Tnp-470 polymer conjugates and use thereof

In recent years, it has become clear that angiogenesis, the growth of new capillary blood vessels from pre-existing vasculature, is important not only in physiological processes such as embryonic development, the female reproductive cycle, wound healing, and organ and tissue regeneration, but also in pathological processes such as tumor progression and metastasis¹. Angiogenesis is now recognized as a critical process for all malignancies^2,3. As a result, the microvascular endothelial cell, which is recruited by tumors, has become an important second target in cancer therapy. It is widely accepted that the endothelial cell target, unlike the tumor cells themselves, is genetically stable¹. Antiangiogenic agents have recently emerged as a new class of drugs; however, the optimal means to use these agents alone or in combination with drug delivery systems and with conventional chemotherapy have not yet been fully elucidated.

The hypothesis that tumor growth is angiogenesis-dependent is supported by biological and pharmacological evidence⁴ and confirmed by genetic evidence^3,5-7. Both types of evidence provide a scientific basis for current clinical trials of angiogenesis inhibitors. Increased tumor angiogenesis^4,8 and elevated levels of proangiogenic factors such as vascular endothelial growth factor (VEGF/VPF)^8,9, basic fibroblast growth factor (bFGF)⁸, and interleukin-8 (IL-8)¹⁰ correlate with decreased survival and increased risk of relapse in studies of patients with malignant solid tumors. The importance of angiogenesis is further supported by the observation that antiangiogenic agents inhibit tumor growth in a variety of animal models.

In the U.S. there are currently more than 30 angiogenesis inhibitors in various clinical trials for late-stage cancer. One of these angiogenesis inhibitors, O-(chloracetyl-carbamoyl) fumagillol (TNP-470), is a low molecular weight synthetic analogue of fumagillin¹¹, a compound secreted by the fungus Aspergillus fumigatus fresenius. TNP-470 is a potent endothelial inhibitor in vitro¹². Recently, TNP-470 has been tested as a potential new anticancer agent. In animal models, TNP-470 has the broadest anticancer spectrum of any known agent^4,13. TNP-470 inhibited the growth of murine tumors up to 91%, human tumors up to 100% and metastatic tumors up to 100% in mice (reviewed in ref.¹³). In most studies, mice were treated at the same optimal dose of 30 mg/kg subcutaneously every other day. In clinical trials TNP-470 has shown evidence of antitumor activity when used as a single agent, with a number of objective responses reported with relapsed and refractory malignancies^14-16. It has also shown promise when used in combination with conventional chemotherapy^17,18. However, many patients experience neurotoxicity (malaise, rare seizures, asthenia, anxiety and dysphoria)^16,17,19,20 at doses where antitumor activity has been seen. Because of dose-limiting neurotoxicity, TNP-470 has been tested using multiple dosing regimens, but these attempts to limit its toxicity have been unsuccessful. With few exceptions, weight loss or failure to gain weight was observed in animals receiving TNP-470²¹, and two reports noted a decrease in splenic weight^22,23. Therefore, modifications of TNP-470 that can retain or increase its activity while reducing its toxicity are highly desirable.

The present invention relates to conjugates of water-soluble polymers and o-(chloracetyl-carbamoyl) fumagillol (TNP-470) and use of those conjugates as specific intracellular carriers of the TNP-470 into tumor vessels. The present invention further relates to use of those conjugates to lower the neurotoxicity of TNP-470. Preferably, the polymer has a molecular weight in the range of 100 Da to 800 kDa. More preferably, the polymer has a molecular weight no greater than 60 kDa. Most preferably, the polymer has a molecular weight in the range of 15 kDa to 40 kDa.

Preferred polymers are HPMA copolymers. HPMA copolymers are biocompatible, non-immunogenic and non-toxic carriers that enable specific delivery into tumor endothelial cells overcoming limitations of drug-related toxicities (Duncan, et al., Hum Exp Toxicol, 17:93-104 (1998)). Moreover, their body distribution is well characterized and they are known to accumulate selectively in the tumor site due to the enhanced permeability and retention (EPR) effect (Maeda, et al., J Controlled Release, 65:271-284 (2000)). The conjugate can also include a targeting moiety to direct the conjugate to sites of endothelial cell proliferation or cancer cells or to specific receptors or markers associated with proliferating endothelial cells.

The data presented herein demonstrate that TNP-470 conjugated to an HPMA copolymer: (i) avoid high peak drug levels in the circulation (ii) avoid penetration of TNP-470 to the cerebrospinal fluid and thus prevent the problem of neurotoxicity; (iii) prolong its half-life; (iv) facilitate the accumulation of TNP-470 in tissues involving neovascularization; (v) convert TNP-470 to a highly effective and widely useful angiogenesis inhibitor. We have also surprisingly discovered that conjugating TNP-470 to HPMA results in a water soluble composition.

The present invention further relates to use of the conjugates in methods of treating angiogenic diseases and decreasing neurotoxicity of TNP-470. Angiogenic disease amenable to treatment with the present invention include but are not limited to diabetic retinopathy, macular degeneration, retrolental fibroplasia, trachoma, neovascular glaucoma, psoriases, angio-fibromas, immune and non-immune inflammation, capillary formation within atherosclerotic plaques, hemangiomas, excessive wound repair, solid tumors, metastases, Kaposi\'s sarcoma and the like.

In accordance with the present invention, if polymer a having a molecular weight greater than 60 kDa is used, it is preferred that the polymer be a degradable polymer or inert. As used herein, a “degradable” polymer is one that breaks down in vivo to components having a molecular weight no greater than 60 kD. As defined herein, poly vinyl alcohol (PVA) is not a degradable polymer.

Other aspects of the invention are disclosed infra.