Introduction

Covalent histone modifications are involved in many nuclear processes such as DNA packaging, DNA replication, DNA repair, transcriptional regulation, as well as chromosome condensation, and segregation during mitosis. In transcription, the acetylation of lysine residues particularly in the N-terminal tails of core histones regulates protein–protein interaction and protein access to DNA. In general, acetylation activity is correlated with transcriptional activation, whereas deacetylation activity is accompanied by transcriptional repression (Grunstein 1997; Wade et al. 1997; Peterson 2002). However, recent findings show that deacetylation inhibition may not only lead to gene activation but also to gene repression, depending on the nature of the gene (Glaser et al. 2003). Two types of enzymes, histone acetyltransferases (HAT) and histone deacetylases (HDAC), have been shown to maintain the delicate dynamic equilibrium in the acetylation level of nucleosomal histones and may very well be active in the regulation of other cellular processes as well (Grozinger and Schreiber 2002; Hildmann et al. 2007). Both the activity of HAT and, in particular, HDAC affect angiogenesis, cell-cycle arrest, apoptosis, the terminal differentiation of different cell types, and the pathogenesis of malignant disease (Chung 2002).

HDAC inhibitors in cancer therapy

Particularly, the functions of histone deacetylase inhibitors as anticancer agents are manifold and, in most cases, seem to primarily affect gene expression regulation by either specifically activating the gene expression through the inhibition of histone deacetylation or by blocking the deacetylation of transcription factors. Major consequences frequently observed upon treatment with HDAC inhibitors include the induction of differentiation, cell cycle arrest, apoptosis, and the inhibition of angiogenesis (Liu et al. 2006). In the following, due to space limitations, only some examples of the different modes of HDAC inhibitor action are discussed in some detail (for a more comprehensive review, see Johnstone 2002; Vigushin and Coombes 2002; Bi and Jiang 2006; Huang 2006; Kouraklis et al. 2006; and Quaissi and Quaissi 2006).

Direct structural alterations in HDACs associated with cancers appear to be rare (Ropero et al. 2006), in contrast to translocations, deletions, and point mutations found in HAT genes (Drummond et al. 2005a,b; Linggi et al. 2005). However, HDACs are involved in the function of translocation fusion proteins associated with various forms of leukemia and lymphoma (Ferrick and Hiebert 1998; Rosato and Grant 2003; Marks et al. 2004; Drummond et al. 2005a,b). Translocations affect DNA-binding proteins such as BCL6, PLZF, RARα, RUNX1, and TAL1. A well-studied example is the translocation t(11;17) where promyelocytic leukemia zinc finger protein (PLZF) is fused to the retinoic acid receptor α (RARα; Fenrick and Hiebert 1998). The PLZF–RARα fusion proteins are no longer responsive to physiological levels of retinoic acid, resulting in the repression of retinoic acid-inducible genes that normally would promote myeloid differentiation. The consequence is a clonal expansion of cells arrested in the promyelocyte stage of development, the hallmark of acute promyelocytic leukemia (APL). The treatment of patients with high levels of all-trans retinoic acid (ATRA) has been unsuccessful. Presumably, the reason is that both the PLZF and the RARα part associate with co-repressor complexes (containing HDACs) but only the RARα part releases the co-repressor complex upon ATRA treatment. Consistent with these findings, HDAC inhibitors are capable of restoring RA responsiveness to PLZF–RARα and can result in complete remission in APL patients (Warrell et al. 1998). The mechanism by which HDAC inhibitors disintegrate co-repressor complexes is possibly based on the induction of a conformational change in histone deacetylases upon inhibitor binding (Riester et al. 2007).

Histone deacetylase inhibitors such as suberoylanilide hydroxamic acid (SAHA) 1 or trichostatin A are also capable of inducing cell cycle arrest. One of the most commonly induced genes is that of the cyclin-dependent kinase (CDK) inhibitor p21WAF1, a central regulator of the sequential activation of cyclin/CDK complexes that modulate progression through the cell cycle (Gartel and Tyner 2002). In this connection, HDAC inhibitors target the promoter directly by inducing the hyperacetylation of associated histones and modulate the transcription factors associated with specific binding sites such as Sp1 (Gartel and Tyner 1999; Huang et al. 2000; Gui et al. 2004). As a consequence of elevated levels of p21WAF1, downregulation of cyclinA and cyclinD, hypophosphorylation of Rb, p107, and p130, and thus, cell, growth inhibition as well as G1 and G2/M cell-cycle arrest have been observed in various cancer cells (Archer et al. 1998; Huang and Pardee 2000; Kim et al. 2000; Suzuki et al. 2000).

Experimental evidence also indicates that HDAC inhibitors may induce apoptosis in different cancer cells, activating the death receptor and/or the intrinsic apoptotic pathways (reviewed in Johnstone 2002; Kouraklis et al. 2006). As an example of the latter, the HDAC inhibitors SAHA 1 and trichostatin A (TSA) increase BAX protein levels in human pancreatic adenocarcinoma cells. As a consequence, the excess of free BAX molecules translocate to the mitochondria, followed by the release of an apoptosis-inducing factor (AIF) and Omi/HtrA2 from the mitochondria, the relocalization of AIF into the nuclei with the induction of DNA fragmentation (Garcia-Morales et al. 2005). By contrast, valproic acid 14 was demonstrated to selectively activate the death receptor apoptosis pathway in leukemia cells by upregulating the transcription of TRAI, DRS, FAS, and FASL (Insiga et al. 2005; Nebbioso et al. 2005).

Other major anticancer effects of HDAC inhibitors include the suppression of angiogenesis (Kim et al. 2001; Cao et al. 2006; Qian et al. 2006a,b) and repression of metastasis (Coradini et al. 2004). In this connection, it is noteworthy that there is evidence suggesting that the deacetylation of microtubules by HDAC6 is an important factor for tumor cells to migrate and metastasize (Haggarty et al. 2003). However, somewhat in contrast to these findings, HDAC6 expression is positively correlated with better survival in breast cancer (Zhang et al. 2004). HDAC inhibitors are also capable of stimulating the host immune response (Magner et al. 2000; Mishra et al. 2001). The latter was also recently used as a novel principle to enhance the immune responses to DNA vaccination (Vanniasinkam et al. 2006).

Other functions of HDAC inhibitors

In addition to anticancer effects, HDAC inhibitors have demonstrated pronounced in vitro and in vivo anti-inflammatory effects (Blanchard and Chipoy 2005; Adcock 2006) mainly by modulating NF-κB and STAT transcriptional activity and by suppressing the production of proinflammatory mediators, like cytokines (Huang 2006), and downregulation, particularly of the tumor necrosis factor alpha (TNF-α) receptor (Imre et al. 2006). In line with these findings, thiol-specific antioxidant was reported to attenuate airway inflammation in a mouse asthma model (Choi et al. 2005). However, things may not be that simple. For example, researchers recently found a correlation between the loss of HDAC activity and increased inflammation in the lungs of patients suffering from chronic obstructive pulmonary disorder (COPD; Ito et al. 2005). Previous research had shown that theophylline is able to restore HDAC activity and simultaneously helps to relax the bronchial tubes in COPD patients (Ito et al. 2002). Despite the fact that an in-depth understanding of the role of HDACs in inflammation is still missing, companies such as Chroma Therapeutics have already begun to develop HDAC inhibitors for autoimmune and inflammatory disorders.

HDAC inhibitors such as TSA and sodium butyrate also appear to be effective chondroprotective agents that modulate metalloproteinase gene expression and, thereby, block cartilage destruction (Young et al. 2005). Recent work has also demonstrated that HDAC inhibitors are active modulators of cellular hypertrophy including chondrocyte hypertrophy (Vega et al. 2004) and cardiac hypertrophy (Hoshijima and Chien 2002; Kook et al. 2002). In models of cardiac hypertrophy, HDAC inhibitors were able to modulate cardiomyocyte growth with no evidence of cell death and, therefore, hold promise as potential future therapeutic agents in hypertrophic heart disease (Metzger 2002; Antos et al. 2003; Hamamori and Schneider 2003; Kee et al. 2006; Kong et al. 2006; see also US patent 6946441).

The evasion strategies of tumors and protozoan parasites share some notable similarities (Quaissi and Quaissi 2005). Particularly, HAT and HDACs are also important regulators in the life cycle of parasites including Trypanosoma brucei, Plasmodium falciparum, and Leishmania spp. It is, therefore, not surprising that HDAC inhibitors interfere with parasite development, making them an interesting therapeutic approach in sleeping sickness, malaria, and leishmaniasis (reviewed in Quaissi and Quaissi 2006).

Histone deacetylase inhibitors may also be beneficial in the treatment of neurological diseases. Already a common treatment strategy for schizophrenia and bipolar disorder is the use of HDAC inhibitor valproic acid 14 as an adjunctive to atypical antipsychotics. Recent work suggests that benzamides such as MS275 15 may be more advantageous than valproic acid 14 in association with atypical antipsychotics (Simonini et al. 2006). Furthermore, the HDAC inhibitor phenyl butyric acid 13, particularly in combination with an antioxidant, exerts neuroprotective effects in a transgenic mouse model of amyotrophic lateral sclerosis (Petri et al. 2006). Cytoprotective effects have also been reported in the case of dentatorubral–pallidoluysian atrophy. In this study, HDAC inhibitors significantly reduced polyglutamine-induced cell death (Kariya et al. 2006). Similar results have been obtained in the Drosophila model (Steffan et al. 2001). More recently, a report on the treatment of spinal muscular atrophy, an α-motoneuron disorder with insufficient survival motor neuron protein levels was published (Hahnen et al. 2006). It was demonstrated that SAHA 1 but not MS275 15 was able to activate the survival motor neuron gene 2 and, thus, has the potential of decelerating progressive a-motoneuron degeneration. And on top of that, recent insights into the cAMP-response element-binding protein (CREB)/CREB-binding protein (CBP) pathway suggest that HDAC inhibitors could also work as potential memory enhancers in neurodegenerative disorders like Alzheimer’s disease (Beglopoulos and Shen 2006). For example, long-term potention and memory deficits were improved in Cbp+/− mice after treatment with a HDAC inhibitor (Alarcon et al. 2004).

The design of histone deacetylase inhibitors

The search for new HDAC inhibitors is largely dependent on the existence of bioassays well suited for inhibitor screening (reviewed in Wegener et al. 2003a,b). At present, many researchers favor fluorogenic assays (Wegener et al. 2003a,b) because of their suitability for high-throughput screening. Further developments recently included the design of HDAC subtype-selective fluorogenic substrates (Heltweg et al. 2004; Riester et al. 2004; Riester et al. (2007) Profiling of histone deacetylase specificity based on combinatorial fluorogenic substrate libraries, submitted for publication), the development of the first non-isotopic, competition binding assay for HDAC inhibitors (Riester et al. 2007), and a very elegant noninvasive method to monitor HDAC activity in living cells (Sankaranarayanapillai et al. 2006).

The discovery of histone deacetylase inhibitors dates back to times when even histone deacetylases had not yet been discovered. Over the past years, a large number of natural and synthetic compounds belonging to different structural classes have been discovered. A comprehensive survey of the HDAC inhibitor field certainly would go beyond the scope of this article. For review, the reader should refer to a number of recent publications (Monneret 2005; Moradei et al. 2005; Suzuki and Miyata 2005; Weinmann and Ottow 2005; Minucci and Pelicci 2006). In the following, only HDAC inhibitors that reached clinical trials (Figs. 1, 2, and 3) are described in some detail (Table 1).

Fig. 1
figure 1

HDAC inhibitor structures: hydroxamic acids. 1 SAHA, 2 PDX101, 3 LBH589, 4 CRA-024781, 5 CRA-026440, 6 ITF-2357 (as deduced from US6034096; Leoni et al. 2005), 7 NVP-LAQ824, 8 pyroxamide

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Fig. 2
figure 2

HDAC inhibitor structures: short-chain fatty acids and their derivatives. 9 Butyric acid, 10 tributyrin, 11 pivanex, 12 phenyl acetic acid, 13 phenyl butyric acid, 14 valproic acid

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Fig. 3
figure 3

HDAC inhibitor structures: benzamides and cyclic peptides. 15 MS-275, 16 MGCD0103, 17 CI-994, 18 FK-228 (depsipeptide), 19 trapoxin A

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Table 1 Histone deacetylase inhibitors in clinical trials
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Hydroxamic acids

In October 2006, a very important landmark was reached with the Food and Drug Administration (FDA) approval of the first HDAC inhibitor, vorinostat 1 (SAHA, Zolinza®; Fig. 1; Merck), for cutaneous T-cell lymphoma (CTCL), a rare form of non-Hodgkin’s lymphoma that affects the skin. SAHA was discovered in the pioneering work of Paul Marks (Memorial Sloan–Kettering Cancer Center, New York; Marks and Beslow 2007). SAHA had previously been tested in several clinical studies (Acharya et al. 2005). The major trial supporting approval was conducted with patients who had failed two systemic therapies and oral administration of the drug. In this study, skin disease was monitored using a severity-weighted assessment tool (SWAT) based on the total body surface area involved in patch, plaque, or tumor lesions. A total of 30% of the patients experienced responses (≥50% decrease in SWAT). Major adverse events were reported to be diarrhea, fatigue, nausea, dehydration, and anorexia, as well as hematologic laboratory abnormalities like anemia, thrombocytopenia, low white blood cell, and low neutrophil count. In most patients, these adverse events were of low grades in severity (Merck 2006; O’Connor 2006; Duvic et al. 2007). Previously, a phase I trial had revealed no dose-limiting toxicities (Kelly et al. 2003). As the major mechanism of therapeutic action, SAHA 1 induces apoptosis in CTCL cells (Zhang et al. 2005). Meanwhile, SAHA also proved effective in other cancer cells (Munster et al. 2001; Butler et al. 2002; Cohen et al. 2002; Hsi et al. 2004; Reddy et al. 2004; Eyüpoglu et al. 2005; Garcia-Manero et al. 2005; Luong et al. 2006; O’Connor et al. 2006; Sonnemann et al. 2006a,b; Xu et al. 2006). Therefore, it is also being investigated in other indications including myeloma, mesothelioma, and a variety of other cancers that have entered phase I, II, and III clinical trials since 2005.

Besides SAHA 1, other drug candidates in this class will not be far behind. PDX101 2 (TopoTarget/CuraGen) is currently in phase II trials for multiple myeloma, T-cell lymphoma, myelodysplastic syndrome, and ovarian cancer after already promising preclinical data (Plumb et al. 2003; Qian et al. 2006a,b). It is also in phase I trials for acute myeloid leukaemia (AML), liver, and colorectal cancer. PXD101 2 showed very low penetration into the cerebrospinal fluid of rhesus monkeys after intravenous administration (Warren et al. 2006).

LBH589 3 (Novartis) is a cinnamic hydroxamic acid analog that has been studied as an intravenous drug in a phase I trial in patients with advanced solid tumors. Six out of 13 patients were reported to show stable disease (Acharya et al. 2005). The drug also was well tolerated in phase I studies with consistent antileukemic effects in patients with refractory hematologic malignancies (Giles et al. 2006). In this study, LBH589 3 was administered intravenously. However, preliminary results using an oral formulation of the drug were also reported to be clinically active. Previously, LBH589 3 was shown to be an efficient antimyeloma agent inducing apoptosis (Maiso et al. 2006). In addition, the drug also affects tumor angiogenesis (Qian et al. 2006a,b). It is now planned to start a pivotal phase II registration study in patients with CTCL.

CRA-024781 4 (PCI-24781; Pharmacyclics/Celera) is another hydroxamic acid-based HDAC inhibitor with antitumor activity in vitro and in vivo (Buggy et al. 2006). After efficacy was shown in xenograft cancer models, CRA-024781 4 is now under evaluation in phase I clinical trials in patients with refractory solid malignancies (Celera 2005). The substance inhibits many class 1 and 2 enzymes in the nanomolar range and induces apoptosis in a whole variety of tumor cell lines. Additionally, Celera Genomics has other programs in the lead optimization stage to develop other variants of HDAC inhibitors, preferentially with more selectivity in targeting cancer cells. CRA-026440 5 is among these HDAC inhibitors in early development and was in-licensed by Pharmacyclics in 2006, together with CRA-024781 4. CRA-026440 5 proved to be a broad-spectrum HDAC inhibitor with potent antitumor activity and good efficacy in xenograft cancer models. In addition, CRA-026440 5 was reported to show antiangiogenic activity (Cao et al. 2006).

ITF-2357 (Italfarmaco SpA) was reported to be an orally active, synthetic HDAC inhibitor containing a hydroxamic acid moiety linked to an aromatic ring, as described in patent WO 97/43251, US6034096 (6; Leoni et al. 2005), shown to inhibit proliferation and induce cell death in hepatocellular carcinoma cell lines (Armeanu et al. 2005) without causing cell death in primary human hepatocytes. It also induces apoptosis in leukemia cells but not in mesenchymal stromal cells (Golay et al. 2006). Recently, the drug was also reported to possess anti-inflammatory activities in vivo and in vitro (Leoni et al. 2005). Interestingly, the substance was originally developed as an oral active TNF-α inhibitor for Crohn’s disease and other TNF-α related diseases, and was reported in 2005 to be in phase II clinical trials.

NVP-LAQ824 7 (Novartis), a cinnamic hydroxamic acid derivative, was shown to inhibit HDAC enzymatic activities in vitro and to exhibit anti-proliferative effects in a number of cancer cells including leukemia (Weisberg et al. 2004). In many of these cancer cells, but not in normal cells, apoptosis was induced (Atadja et al. 2004). Furthermore, tumor cells treated with NVP-LAQ824 7 showed acetylation of HSP90 and degradation of its cargo oncoproteins (Atadja et al. 2004; Kristeleit et al. 2004). The drug also exhibited antitumor effects in colon, lung, and breast tumor xenografts (Remiszewski et al. 2003; Drummond et al. 2005a,b). In 2004, promising results of a phase I trial in patients with advanced solid tumors for intravenously administrated NVP-LAQ824 7 were published (e.g., Kristeleit et al. 2004; Acharya et al. 2005). In this study, 3 out of 28 patients showed stable disease. In another phase I clinical study, in patients with hematologic malignancies, 6 out of 21 patients showed stable disease (Acharya et al. 2005). Many researchers speculate that further studies have been discontinued because of occasional cardiac side effects observed and reported by Novartis.

Pyroxamide 8 (Aton) is a hydroxamic acid derivative that induces terminal differentiation in murine erythroleukemia (MEL) cells and caused growth inhibition by cell-cycle arrest/apoptosis in MEL, prostate carcinoma, bladder carcinoma, and neuroblastoma cells (Butler et al. 2001). In 2002, a phase I study in patients with advanced malignancies was conducted. However, no active clinical trials have been reported since then.

Among the most promising hydroxamic acid inhibitors in preclinical development is presumably (S)-HDAC-42, which was reported to be orally bioavailable and significantly more potent than SAHA in suppressing the viability of various cancer cell lines and the growth of prostate tumor xenografts (Kulp et al. 2006). Another interesting candidate is M344, a promising candidate for a therapy of spinal muscular atrophy (Riesland et al. 2006). However, no clinical data is available so far.

Short-chain fatty acids and derivatives thereof

Short-chain fatty acids (Fig. 2) have become a favorite research topic in the cancer field because they are believed to emerge from bacterial fermentation of dietary fiber and may protect against colon cancer (Archer et al. 1998). Already two decades before, short-chain fatty acids had been shown to increase histone acetylation, although the molecular mechanism was not clear at this time (Riggs et al. 1977). Particularly, butyric acid 9 and its prodrug, tributyrin 10 (Gaschott et al. 2001), have since been used in a variety of in vivo tumor models and clinical studies. The drug is known to cause histone hyperacetylation and p21WAF1 induction (Archer et al. 1998), as well as induction of malignant cell differentiation followed by arrest of cell growth and apoptosis (Schröder and Maurer 2002; Kuefer et al. 2004; Entin-Meer et al. 2005). Reports from phase I studies in patients with solid tumors indicate that tributyrin 10 is well tolerated at levels associated with in vitro activity (Conley et al. 1998; Edelman et al. 2003). However, no active clinical trials have since been reported. For another prodrug of butyric acid (Entin-Meer et al. 2005), pivaloyloxymethyl butyrate 11 (Pivanex, AN-9; Titan Pharmaceuticals), the results of a phase I trial in patients with solid tumors and a phase II clinical study for patients with refractory non-small cell lung cancer have been reported. In the latter, 2 out of a total of 47 patients had partial responses and 18 had stable disease (Kelly et al. 2002; Acharya et al. 2005). In a phase II study (combination with docetaxel) in patients with advanced non-small cell lung cancer, 3 out of 12 patients achieved a partial response (Acharya et al. 2005). Another phase II clinical study for patients with refractory chronic lymphocytic leukemia and refractory malignant melanoma has been announced in 2004 (Farrel 2004). However, no information on active clinical trials have been reported since then. VX-563 (Vertex) is another butyrate prodrug (Archer et al. 2001). It was reported to induce differentiation and cell cycle arrest in colon cancer cells. Preclinical studies have also demonstrated that the drug can stimulate embryonic or fetal globin gene expression, suggesting that it may have potential for the treatment of sickle cell disease. VX-563 was reported to have entered phase I clinical trials. No detailed clinical results were disclosed so far. However, the company finally announced not to proceed with additional trials. Structural variants of butyrate 9 generally appeared to be of little advantage. An exception is maybe HA-But, a hyaluronic acid esterified with butyric acid. HA-But tends to concentrate in the liver and spleen and appears to be a promising new drug for the treatment of intrahepatic tumor lesions (Coradini et al. 2004).

Phenyl acetic acid 12 and phenyl butyric acid 13 are both also differentiation inducers (Bar-Ner et al. 1999; Miller et al. 1999). For phenyl acetic acid 12, two-phase I trials in patients with solid tumors have been reported. An additional phase II trial in patients with recurrent malignant glioblastoma revealed that 3 out of 40 patients achieved partial response, whereas 7 had stable disease (Kelly et al. 2002; Acharya et al. 2005). However, no further active clinical trials have been reported. For phenyl butyric acid 13, phase I trials for solid tumors and myeloid malignancies, a phase I trial (in combination with azacitidine) for solid tumors, and a phase I trial (in combination with retinoic acid) for APL have been reported (Kelly et al. 2002; Acharya et al. 2005). Although, no further information on clinical trials in the cancer field has been disclosed, data on a trial of phenyl butyric acid 13 in spinal muscular atrophy has been published (Mercuri et al. 2007) recently. However, at the conditions used in this study, efficacy was low.

Valproic acid 14 (G2M-777, Depakote, Depakene), an extensively used anti-epileptic, was reported to be a moderately active histone deacetylase inhibitor that causes hyperacetylation of histones, inhibition of proliferation, and induction of apoptosis (Tang et al. 2004; Armeanu et al. 2005) or cellular differentiation (Göttlicher et al. 2001; Gurvich et al. 2004). Its different formulations, savicol (orally available), Baceca, Avugane, and pulse-enhanced acetylation (PEAC; TopoTarget) are in clinical investigations. Savicol is currently in phase II studies for familial adenomatous polyposis. Baceca (in combination with all-trans retinoic acid) is in phase II trials for basal cell carcinoma (topical treatment). Avugane is in phase II trials for acne vulgaris. PEAC has entered phase II evaluation for patients with genetic predisposition to develop colon cancer. Different publications describe the first results of clinical evaluations of valproic acid. For patients with advanced leukemia, the results from a phase I/II trial (in combination with 5-aza-2′-deoxycitidine; Garcia-Manero et al. 2006) indicate that the combination was found safe and active with 19% of the patients showing complete remission. Promising results were also reported from other clinical trials (in combination with all-trans retinoic acid; Bug et al. 2005; Kuendgen et al. 2005; Cimino et al. 2006; Stapnes et al. 2006). For patients with advanced solid tumors, the results of a clinical phase I trial (in combination with epirubicin) have been reported, indicating that plasma levels of the drug needed for biological effects are easily achieved with minimal toxicity (Munster et al. 2006). In line with these findings, a remarkable response of a radiochemotherapy–refractory glioblastoma multiforme to valproic acid was recently published. After ambulant therapy with oral valproic acid 14 (at a final serum concentration of 140 mg/l), the clinical condition of the patient improved with a documented complete remission after 10 months of treatment (Witt et al. 2004).

Benzamides

A number of companies developed o-amino benzamides as yet another class of HDAC inhibitors (Fig. 3. MS-275 15 (MS-27-275; Schering), the best-known member of this class, is an orally active synthetic inhibitor with selectivity for class 1 HDACs (Hildmann et al. 2006). Unlike many other potent HDAC inhibitors, MS-275 15 is a brain region-selective inhibitor (Eyüpoglu et al. 2006; Simonini et al. 2006) with a reported half-life of up to 80 h in human (results from a phase I study; Ryan et al. 2005). Chemotherapeutic efficacy in vivo against malignant gliomas has been demonstrated (Eyüpoglu et al. 2006). One phase I study for patients with hematologic malignancies and two phase I trials for patients with solid tumors and lymphomas were conducted. In one of the latter, one melanoma patient with partial response was observed (Acharya et al. 2005). A phase II study for patients with metastatic melanoma was recently completed. Preliminary results from this study suggest that MS-275 15 is well tolerated and shows long-lasting tumor stabilization (Hausschild et al. 2006). However, objective tumor responses were not observed with this single-agent treatment. Currently, MS-275 15 (in combination with azacitidine) is in a clinical phase I/II trials for myelodysplastic syndromes, chronic myelomonocytic leukemia, and acute myeloid leukemia with multilineage dysplasia. MS-275 15 (in combination with azacitidine) is also in clinical phase I/II trials for patients with recurrent advanced nonsmall-cell lung cancer.

Researchers at MethylGene have also extensively explored benzamides as HDAC inhibitors. The most promising candidate, MGCD0103, is further developed together with MethylGene’s partners, Pharmion and Taiho Pharmaceuticals. MGCD0103 16 is a rationally designed, oral, isotype-specific HDAC inhibitor. It showed complete bone marrow responses in three of nine patients in a phase I trial for myelodysplastic syndromes or acute myelogenous leukemia. The drug is currently in phase II clinical trials for patients with refractory or relapsed diffuse large B-cell lymphoma and follicular lymphoma. In addition, a single-agent phase II trial and a combination phase I/II trial (combination with azacitidine) for patients with high-risk myelodysplastic syndromes or relapsed or refractory acute myelogenous leukemia was initiated. Furthermore, a phase I/II combination trial (combination with gemcitabine) for patients with solid tumors and pancreatic cancer, and a phase II single-agent trial in patients with refractory or relapsed Hodgkin’s lymphoma was announced recently.

CI-994 17 (acetyldinaline, PD-123654) is an orally available, acetylated derivative of dinaline. The latter originally was developed as an anticonvulsant agent and later was reported to have antitumor activity (Berger et al. 1985). CI-994 17 is a relatively weak HDAC inhibitor that has broad preclinical antitumor activity (LoRusso et al. 1996). In a single-agent phase I trial for patients with solid tumors, 1 of 53 patients achieved a partial response. In a single-agent phase I trial for patients with nonsmall-cell lung cancer, 2 of 32 patients showed a partial response (Kelly et al. 2002; Acharya et al. 2005). Phase II studies were conducted in patients with nonsmall-cell lung cancer, renal cell carcinoma, and pancreatic cancer. Two out of 32 patients with nonsmall-cell lung cancer had a partial response. Stable disease was observed in 28 and 58% of the patients with nonsmall-cell lung cancer and renal cell carcinoma, respectively (Kelly et al. 2002). In contrast, patients with pancreatic cancer achieved no objective responses (Zalupski et al. 2000). In addition to monotherapy, combinations with other drugs proved to be efficacious (Acharya et al. 2005). In a phase I trial (combination with gemcitabine), in 20 patients with advanced cancer, 2 patients achieved minor response, whereas 12 had stable disease (Nemunaitis et al. 2003). In a phase I trial (combination with capecitabine), in 14 patients with refractory solid tumors, 1 patient with colorectral cancer achieved a partial response (Acharya et al. 2005). In a phase I trial (combination with carboplatin and paclitaxel), 24 patients received more than one cycle of treatment. Of these, five achieved partial response and two achieved complete response (esophageal and bladder cancer) (Pauer et al. 2004). In contrast, the combination with gemcitabine offered no advantage over gemcitabine alone (Richards et al. 2006).

A number of other benzamides have been disclosed that are expected to enter clinical trials soon (Weinmann and Ottow 2005).

Cyclic peptides

A structurally diverse class of HDAC inhibitors is constituted by compounds with cyclic peptide structures such as natural products like FK-228 18 (Fig. 3), the trapoxins A 19 and B, apicidin, and the cyclic hydroxamic acid-containing peptides (CHAPs). FK-228 18 (depsipeptide, FR901228, NSC 630176, romidepsin; Gloucester), a unique bicyclic peptide isolated from Chromobacterium violaceum strain WB968, so far, is the only notable inhibitor of this class that is currently in clinical trials. FK-228, however, already has a checkered career. After being dropped because of cardiac toxicity observed in dogs, it came back into the race when the National Cancer Institute (NCI) devised a new dosing regimen. In a single-agent phase I trial comprising 37 patients with refractory neoplasms, one patient with renal cell carcinoma achieved a partial response and 8 patients had stable disease (Sandor et al. 2002; Acharya et al. 2005). In a small phase I trial in four patients with CTCL/peripheral T-cell lymphomas, three partial and one complete response were noted (Kelly et al. 2002). In contrast, two recent phase I trials in patients with chronic lymphocytic leukemia/acute myeloid leukemia (Byrd et al. 2005) as well as in pediatric patients with refractory solid tumors (Fouladi et al. 2006) revealed no partial or complete responses, whereas the drug was generally well tolerated. Currently, FK-228 is in clinical phase II studies for patients with CTCL and for patients with hormone refractory prostate cancer with interim results reported at the 2006 annual meeting of the American Society of Clinical Oncology. In the study on CTCL, 2 out of 28 patients experienced complete responses, 7 achieved partial responses, and 16 patients had stable disease. In the study on metastatic hormone refractory prostate cancer, 1 patient out of 22 achieved a partial response; in up to 7 patients, stable disease was observed. As far as cardiac toxicity is concerned, the outcome of clinical trials is somewhat contradictory. One recent study revealed no significant myocardial damage or impaired cardiac function associated with the administration of depsipeptide (Piekarz et al. 2006). In contrast, electrocardiogram changes and even sudden deaths are reported from other trials (Garber 2007). Nevertheless, Gloucester intends to file FK-228 for FDA approval in late 2007.

Potential class 3 therapeutics

Finally, it is noteworthy that the year 2006 not only yielded the FDA approval of the first inhibitor of class 1, 2, and 4 histone deacetylases. Also, the first class 3 or sirtuin therapeutic, a resveratrol derivative and SIRT1 activator, was announced to have successfully finished phase I trials (Elliott 2006). Furthermore, several sirtuin inhibitors are currently under development (Weinmann and Ottow 2005; Heltweg et al. 2006).

Monotherapy versus combination therapy

In addition to being used as anticancer agents for monotherapy, several studies have shown that HDAC inhibitors may act synergistically in combination therapy. It is for example obvious to simultaneously attack the cellular cytoskeleton through two different pathways, combining the inhibition of HDAC6-dependent tubulin deacetylation and the taxane-based targeting of mitotic structures. Exactly, this strategy is now followed by Merck by combining SAHA with docetaxel. Other recent examples include combinations of HDAC inhibitors with ATRA (Bug et al. 2005; Kuendgen et al. 2005; Cimino et al. 2006; Stapnes et al. 2006), classical chemotherapeutics (5-fluorouracil, oxaliplatin, irinotecan, idarubicin, epirubicin, doxorubicin, melphalan; Kim et al. 2003; Khan et al. 2006; Munster et al. 2006; Piacentini et al. 2006; Sanchez-Gonzalez et al. 2006; Sato et al. 2006; Zhang et al. 2006), different kinase inhibitors (gefitinib; Piacentini et al. 2006), dasatinib (Fiskus et al. 2006a,b), AMN107 (Fiskus et al. 2006a,b), staurosporine, UCN-01 (Yeow et al. 2006), flavopiridol (Dashmahapatra et al. 2006), PKC412 (Bali et al. 2004), methyltransferase inhibitors (gemcitabine; Zhang et al. 2006), 5-aza-2′-deoxycitidine (Garcia-Manero et al. 2006; Gore et al. 2006; Hurtubise and Momparler 2006), HAT inhibitors (curcumin; Chen et al. 2006), chaperone inhibitors (17-AAG; George et al. 2005), proteasome inhibitors (bortezomib; Catley et al. 2006; Horton et al. 2006; Nawrocki et al. 2006; Sutheesophon et al. 2006), P-gp/MRP1 inhibitors (verapamil, MK571; Okada et al. 2006a,b), apoptosis inducer [(TNF)-related apoptosis-inducing ligand (TRAIL); Butler et al. 2006; Sonnemann et al. 2006a,b], antibodies (Ten Cate et al. 2007), oncolytic adenoviral agents (Bieler et al. 2006; Okada et al. 2006a,b; Watanabe et al. 2006). Furthermore, HDAC inhibitors proved to selectively radiosensitize various human cancer cells (Miller et al. 1999; Entin-Meer et al. 2005; Chinnaiyan et al. 2006; Karagiannis and El-Osta 2006; Kim et al. 2006). It is noteworthy that the effect can be reversed upon too-short pretreatment with HDAC inhibitors (Miller et al. 1999).

There is also evidence for antagonist effects of combinations of HDAC inhibitors and other antitumor agents. For example, neuroblastoma cells that have been driven into cell-cycle arrest and differentiation by HDAC inhibitor BL1520 no longer appear to be sensitive to gemcitabine (De Ruijter et al. 2006). FK228 (18)/ATRA induced the expression of the multidrug resistance 1 gene product P-gp, resulting in the prevention of growth inhibition and apoptosis in leukemia cells (Tabe et al. 2006). P-gp and the multidrug resistance-associated protein also play a role in the resistance of certain osteosarcoma and Ewing’s sarcoma cell lines against FK228 18 and apicidin. Interestingly, resistance could be reversed by the application of P-gp inhibitor verapamil and MRP1 inhibitor MK571 (Okada et al. 2006a,b).

Perspectives and trends in HDAC inhibitor discovery

Currently, the excitement about the initial clinical results of HDAC inhibitors in cancer patients predominates the judgement of many researchers active in the field. Indeed, promising results have been obtained for cancer treatment in a number of clinical trials with different drugs. Nearly each clinical trial revealed a clinical effect in patients, and even long-term and sometimes dramatic responses have been described several times. Many HDAC inhibitors are, generally, rather well tolerated. Only some HDAC inhibitors such as TSA or trapoxin are of limited therapeutic use due to toxic side effects, or other factors such as poor bioavailability in vivo, poor stability, and mutagenicity (as reported for apicidin; Yoo and Lee 2005). Nevertheless, chronic constitutional symptoms (e.g., fatigue, nausea, and electrolyte disturbances) have been observed for different clinical HDAC inhibitors (summarized in Byrd et al. 2005) and, therefore, possibly are general clinical manifestations of HDAC inhibition (or our present state-of-the-art of HDAC inhibitors). There might be other drawbacks of a HDAC inhibitor-based therapy. In a recent paper, Moreth et al. (2006) presented experimental evidence that HDAC inhibitors such as SAHA 1 or MS-275 15 may also block lipase and liver esterase activity. These results suggest that nonselectivity of our present HDAC inhibitors may be responsible for some of their adverse effects. On the other hand, additional functions besides direct HDAC modulation may play a significant role in the ability of HDAC inhibitors to induce differentiation or apoptosis, e.g., in cancer cells (Newmark and Young 1995; Ma et al. 2006; Mora et al. 2006). Another issue to be considered is the acquired resistance of cancer cells to HDAC inhibitors (Xiao et al. 2005; Okada et al. 2006a,b; Ropero et al. 2006). However, at least in some cases, resistance can be reversed or prevented through combination therapy, e.g., with P-gp inhibitor verapamil or MRP1 inhibitor MK571 (Okada et al. 2006a,b). Notwithstanding, caution should be employed when considering combination therapy. Despite the fact that a large number of drugs synergize with HDAC inhibitors, there are also examples of antagonist effects of combinations of HDAC inhibitors and other antitumor agents (De Ruijter et al. 2006; Tabe et al. 2006), not to speak of possible side effects that only become apparent under conditions of drug combination.

Despite the fact that we are close to understanding the catalytic mechanism of histone deacetylases already enabling structure-based inhibitor design (Lu et al. 2005; Wang et al. 2005; Hildmann et al. 2006; Shinji et al. 2006), our knowledge of HDAC biology is still very limited. With the increasing number of proteins known to be deacetylated by HDACs, it can even be questioned whether histone acetylation is a good measure to predict anticancer activity of a drug. Furthermore, the role of individual HDAC isoforms, for example, is still under investigation, and it is an open question whether subtype-specific inhibitors would be more useful in therapy, e.g., by minimizing adverse effects. First examples of such compounds that are at least class-selective (e.g., Mai et al. 2003, 2006; Wang et al. 2005; Shinji et al. 2006; Suzuki et al. 2006) have been reported recently and more and more researchers are focussing now on this topic. At least, these selective inhibitors are welcome as biochemical tools to dissect the roles of specific HDAC subtypes. Another line of research focuses on new warheads and, particularly substitutes of hydroxamic acids which would better meet criteria of ‘druglikeness’, i.e. favorable physicochemical and pharmacokinetic properties (reviewed in Moradei et al. 2005; Suzuki and Miyata 2005; Weinmann and Ottow 2005). Whereas many new structures cannot compete with the existing Zn-binding motifs in regard of HDAC inhibitory and antitumor activity (Hanessian et al. 2006), particularly electrophilic ketones, which have been shown to be inhibitors of various hydrolytic enzymes, appear to be attractive HDAC inhibitor structures (Frey et al. 2002; Wada et al. 2003). More recent examples of this promising class of HDAC inhibitors include α-mercaptoketones or α-thioacetoxyketone (Gu et al. 2006), bromomethylketones, and hydroxymethylketones (Bhuiyan et al. 2006). In this context, it might be worth to reconsider the class of trifluoroketones that already had demonstrated antiproliferative activity but, unfortunately, also showed significant metabolic liabilities (Frey et al. 2002). With the first nonhydroxamate crystallographic structure, a trifluoroketone inhibitor in complex with FB188 HDAH, now available (Nielsen et al. 2006), the structure-based design of derivatives with optimized properties is now largely facilitated. Most of the aforementioned electrophilic ketones had given up the principle of bidentate zinc binding and rely on additional contacts, e.g., through fluor atoms that participate in the interactions to the enzyme (Nielsen et al. 2006). In contrast, other inhibitors such as mercaptoamides (Anandan et al. 2005) were designed as bidentate zinc chelators similar to hydroxamates. It remains an open question whether these reactive compounds can be turned into therapeutically useful drugs.

The diversity of natural products always has been a rich source for new pharmaceutically active compounds including HDAC inhibitors. Recent studies in this field particularly focus on dietary HDAC inhibitors. Although it is still a matter of debate if blood concentrations achievable through diet are sufficient to affect HDAC activity in vivo, particularly natural isothiocyanates from cruciferous vegetables and their synthetic counterparts appear to be a promising class of HDAC inhibitors. For example, sulforaphane, an isothiocyanate from broccoli was recently reported to inhibit HDACs in vivo and suppress tumorigenesis in Apcmin mice (Myzak et al. 2004, 2006). In addition, phenylhexyl isothiocyanate was shown to inhibit HDACs and induce growth arrest in human leukemia and prostate cancer cells, as well as in xenografted prostate tumors (Ma et al. 2006). However, the exact mechanism of antitumor activity still has to be elucidated. Our group has screened a natural product library for inhibitors of FB188 HDAH, a model of class 2 HDACs, and identified a number of novel inhibitor structures including disaccharide and benzochinone derivatives that possibly exhibit unusual modes of binding to the enzyme (D. Wegener and A. Schwienhorst, personal communication). Furthermore, with the new binding assay for direct HDAC inhibitors (Riester et al. 2007), it is now even conceivable to specifically screen for nonactive-site versus active-site binders. Thus, it might be possible to identify HDAC inhibitors that do not depend on canonical active-site binding, e.g., zinc coordination, but instead block for example the internal cavity of the enzymes. However, progress in inhibitor design should not make us forget that it is equally important to identify biomarkers that predict tumor sensitivity to HDAC inhibitors and, thus, susceptible patients.

In conclusion, with our recent progress in understanding the role of histone modification (and nonhistone protein modification) in cancer, we are now on the edge of turning epigenic mechanisms of gene regulation into powerful tools of therapeutic intervention in malignant and other diseases.