Cancer Intergroup (GICG) CA125 criteria in the case of patients with ovarian cancer. Schedules comprised cisplatin, carboplatin, paclitaxel, vinorelbine, gemcitabine, pemetrexed, topotecan, doxorubicin, cyclophosphamide and anastrozole. A total of 17 patients whose primary tumors sites were cervix (3), breast (3), lung (1), testis (1) and ovarian (7) carcinomas were evaluable for toxicity, and 15 for response. Most patients were heavily treated and had received two and three previous lines of treatment. After entering the protocol, of note, patients restarted the schedule employing the same dose they received previously. Clinical benefit (complete or partial response and disease stabilization) was observed in 12 (80%) of the 15 patients, four partial and eight stabilization of disease. Among partial responses, three were in ovarian cancer according to IGCG CA125 criteria, and a PR was observed in one patient with cervical cancer as evaluated by MRI of a supraclavicular lymph node. Regarding patients with stable disease, four occurred in patients with ovarian cancer and one each in patients with cervix, lung, testis and breast cancers. The majority of patients who exhibited response or 4.2.2

6

stabilization also demonstrated improvement in symptoms such as dyspnea, cough and pain. Regarding evaluation of response according to the total number of evaluable disease sites, there were 16 evaluable lesions in the 8 cases excluding ovarian malignancies. Of these, one (6.6%) complete and three (18.75%) partial responses were achieved, whereas nine (56.2%) had disease stabilization. These antitumor effects resulted in a median progression-free survival of 3.3 months (range 2.4 -- 5.7 months) and median survival was 6.1 months (range 3.8 -- 12.8 months). Regarding pharmacodynamic end points, there was a mean reduction of 23.3% in HDAC activity at day 8 of treatment with the epigenetic agents. These effects were seen at a mean concentration of 86.3 µg/ml of valproic acid in plasma. This study was not intended to have post-treatment samples of tumors; instead, the promoter methylation of three genes (hMLH, RARb and DAPK) were analyzed from serum DNA. Overall, of 15 informative MSP reactions, 5 (33.3%) showed promoter demethylation. As for patients, eight of nine were informative and 4 (50%) had demethylation of at least one gene. These results suggest that the response and disease stabilization rates observed with valproate and hydralazine may have resulted from the overcoming of epigenetic changes, mediating chemotherapy resistance [54]; however, the limited sample size cautions against the generalizability of these results. Cervical cancer In this Phase II study in cervical cancer, hydralazine and valproate were added to standard cisplatin chemoradiation in FIGO stage IIIB patients under the rational that hydralazine and valproate increase the cytotoxic effect of cisplatin and radiation [55,56], and upregulate human leukocyte antigen class-I antigen expression and antigen-specific cytotoxic T-lymphocyte response as well as natural killer cell activity [57,58]. Eligible patients were FIGO stage IIIB, aged more than 18 years, with an ECOG performance status, 0 -- 2; and adequate organ function. Treatment included the combination of hydralazine valproate as described above starting 7 days before weekly cisplatin at 40 mg/m2 and external and intracavitary pelvic radiation. The results demonstrate that out of 18 patients evaluable for response, all had a clinical complete response at the end of external radiation. The comparison between pre- and post-treatment biopsies (after the 7 days of treatment with hydralazine and valproate) showed moderate-to-intense infiltration of tumor and stroma, composed mainly of lymphocytes as well as an increase in connective tissue with fragmentation of solid malignant nests, with a trabecular pattern, indicating that these drugs exert by their own a modest antitumor effect. The mean concentrations in plasma of valproic acid at weeks 1, 4 and 7 were 66.4, 63.7 and 63.5 µg/ml, respectively, for an overall mean of 64.5 µg/ml [59]. Ten pairs of tumor samples before and after hydralazine valproate (at day 7) could be analyzed for global gene expression by microarray analysis. There were 964 genes 4.2.3




upregulated. The two pathways possessing the highest number of upregulated genes comprised the ribosome protein and the oxidative phosphorylation pathways, followed by MAPK signaling, tight junction, adherens junction, actin cytoskeleton, cell cycle, focal adhesion, apoptosis, proteasome, Wnt signaling, and antigen processing and presentation pathways. Upregulated genes by hydralazine valproate clustered with downregulated genes in untreated primary cervical carcinomas and were more alike as compared with upregulated genes from untreated patients in terms of gene ontology. Increased acetylated p53 was also observed [60]. 4.3

Phase III studies Advanced cervical cancer

4.3.1

Preliminary results of a placebo-controlled Phase III study. The reversing of epigenetic aberrations using the inhibitors of DNA methylation and HDACs may have therapeutic value in cervical cancer. In this randomized Phase III study, either placebo or hydralazine valproate was added to cisplatin topotecan in advanced cervical cancer. Patients received hydralazine at 182 mg for rapid, or 83 mg for slow acetylators, and valproate at 30 mg/kg, beginning a week before chemotherapy and continued until disease progression. Response, toxicity and PFS were evaluated. A total of 36 patients (17 hydralazine valproate and 19 placebo) were included. The median number of cycles was six. There were 4 (23.5%) and 1 (5%) partial responses, whereas 8 (47%) and 12 (63%) patients progressed in the hydralazine valproate and placebo groups, respectively. At a median follow-up time of 7 months (1 -- 22), the median PFS was 6 months for chemotherapy and placebo and 10 months for chemotherapy and hydralazine valproate (p = 0.0384, two-tailed). Although preliminary, this study represented the first randomized clinical trial to demonstrate a significant advantage in progression-free survival for epigenetic therapy over one of the current standard combination chemotherapies in cervical cancer [61]. This study was terminated because of administrative reasons; however, a randomized double-blind Phase III trial comparing hydralazine valproate versus placebo is ongoing using the commonly used regimen of carboplatin paclitaxel, which is noninferior to cisplatin paclitaxel [62]. 5.

Safety and tolerability

The Phase I studies of hydralazine and valproate separately showed as expected a remarkable low toxicity with no grade 3 or 4 events. For hydralazine, nausea, dizziness, fatigue, headache and palpitations were observed in around 50% of patients regardless of the dose (50, 75, 100 or 150 mg at day) [50]. For magnesium valproate, the most common event was grade 2 depressed level of consciousness in 9 out of 10 patients, and grade 1 in one (grade 2 is defined as somnolence or sedation interfering with function but not interfering with activities of daily living). Grade 2 fatigue was observed in four patients [51]. There were no changes in the values of

nonhematological or hepatic parameters except by lymphopenia grade 1 in a patient receiving the lowest dose level of magnesium valproate. Due to the limited sample size in both studies, there was no correlation between the dose level and toxicity. Hydralazine and valproate in combination with cytotoxic chemotherapy

5.1

In the breast cancer study, hematological toxicity was within ranges reported by anthracyclin-based schemas, with no grade 3/4 thrombocytopenia; nevertheless, grades 3/4 anemia and neutropenia in 15 and 35% appears to be slightly higher than that reported [63,64]. Among nonhematological toxicities, drowsiness was the most frequent side effect; it was observed in 31% of cycles, nonetheless in the vast majority of grade 1 severity. Other side effects were tremor, edema, fatigue, nausea/vomiting and headache, mainly grades 1 and 2. Hematological toxicity, however, is increased when hydralazine valproate is used in heavily pretreated patients. In the Phase II study to overcome resistance to chemotherapy, grade 3/4 anemia, leukopenia, neutropenia and thrombocytopenia were 23, 35, 41 and 47%, respectively. In line with this, valproic acid is known to induce tri-l6ineage hematological toxicity [65] and to increase the hematological toxicity of fotemustine and cisplatin [66]. The only toxicity that could be attributed to valproate was drowsiness grades 1 and 2, and regarding hydralazine, no flushing, hypotension, palpitation, tachycardia, dizziness or angina pectoris were observed most likely due to the fact that slow-release formulation decreases hydralazine plasma peaks. When hydralazine valproate is used with cisplatin chemoradiation, the most important toxicity is hematological with grade 3 anemia, leukopenia, neutropenia and thrombocytopenia of 9, 45, 45 and 9%, respectively, which is higher than historical controls treated with cisplatin chemoradiation [67]. 6.

Postmarketing surveillance

Currently, hydralazine valproate is marketed in Mexico, and since its commercialization in 2009, seven safety reports have been sent to the Mexican Regulatory Agency Comisioń Federal para la Proteccioń contra Riesgos Sanitarios (COFEPRIS) . No serious or unexpected adverse events have occurred and no adverse regulatory actions have been taken by COFEPRIS against hydralazine valproate. 7.

Regulatory affairs

Approval in Mexico. This drug has been approved in Mexico by the Health Regulatory Agency COFEPRIS, registry number 091M2009 SSA IV, which was granted on May 2009. The approval was based on the preliminary results of a double-blind randomized Phase III trial with the indication against advanced cervical cancer associated with chemotherapy as first-line therapy [61].


7



Orphan drug designation for MDS and CTCL by the FDA. The success of DNMT and HDACi represents a proof of concept for the use of epigenetic agents for the treatment of hematological malignancies, specifically MDS and CTCL [68]. This fact led us to evaluate MDS as it exhibits a high frequency of tumor suppressor gene methylation, and in addition, deacetylation by increased HDAC activity may also lead to epigenetic silencing of tumor suppressor genes. Combining DNMTi with HDACi in vitro results in synergistic tumor suppressor gene re-expression [69]. In an open Phase II study for previously treated patients with MDS, hydralazine was given according to 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 Toxicity 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 treatments was 2 (range 1 -- 6). Refractory cytopenia with multilineage dysplasia was diagnosed in 10 cases and refractory anemia with excess of blasts in 2. Overall response was documented in 6 (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 1.1 103 to 2.0 103 (in 3 weeks) and platelets from 66 109 to 72 109/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 nontoxic and effective therapy for MDS [70]. Regarding CTCL, this neoplasia overexpresses HDACs and is very sensitive to HDACi, suggesting that this sensitivity extends to all agents belonging to this drug class. In addition, valproic acid has shown strong antitumor effects in a number of lymphoid cell lines [71,72]. On the other hand, although preclinical and clinical data are scarce regarding the effect of DNMTi in CTCL or any other malignant lymphoid neoplasm, the fact that there is extensive promoter methylation in CTCL [73] provides a strong rationale for the use of hydralazine and valproate. In fact, in a case report, a pretreated patient with mycosis fungoides the patient achieved a very fast response, which was maintained for > 18 months [74]. On this basis, the FDA granted orphan drug designation for hydralazine magnesium valproate for both MDS and CTCL [75,76]. 8.

Conclusion

Among targeted cancer therapy, epigenetic agents that inhibit DNMTi and HDACi are of proven efficacy in hematological malignancies. On the other hand, epigenomics has revealed an 8

unanticipated level of epigenetics complexity with the discovery of new epigenetic players. This knowledge has led to the development of new epigenetic drugs beyond ‘first generation’ DNMTi and HDACi drug classes. Nevertheless, the potential of currently FDA-approved epigenetic drugs is not yet fully realized, particularly for the treatment of solid tumors and additional hematological neoplasias. In this context, drug repositioning adds value to the portfolio of pharmaceutical companies to develop innovative uses of existing drugs for cancer. The combination of hydralazine, a blood pressurelowering agent, and the antiepileptic drug valproate are repositioned as DNMTi and HDACi respectively and marketed in Mexico. This is the first epigenetic agent combining these two drug classes that are known to be synergistic in the reactivation of tumor suppressor genes and for antitumor effects in a number of model systems. Because of its safety profile, this agent is well suited for long-term administration as well as for its use in combination with cytotoxic chemotherapy. Preliminary Phase II studies in combination with chemotherapy and/or chemoradiation show promising activity in a number of solid tumors, including cervical, breast and ovarian cancer. The preliminary results of a randomized Phase III placebo-controlled study in advanced cervical cancer demonstrated a statistically significant increase in PFS and trend for better response rate and overall survival; nevertheless, the preliminary nature of these results does not allow to firmly conclude on its efficacy. The rational for the use of this combination in MDS and CTCL is particularly noticeable. In fact, early indications of its potential efficacy stem from a small Phase II study in MDS and a case report of CTCL. These data led the FDA to design hydralazine--valproate as an orphan drug for these two hematological conditions. This agent deserves to be developed to increase the armamentarium of epigenetic drugs for cancer treatment. 9.

Expert opinion

The use of existing drugs for new therapeutic applications in oncology commonly referred as drug repositioning is a way for fast and cost-efficient drug discovery. A the time of this writing, a simple search in PubMed using the words ‘cancer repositioning’ yielded 739 publications. This simple fact underscores the importance of this field; however, no other drug than thalidomide -- approved by the FDA in 2006 for the treatment of multiple myeloma -- has gained FDA approval for cancer treatment [77]. This not necessarily speaks on the lack of or lower efficacy of drugs being studied for repurposing but on purely economical issues. Mailankody and Prasad recently provided a nice perspective on this [78]. The approval of abiraterone touted as having more specific 17-a-hydroxylase inhibitory activity that ketoconazole was based on a randomized trial comparing it with placebo because ketoconazole has never been studied in a powered trial to demonstrate its efficacy on survival. They estimated $68,882,000 just on purchasing abiraterone to compare it




head-to-head with ketoconazole in a noninferiority study [78]. Of course, such a study is unlikely to be performed. These facts suggest that in order to reposition in cancer therapy, innovative models and players other than big pharma for sponsoring clinical trials are needed. The epigenetic therapy of cancer is on need to have increased armamentarium. There is ample experimental evidence and clinical suggestions that combining DNMTi with HDACi would be more efficacious than each agent by its own [79,80]. The combination of hydralazine and valproate is the first therapeutic agent combining these two drug classes, which has been shown to be safe either when used alone or in combination with chemotherapy or chemoradiation. The fact that these drugs are orally administered is another advantage over current epigenetic drugs as there are no approved DNMTi for oral administration. Of course, a much lower price than that for approved DNMTi and HDACi is a factor that should be taken into account should the combination of hydralazine and valproate is eventually approved by main regulatory agencies worldwide. So far there is not enough information on the efficacy of the combination of hydralazine and valproate to define its place among current treatment strategies. In cervical cancer, the only locally approved (Mexico) indication for advanced cervical cancer as an adjuntive to chemotherapy; a 4-month increase in PFS, from 6 to 10 (p = 0.0384), was obtained, whereas there was nonstatistical significant difference in median survival (12 vs 19 months) in favor of the experimental arm. These differences in median survival appear to be in range obtained when bevacizumab is added to chemotherapy in advanced cancer 13.3 versus 17 months [81]. Thus, in the future scenario, hydralazine and valproate plus chemotheray could be as effective as the bevacizumab combination now regarded by some as the new standard of treatment for cervical cancer. The use of hydralazine and valproate plus chemoradiation is also promising with a clinical complete response rate of 100% that deserves further evaluation. On the other hand, the results obtained when hydralazine--valproate were added to the same chemotherapy scheme

to which patients were actively progressing in solid tumors are remarkable with a 80% clinical benefit (PR + SD) at the expense of increased but manageable hematological toxicity, suggesting that epigenetic events are at least partly responsible for drug resistance [53]. Interestingly, these results are supported by a recent Phase I study in which 27 patients with refractory solid tumors received hydralazine and valproate (with no chemotherapy). One partial and five stable disease for 3 -- 6 months (melanoma, soft-tissue sarcoma, head and neck, ovarian and breast) were observed with 3 survivors at 16, 18 and 18 months. No grade 3 or 4 toxicity events occurred [82]. Regarding hematological neoplasias, again, yet preliminary, in MDS the overall response rate of 50%, including cytogenetic responses and improvements in transfusion requirements, are remarkable due to the tolerability of this regimen with essentially no grade 3 and 4 adverse events. The efficacy appears similar to other trials combining DNMTi and HDACi [83-85]. Following the rapid complete response in a patient with mycosis fungoides [74], a Phase II study of this combination is ongoing in CTCL. The results are very encouraging with objective response rates above 60% as well as rapid improvement in pruritus in most patients. Altogether, it seems that this combination could show similar efficacy than current epigenetic drugs but with lower toxicity. In summary, the hydralazine--valproate combination seems to be promising, but larger studies are needed as Phase III studies do not always confirm the promising results of earlier studies.

Declaration of interest A Duenas-Gonzalez has received research funding from Alpharma. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.


9


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Affiliation

Alfonso Duenãs-Gonzalez†*1,2, Jaime Coronel1, Lucely Cetina1 MD, Aurora Gonza´lez-Fierro1, Alma Chavez-Blanco1 & Lucia Taja-Chayeb1 † Author for correspondence *On behalf of the Tumor Study Group 1 Instituto de Investigaciones Biomedicas UNAM/Instituto Nacional de Cancerologıá Mexico, Unit of Biomedical Research on Cancer, Mexico City, Mexico 2 ISSEMyM Cancer Center, Toluca, Mexico E-mail: [email protected]

Epigenetic therapy in gastrointestinal cancer: the right combination.

Nanoparticles for combination drug therapy.

Combination of small molecule prodrug and nanodrug delivery: amphiphilic drug-drug conjugate for cancer therapy.

Hypoxia as a target for drug combination therapy of liver cancer.

Combination drug therapy for the treatment of status epilepticus.

True combination therapy using synergistic drug combination.

Drug combination therapy increases successful drug repositioning.

Novel epigenetic target therapy for prostate cancer: a preclinical study.

Epigenetic drug therapy based on bromodomain inhibition.

Hybrid nanoparticles for combination therapy of cancer.

Nanoparticles for Effective Combination Therapy of Cancer.

Combination drug therapy for treatment of hyperthyroid Grave's disease.

Drug therapy for cancer pain.

Stimuli-sensitive nanopreparations for combination cancer therapy.

Epigenetic polypharmacology: from combination therapy to multitargeted drugs.

Nano-assemblies of J-aggregates based on a NIR dye as a multifunctional drug carrier for combination cancer therapy.

Quantifying the pharmacology of antimalarial drug combination therapy.

Amodiaquine-Ciprofloxacin: a potential combination therapy against drug resistant malaria.

Epigenetic therapy for the treatment of epithelial ovarian cancer: A clinical review.

Combination therapy in combating cancer.

A Novel Epi-drug Therapy Based on the Suppression of BET Family Epigenetic Readers.

Drug Carrier for Photodynamic Cancer Therapy.

Top2a identifies and provides epigenetic rationale for novel combination therapeutic strategies for aggressive prostate cancer.

Doxorubicin-loaded magnetic gold nanoshells for a combination therapy of hyperthermia and drug delivery.