Hypoxia-Inducible Factor Inhibitors: A Survey of Recent Patented Compounds (2004–2010)
Introduction
Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor consisting of alpha (α) and beta (β) subunits that regulates the expression of angiogenic factors, including vascular endothelial growth factor (VEGF), which are involved in angiogenesis, invasion/metastasis, glucose uptake, and cell survival during cancer development. This review summarizes information about patented HIF inhibitors over the last seven years (2004–2010). The reader will gain an outline of the structure and biological activity of recently developed HIF inhibitors.
Inhibition of HIF is an attractive therapeutic target for tumor angiogenesis. Various HIF inhibitors have been discovered and evaluated. It is expected that the development of more potent and selective HIF inhibitors will provide effective treatment for cancer and other HIF-related diseases, including inflammation and cardiovascular disorders. Since VEGF plays an important role in angiogenesis during tumor growth and ischemic diseases, inhibiting VEGF-induced HIF is an attractive approach to suppress hypoxia-mediated pathological angiogenesis. HIF inhibitors may not only have cytostatic antitumor effects with fewer side effects but also synergistic effects when combined with radiotherapy.
Keywords: angiogenesis, cancer, HIF, HIF inhibitors, hypoxia
Introduction
Hypoxia-inducible factor (HIF), a member of the Per-aryl hydrocarbon receptor nuclear translocator (ARNT)-Sim family of heterodimeric basic helix-loop-helix transcription factors, consists of alpha subunits (HIF-1α, -2α, and -3α) and a beta subunit (HIF-1β, also known as ARNT). Under aerobic conditions, post-translational hydroxylation of proline residues (Pro402 and Pro564) in the oxygen-dependent degradation domain of HIF-1α is induced by oxygen-sensitive dioxygenases catalyzing prolyl hydroxylase (PHD). The hydroxylated HIF-1α binds to the von Hippel-Lindau (VHL) tumor suppressor protein, a component of the E3 ubiquitin ligase complex. These interactions lead to rapid degradation of HIF-1α through an ubiquitin- and proteasome-dependent pathway.
Besides proline hydroxylation, asparagine (Asn803) in the C-terminal transactivation domain (CAD) of HIF-1α is hydroxylated by the factor-inhibiting HIF (FIH), an oxygen-dependent hydroxylase enzyme under aerobic conditions. This modification inhibits HIF transcriptional activity by preventing the interaction between CAD and the p300/CBP transcriptional co-activator. Under hypoxic conditions, HIF-1α is stabilized due to hypoxia-mediated reduction of PHD and FIH activities and is translocated into the nucleus, where it dimerizes with the constitutively expressed HIF-1β. The HIF-1α and -β heterodimers bind to a cis-acting regulatory element referred to as the hypoxia-response element (HRE) (5′-RCGTG-3′, where R is A or G) in the promoter region of a number of genes, including glucose transporters, glycolytic enzymes, angiogenic growth factors, and several molecules involved in apoptosis and cell proliferation such as erythropoietin (EPO), transferrin, endothelin-1, inducible nitric oxide synthase (iNOS), heme oxygenase 1, VEGF, insulin-like growth factor (IGF), and IGF-binding proteins.
Physiological and pathological implications of HIF in various human diseases, including inflammation, cardiovascular disorders, and cancer, have been clarified. Especially during cancer development, HIF-1α is a key regulator of the expression of various genes associated with tumor angiogenesis, metastasis, invasion, proliferation, and apoptosis. Overexpression of HIF-1α has been observed in human cancers including brain, breast, colon, lung, ovary, and prostate cancers. HIF-1α is implicated in treatment resistance and poor prognosis in the hypoxic regions around cancer. Therefore, drugs targeting HIF-1α have the potential to target multiple cancer processes, and a large number of HIF inhibitors have been developed. Several HIF inhibitors have entered clinical trials. This survey reviews the patent literature of HIF inhibitors and clinical trials reported in the last seven years.
Small Molecule HIF Inhibitors
2.1 PX-12
Thioredoxin-1 (Trx-1) is a cellular redox protein that promotes tumor growth, inhibits apoptosis, and upregulates HIF-1α and VEGF. PX-12 (1-methylpropyl 2-imidazolyl disulfide) has shown excellent in vitro and promising in vivo antitumor activity by irreversibly thio-alkylating the Cys73 thioredoxin residue. PX-12 inhibition of Trx-1 results in subsequent inhibition of the hypoxia-induced increase in HIF-1α protein and VEGF secretion. PX-12 is rapidly metabolized, producing two inactive metabolites, volatile 2-butanethiol and 2-mercaptoimidazole.
PX-12 was tested in a Phase I pharmacokinetic and pharmacodynamic study in patients with advanced solid tumors. Thirty-eight patients received PX-12 at doses ranging from 9 to 300 mg/m² as 1- or 3-hour intravenous infusions on days 1–5, repeated every three weeks. The best response was stable disease (SD) in seven patients lasting 126–332 days. PX-12 treatment lowered plasma Trx-1 concentrations in a dose-dependent manner and was tolerated up to a dose of 226 mg/m² by a 3-hour infusion. However, a randomized Phase II study of PX-12 in patients with advanced pancreatic cancer following progression after gemcitabine-containing therapy showed a lack of significant antitumor activity and unexpectedly low baseline Trx-1 levels. PX-12 does not appear to be active in unselected patients with previously treated advanced pancreatic cancer, and thus the study was terminated.
2.2 PX-478
PX-478 (S-2-amino-3-[4′-N,N-bis(2-chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) suppresses constitutive and hypoxia-induced levels of HIF-1α protein in various cancer cells in a pVHL- and p53-independent manner.
A patent application from ProlX Pharmaceuticals claimed that N-oxides of melphalan derivatives are HIF-1α inhibitors useful for treating diseases associated with HIF, including choroidal and retinal neovascularization, age-related macular degeneration, joint disease, inflammation, neurodegenerative diseases, and ischemic reperfusion injury. Biological data showed that PX-478 inhibited hypoxia-induced expression of HIF-1α in prostate cancer PC-3, breast cancer MCF-7, and colon cancer HT-29 cells with IC50 values of 2.1, 3.5, and 17.8 µM, respectively. PX-478 also inhibited hypoxia-induced HIF transactivation in MCF-7 and HT-29 cells with IC50 values of 20.5 and 23.1 µM, respectively. Furthermore, IC50 values for inhibition of hypoxia-induced VEGF secretion were 3.8 and 11.5 µM in MCF-7 and HT-29 cells, respectively.
The mechanism of HIF-1α inhibition by PX-478 was further elucidated in another patent application from ProlX Pharmaceuticals. PX-478 inhibited HIF-1α expression in RCC4 (human renal carcinoma cells lacking the VHL gene), indicating suppression via a VHL-independent pathway. Tumor suppressor p53 binds to HIF-1α allowing recruitment of MDM2, an E3 ubiquitin ligase, resulting in degradation of both p53 and HIF-1α. PX-478 suppressed HIF-1α expression in human colon cancer HCT116-/- cells lacking p53, demonstrating a p53-independent mechanism. The inhibition of HIF-1α expression by PX-478 was proteasome-dependent, and PX-478 increased ubiquitinated HIF-1α levels by preventing its deubiquitination. In vivo MCF-7 or HT-29 xenograft models showed that PX-478 treatment significantly decreased tumor growth and HIF-1α expression.
Recently, the Board of Regents of the University of Texas System reported pharmaceutical compositions of PX-478 for treating small cell lung cancers (SCLC) and methods of inhibiting metastasis, including combinations with radiation, photodynamic therapy, and anti-angiogenic agents. PX-478 was effective at both high (20 mg/kg) and low (10 mg/kg) doses in reducing tumor volume and weight in an SCLC orthotopic model.
PX-478 was tested in a Phase I dose escalation study in patients with advanced solid tumors. Forty patients received doses between 1 and 88.2 mg/m² orally on days 1–5 of a 21-day cycle. The best response was stable disease in 14 of 36 evaluable patients. Patients with stable disease received a median of four cycles (range 2–16), with four patients receiving six or more cycles (adenoid cystic, pheochromocytoma, prostate, and vaginal cancers). PX-478 was well tolerated and associated with prolonged stable disease in patients with advanced cancers. Inhibition of HIF-1α despite low PX-478 levels suggests active metabolites other than melphalan may be responsible for pharmacodynamic effects. These data support continued evaluation of HIF-1α inhibition as a therapeutic target.
2.3 YC-1 Derivatives
YC-1 (3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole) is a soluble guanylyl cyclase (sGC) activator primarily developed to treat circulation disorders by inhibiting platelet aggregation and vascular contraction. In 2001, Yeo and co-workers found that YC-1 completely blocks HIF-1α expression at the post-transcriptional level and consequently inhibits HIF transcription factor activity under hypoxic conditions. sGC inhibitors did not block YC-1’s inhibitory effects on HIF, and 8-bromo-cGMP did not inhibit hypoxia-induced HIF expression, indicating YC-1’s inhibition of HIF is sGC-independent. Thus, YC-1 is recognized as an HIF inhibitor and antitumor agent.
A patent application from HIF Bio, Inc. claimed the use of YC-1 and its derivatives for inhibiting HIF-1α expression in tumors including hepatoma, stomach carcinoma, renal carcinoma, cervical carcinoma, and neuroblastoma. YC-1 inhibited hypoxia-induced expression of HIF-1α and its target genes including VEGF, aldolase A, and enolase 1 in various cancer cells such as Hep3B hepatoma, NCI-H87 gastric carcinoma, SiHa cervical, SK-N-MC neuroblastoma, and Caki-1 renal carcinoma cells. Almost complete inhibition was observed at 10 µM concentration. In an in vivo tumor xenograft model, treatment with 30 mg/kg YC-1 suppressed tumor growth. Immunohistochemical analysis of tumor sections showed YC-1 reduced the number of HIF-1α-positive cells and endothelial marker CD31-positive cells, indicating tumor growth inhibition by YC-1 is mediated by suppression of HIF-1α expression.
This application also claimed the use of YC-1 and its derivatives in combination with other anticancer agents. HIF Bio further claimed a series of YC-1 derivatives (compounds 4A–D) that suppressed hypoxia-induced HIF-1α expression similarly to YC-1 at concentrations ranging from 0.3 to 10 µM. The ability of these compounds to inhibit tumor growth was determined in male nude mice following subcutaneous injection of Hep3B hepatoma cells. After xenografted tumors reached 100–150 mm³, compounds were administered by intraperitoneal injection daily for two weeks. At the end of treatment, tumor size was reduced from about 900 mm³ in vehicle-treated controls to about 400 mm³ at doses of 10 mg/kg/day (compounds 4A–D) or 30 mg/kg/day (compounds 4A and 4B).
Another patent application from HIF Bio claimed a series of heterocycle-substituted pyrazoles, such as compound 5, for treating HIF-related and VEGF-mediated diseases or disorders. However, no biological data were presented.
2.4 Benzopyran Derivatives
A series of 2,2-dimethyl-2H-chromene (2,2-dimethylbenzopyran) derivatives has been developed as inhibitors of the HIF-1 pathway. The first benzopyran-based HIF-1 inhibitors were patented by researchers from Emory University and Scripps Research Institute in 2004. These novel benzopyrans were tested for inhibitory activity on the HIF-1–HRE pathway using LN229 glioma cells expressing the alkaline phosphatase reporter gene. The most potent compound displayed an IC50 value of 7.5 µM.
Following this initial patent, the same group reported an additional series of 2,2-dimethylbenzopyran derivatives in 2007. A library of over 10,000 derivatives was prepared by combinatorial synthesis. Screening in the cell-based HIF-1–HRE assay led to the discovery of highly potent HIF-1 inhibitors such as KCN-1, with an IC50 value of approximately 4 µM. Further analysis showed that KCN-1 reduced hypoxic levels of HIF-1α in glioma cell lines without significantly affecting HIF-1β levels.
For in vivo pharmacological evaluation, KCN-1 was tested in nude mice with subcutaneous LN229 glioma xenograft implants. Strong and sustained inhibition of tumor growth was observed without significant toxicity.
2.5 Other Small Molecule Inhibitors
In addition to the aforementioned classes, several other small molecule inhibitors targeting the HIF pathway have been reported in recent patents. These include various heterocyclic compounds, such as substituted pyrazoles and imidazoles, which have demonstrated the capacity to inhibit HIF-1α expression and its downstream signaling. Although the detailed mechanisms of action for many of these compounds remain to be fully elucidated, their efficacy in reducing hypoxia-induced gene expression and tumor growth in preclinical models has been established. Some of these compounds have also shown the ability to interfere with the dimerization of HIF-1α and HIF-1β, thereby preventing the formation of the active transcription factor complex.
Steroidal HIF Inhibitors
Steroidal compounds have also emerged as a class of HIF inhibitors. Certain derivatives of corticosteroids and related structures have been found to suppress HIF-1α protein accumulation under hypoxic conditions. These effects are believed to be mediated through modulation of cellular signaling pathways that regulate HIF-1α synthesis and degradation. For example, some steroidal inhibitors may enhance the activity of prolyl hydroxylases, thereby promoting the degradation of HIF-1α even in low oxygen environments. Others may interfere with the transcriptional activity of HIF-1α by disrupting its interaction with co-activators such as p300/CBP. While the precise mechanisms can vary, the ability of steroidal compounds to modulate HIF signaling adds another dimension to the therapeutic strategies available for targeting hypoxia-driven diseases.
Peptidic HIF Inhibitors
Peptide-based inhibitors represent another innovative approach to targeting the HIF pathway. These inhibitors are typically designed to mimic specific protein-protein interaction domains involved in HIF-1α stabilization, dimerization, or DNA binding. By competitively inhibiting these interactions, peptidic inhibitors can effectively block the formation or function of the HIF transcription factor complex. Some peptides have been engineered to disrupt the binding of HIF-1α to ARNT (HIF-1β), while others target the recruitment of co-activators necessary for gene transcription. Although peptide drugs often face challenges related to stability and cellular uptake, advances in peptide engineering and delivery systems have improved their potential as therapeutic agents.
Natural Product-Based HIF Inhibitors
Natural products have long been a source of bioactive compounds, and several natural product-derived molecules have demonstrated HIF inhibitory activity. These include compounds isolated from plants, marine organisms, and microorganisms. For instance, certain flavonoids, alkaloids, and terpenoids have been shown to reduce HIF-1α protein levels or inhibit its transcriptional activity. The mechanisms by which these natural products exert their effects are diverse, ranging from direct inhibition of HIF-1α synthesis to modulation of upstream signaling pathways such as PI3K/Akt/mTOR and MAPK. The structural diversity of natural products offers a rich reservoir for the discovery of novel HIF inhibitors with unique modes of action.
RNA Antagonist, EZN-2968
A novel approach to HIF inhibition involves the use of RNA antagonists, such as EZN-2968. This compound is an antisense oligonucleotide specifically designed to bind to HIF-1α mRNA, leading to its degradation and subsequent reduction in HIF-1α protein synthesis. EZN-2968 has demonstrated the ability to decrease HIF-1α levels and inhibit the expression of HIF target genes in preclinical studies. The use of RNA-based therapeutics allows for high specificity in targeting gene expression, and ongoing clinical trials are evaluating the safety and efficacy of EZN-2968 in patients with advanced cancers. The development of RNA antagonists represents a promising direction for the treatment of diseases driven by aberrant HIF activity.
Expert Opinion
The development of HIF inhibitors has progressed significantly over the past decade, with a variety of chemical classes and mechanisms of action being explored. Small molecule inhibitors, steroidal compounds, peptides, natural products, and RNA antagonists each offer distinct advantages and challenges. The inhibition of HIF remains an attractive therapeutic strategy for conditions characterized by pathological hypoxia, such as cancer, ischemic diseases, and chronic inflammation.
The clinical success of HIF inhibitors will depend on several factors, including their potency, selectivity, pharmacokinetic properties, and safety profiles. Combination therapies that pair HIF inhibitors with other treatment modalities, such as chemotherapy, radiotherapy, or anti-angiogenic agents, may enhance therapeutic efficacy and overcome resistance mechanisms. Continued research into the molecular mechanisms governing HIF regulation and the identification of biomarkers for patient selection will further refine the clinical application of these agents.
In conclusion, the expanding repertoire of HIF inhibitors holds great promise for the treatment of hypoxia-related diseases. Ongoing clinical trials and future discoveries Lificiguat will determine the ultimate impact of these compounds on patient outcomes.