Patent Application
20120225849
The present invention is generally directed to cancer prevention and therapy, and more particularly to one or more prodrugs of 2-methoxyestradiol (2-ME2) that have enhanced solubility and/or bioavailability.
Prostate cancer is one of the most commonly diagnosed cancers in the United States. Among men, prostate cancer is the most common cancer among men of all races and Hispanic origin populations. It is also the leading causes of cancer deaths among men of all races and Hispanic origin populations. It is estimated that nearly 200,000 men are diagnosed and nearly 30,000 will die of prostate cancer annually in the United States. The age-adjusted incidence rate is approximately 159 cases per 100,000 men annually. Additionally, in the United States there were approximately 2.2 million men alive who had a history of prostate cancer (Reference 15).
The global prostate cancer therapy market was estimated at $5.4 billion in 2009. It is one of the largest segments of the oncology market, alongside breast, non-small cell lung and colorectal cancers. The market is forecasted to grow at a compounded annual growth rate of 6 percent to reach $7.8 billion by 2015. This high growth forecast is mainly due to the strong pipeline landscape with innovative first in class drugs and also due to high population growth. Prospective market entrants will face significant challenges including: low treatment seeking rate, low diagnosis rate, low prescription rates and the availability of generics with better efficacy and safety profiles (Reference 16).
Aside from non-melanoma skin cancer, breast cancer is the most common form of cancer in women. Breast cancer is the number one cause of cancer death in Hispanic women and the second most common cause of cancer death in white, black, Asian/Pacific Islander, and American Indian/Alaska Native women. It is estimated that over 191,000 women are diagnosed annually with breast cancer in the United States and over 40,000 women die annually from the disease (Reference 17). The global breast cancer market was estimated at $8.7 billion in 2009 and is forecast to grow at a compounded annual growth rate of 9.6 percent for the next seven years to reach $16.5 billion by 2016. The high projected growth rate is primarily attributable to a strong pipeline. Increases in the treatment seeking population, the diagnosis population and the availability new first-in-class therapies with better safety and efficacy are expected to drive the growth of the breast cancer market (Reference 18).
Rheumatoid arthritis affects an estimated 2.1 million adults in the United States. The disease occurs in all races and ethnic groups but is much more common in women than in men. The global rheumatoid arthritis therapeutics market was valued at $16.8 billion in 2008 and will be driven by the increasing aging population and the steady increase in incidence rates of autoimmune disorders. The market is expected to grow to $26.7 billion with a compounded annual growth rate of 6.8 percent by year 2015. The rheumatoid arthritis market is increasingly becoming more competitive with introduction of novel therapeutics (Reference 19).
In the year 2000, esophageal cancer (EC) was the eighth most common cancer worldwide, with 412,000 new cases, and sixth most common cause for cancer death with 338,000 deaths. In 2002, the number for new cases increased to 462,000, with 386,000 deaths.
There is currently a great demand for developing new treatments for prostate cancer and breast cancer. There are nearly 100 drugs estimated to be in clinical development for prostate cancer. The majority of these are new targeted therapies, including small-molecule tyrosine kinase inhibitors, monoclonal antibodies and therapeutic vaccine candidates. New agents with novel modes of action are also being evaluated in clinical trials, including Dendreon's Provenge, which is likely to be the first therapeutic cancer vaccine to market, and Bristol-Myers Squibb's fully-human monoclonal antibody, ipilimumab. In addition to the development of novel products, some companies are seeking to optimize the life-cycle of drugs already approved in other indications such as Roche's angiogenesis inhibitor, Avastin (bevacizumab), Pfizer's Sutent (sunitinib), Novartis' Gleevec (imatinib), and GlaxoSmithKline's preventative treatment, Avodart (dutasteride), as the most notable examples (Reference 20).
Additionally, there are currently more than 30 marketed products for the treatment of breast cancer, which include chemotherapies, combinations and targeted therapies. Furthermore, the pipeline for breast cancer consists of more than 1,500 molecules currently in development for various disease segments. Approximately 15 percent of the breast cancer pipeline is accounted for by first-in-class molecules (Reference 21).
The present disclosure is directed to various aspects of the present invention.
One aspect of the present invention includes a prodrug of an estradiol derivative.
Another aspect of the present invention includes a prodrug of 2-methoxyestradiol (2-ME2).
Another aspect of the present invention includes a novel chemotherapy agent which could inhibit tumor cell growth and/or proliferation without any of the usual chemotherapy-induced side effects.
Another aspect of the present invention includes a novel chemotherapy and/or a chemopreventive agent which has enhanced bioavailability, aqueous solubility, and/or bio-efficacy.
Another aspect of the present invention includes a prodrug of 2-ME2, which has enhanced bioavailability than the native 2-ME2. The prodrug of 2-ME2 would be metabolized in vivo to release its active metabolite 2-ME2, which would increase the selectivity of 2-ME2 for an intended tumor target and improve its anticancer potential and/or properties.
Another aspect of the present invention includes a prodrug of 2-ME2, which can be used for prophylaxis or treatment of esophageal cancer, prostate cancer, breast cancer, rheumatoid arthritis, and/or pre-clampsia.
Another aspect of the present invention includes a compound having the following general structure:
Another aspect of the present invention includes a compound having the general formula C22H29Na2O8P.
Another aspect of the present invention includes a chemotherapy agent for prophylaxis or treatment of cancer, or a non-cancerous condition, including a compound having the following general structure:
Another aspect of the present invention includes a chemotherapy agent for prophylaxis or treatment of cancer, or a non-cancerous condition, including a compound having the general formula C22H29Na2O8P.
Another aspect of the present invention includes a prodrug including 2-methoxyestradiol (2-ME2) with a hydrophilic moiety at the 3-position.
Another aspect of the present invention includes a prodrug including 2-methoxyestradiol (2-ME2) with an ester moiety at the 17-position.
Another aspect of the present invention includes a prodrug including 2-methoxyestradiol (2-ME2) with a bioreversible hydrophilic moiety at the 3-position and an ester moiety at the 17-position.
Another aspect of the present invention includes a method of enhancing bio-efficacy and/or bioavailability of 2-methoxyestradiol (2-ME2) in a living being, which includes: providing a prodrug including 2-methoxyestradiol (2-ME2) with a bioreversible hydrophilic moiety at the 3-position and an ester moiety at the 17-position, administering the prodrug to the living being, cleaving off the hydrophilic moiety from the 3-position pre-systemically and/or systemically, and masking the 17-position during a first-pass through the intestinal epithelium and liver.
Another aspect of the present invention includes a method for prophylaxis or treatment of cancer, or a non-cancerous condition, which includes administering a predetermined dose of a medicinal agent to a living being in need thereof, wherein the medicinal agent includes a prodrug of 2-methoxyestradiol (2-ME2).
In summary, the present invention is directed to an improvement over an existing drug, 2-methoxyestradiol (2-ME2). It involves the design of one or more prodrugs of 2-ME2 to overcome the poor bioavailability associated with native 2-ME2. The prodrug version(s) of 2-ME2 (henceforth referred to as Pro-2ME2 or 2ME2-PD) would preferably be metabolized in vivo to release its active metabolite-2-ME2, which would thereby increase the selectivity of the 2-ME2 for its intended tumor target and ultimately improve its anticancer potential.
One of the above and other aspects, novel features and advantages of the present invention will become apparent from the following detailed description of the non-limiting preferred embodiment(s) of invention, illustrated in the accompanying drawings, wherein:
Estrogens occurring naturally in the body are metabolized to catecholestrogens (2- and 4-hydroxyestradiol) by the cytochrome P450 enzymes. 2-Hydroxy catecholestrogens are further metabolized by catechol-O-methyltransferase to 2-methoxyestradiol (2-ME2 or 2-ME2), which is known to be protective against tumor formation (Reference 13). 2-Methoxyestradiol exhibits potent apoptotic activity against rapidly growing tumor cells and inhibits angiogenesis by reducing endothelial cell proliferation and inducing endothelial cell apoptosis. This agent also inhibits tumor cell growth by binding to tubulin, resulting in antimitotic activity, and by inducing caspase activation, resulting in cell cycle arrest in the G2 phase, DNA fragmentation, and apoptosis (Reference 14). The exact mechanism of action of 2-ME2 is still unclear, but it has been shown to be effective in preventing tumor growth in a variety of cell lines.
The present invention is an improvement of the existing drug, 2-ME2. It involves the design of one or more prodrugs of 2-ME2 to overcome the poor bioavailability of native 2-ME2. The prodrug is directed at increasing aqueous solubility/dissolution rates through addition of a 1) bioreversible hydrophilic group at the 3-position of the molecule, and altering metabolism by masking the 17-position through 2) covalent addition of an ester moiety. The 3-position promoiety is designed to preferably be cleaved pre-systemically at the brush-border of the intestinal epithelium providing high local concentrations of the prodrug intermediate for intestinal absorption. (The 3-position promoiety may additionally or alternatively be cleaved-off systemically.) On the first-pass through the intestinal epithelium and liver, the 17-position will be masked and undergoing de-esterification. The design of the prodrug will result in increased systemic exposure to 2-ME2.
2-Methoxyestradiol (2-ME2) is an estradiol derivative that acts as a microtubule destabilizing agent at pharmacological doses (References 1 and 2). Recent data suggests that 2-ME2 is effective against different tumor subtypes and has demonstrated potent antiproliferative and pro-apoptotic properties both in vitro and in vivo settings (References 1-7). Encouraged by the preclinical experience, 2-ME2 was tested in phase I studies involving patients with solid tumors (Reference 8) and breast cancer (Reference 9) and, in a phase II setting, in prostate cancer patients (Reference 10). One of the consistent end-points in all three studies included pharmacokinetic testing of 2-ME2, when administered orally. Generally, irrespective of the tumor types tested, in the majority of the patients, large interpatient and intrapatient variability of 2-ME2 pharmacokinetics was reported, which was ascribed to the poor bioavailability of 2-ME2 (References 8-10).
Despite the high level of clinical research activity with 2-ME2, the reasons for poor bioavailability and low systemic concentrations of 2-ME2 observed after oral dosing, even at very high doses in patients, are not well understood. The major barriers to poor oral drug delivery and systemic exposure of 2-ME2 include, but are not limited to, formulation, solubility, permeability, transporter effect, and first-pass metabolism. These are summarized below.
2-ME2 is formulated as 200 mg capsules with lactose, sodium starch glycolate, colloidal silicon dioxide, and magnesium stearate (Panzem®, Entremed Inc.). Due to the limited aqueous solubility of 2-ME2, extremely high doses of this formulation have been given clinically in an attempt to attain systemically useful 2-ME2 plasma levels (References 8-10). In all these studies, the AUC (area under curve) for systemic exposure to 2-ME2 did not correlate with dose. There was no significant increase in exposure with increasing dose. Because of these clinical challenges, Entremed is pursuing a nanocrystalline formulation of 2-ME2, Panzem NCD® (United States). Although initial studies suggest some improvement in oral bioavailability, results indicate there is still significant interpatient variability of 2-ME2 pharmacokinetics with this formulation and the required oral dose needed to reach adequate systemic concentrations is still substantial (Reference 11). In addition, in a phase II study in prostate cancer, in which the daily oral dose was 6000 mg, there was significant gastrointestinal toxicity, raising concerns about the GI tolerability of this particular formulation (Reference 12).
2-ME2 is a poorly soluble compound with a predicted aqueous solubility of 4.8 micrograms/mL (Calculated using Advanced Chemistry Development Software V8.14 for Solaris). 2-ME2 possesses poor aqueous solubility spanning the complete pH profile of the gastrointestinal tract. Since bioavailability has been increased with the nanocrystal formulation (10-K SEC Filing, filed by Entremed Inc. on Mar. 6, 2008), there is evidence that a more soluble form of 2-ME2 may help increase its absolute bioavailability.
The predicted log P of 2-ME2 is 3.84 (Calculated using Advanced Chemistry Development Software V8.14 for Solaris). It is expected that a high fraction of soluble 2-ME2 will permeate from the apical to the basolateral side of the GI epithelial lining and get through to the portal circulation. Permeability of 2-ME2 is thus not believed to be a major factor affecting the bioavailability.
The poor solubility of 2-ME2 can theoretically limit the concentrations entering the enterocytes, thereby preventing the saturation of drug transporters. This phenomenon remains to be further studied.
The first-pass metabolism of an orally administered drug usually occurs within the gastrointestinal (GI) epithelium and liver. In both animal and human studies, only a small fraction of an orally administered 2-ME2 dose (less than 0.1%) and its metabolites (less than 1%) are recovered in the urine (Reference 13). Of the 2-ME2 that reaches the urine, the major metabolite is the glucuronide. The low recovery of 2-ME2 and metabolites in the urine suggests that 2-ME2 is not a high first-pass clearance drug. This is supported by the observed increase in absolute bioavailability observed with the Panzem NCD® formulation.
Clinical studies in humans and in vivo studies in rodents have shown that orally absorbed 2-ME2 is metabolized by oxidation at the 17 position (2-methoxyestrone) and phase II glucuronidation at positions 3 and 17 with clearance through the kidney (References 3 and 13). The conjugated forms of 2-ME2 are inactive, and oxidation to 2-methoxyestrone results in 10-to 100-fold loss in activity in vitro (References 3, 13 and 19). However, approximately only 1% of an orally administered dose of 2-ME2 is recovered in the urine suggesting both solubility and metabolism as barriers to systemic delivery (Reference 13).
To overcome those barriers, we have designed a prodrug of 2-ME2 directed at (i) increasing aqueous solubility/dissolution rate through addition of a bioreversible hydrophilic group at the 3-position, and (ii) altering metabolism by masking the 17-position through covalent addition of an ester moiety. The 3-position promoiety is designed to preferably be cleaved pre-systemically at or adjacent the brush-border of the intestinal epithelium providing high local concentrations of the prodrug intermediate for intestinal absorption. (The 3-position promoiety may additionally or alternatively be cleaved off systemically.) On the first-pass through the intestinal epithelium and liver, the 17-position will be masked and undergoing de-esterification. The result will be increased systemic exposure to 2-ME2.
Generic 2-ME2 Prodrug Synthesis Procedure
The following steps illustrate the synthesis of main compound of the present invention—disodium(((8R,9S,13S,14S,17S)-17-acetoxy-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-3-yl)oxy)methyl phosphate (C22H29Na2O8P).
300 mg (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ol (2-methoxy estradiol; 0.1 mmol) and 0.140 mL (chloromethyl)(methyl)sulfane (1.67 mmol) were dissolved in 20-mL of dry dimethylformamide. 150 mg of 60% sodium hydride was added and the mixture was stirred at room temperature for one hour. After this time the solvent was removed in vacuo and the resulting solids dissolved in ethyl acetate. The organic layer was washed with water then filtered through silica gel. The ethyl acetate was removed in vacuo. The solids were dissolved in a 1:2 v/v mixture of ethyl acetate and hexanes and the major product isolated with elution on a silica gel column with the same solvents (Rf=0.45) to provide 313 mg (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-3-((methylthio)methoxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ol (86% yield).
208 mg (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-3-((methylthio)methoxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ol (0.57 mmol) and 126 mg acetic anhydride (1.23 mmol, 3 eqv.) were dissolved in 10-mL of dry pyridine at 0° C. The reaction was stirred overnight and allowed to come to room temperature. The solvent was removed in vacuo and the resulting solid was dissolved in a 1:2 v/v mixture of ethyl acetate and hexanes and the major product isolated with elution on a silica gel column with the same solvents (Rf=0.81) to provide 157 mg (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-3-((methylthio)methoxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate (68% yield).
157 mg (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-3-((methylthio)methoxy)-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate (0.39 mmol), 325 mg dibenzyl hydrogen phosphate (1.16 mmol, 3 eqv.), and 320 mg N-iodosuccinimide (1.42 mmol, 3.6 eqv.) were dissolved in 5-mL of dry tetrahydrofuran at room temperature. The reaction was stirred one hour after which time the solvent was removed in vacuo and the resulting solid was dissolved in a 1:2 v/v mixture of ethyl acetate and hexanes and the major product isolated with elution on a silica gel column with gradient elution (1:2 v/v ethyl acetate:hexanes to 1:1 v/v ethyl acetate:hexanes; Rf=0.62) to provide 120 mg (8R,9S,13S,14S,17S)-3-(((bis(benzyloxy)phosphoryl)oxy)methoxy)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate (49% yield).
50 mg (8R,9S,13S,14S,17S)-3-(((bis(benzyloxy)phosphoryl)oxy)methoxy)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-yl acetate (0.079 mmol) was dissolved in a mixture of 2 mL water and 25 mL tetrohydrofuran at room temperature. 11 mg disodium carbonate monohydrate and 50 mg 10% palladium on carbon were added and the reaction was stirred two hours under hydrogen at atmospheric pressure. The mixture was then filtered through a 0.45 micron Nylon filter and lyophilized to provide 39 mg of the title compound (100% yield). Identity was confirmed by mass spectroscopy using a Shimadzu 2010 single quadrupole spectrometer in negative ion mode (free acid theoretical mass: 454.45 amu, found 452.95 amu).
It is noted that alkyl anhydride in Step 2 may be replaced by other straight chain, branched chain, and cyclic alkyl anhydrides to produce the analogues of the compound of the present invention with differing position 17 esters. These analogues will have differing rates of esterase cleavage and may provide greater metabolic protection compared to the main compound of the present invention.
The main compound of the present invention (2ME2-PD) and the native compound (8R,9S,13S,14S,17S)-2-methoxy-13-methyl-7,8,9,11,12,13,14,15,16,17-decahydro-6H-cyclopenta[a]phenanthren-17-ol (2-ME2) were dissolved independently in 0.1 M Captisol® at concentrations between 10 mg/mL and 21 mg/mL. The solutions were sterile filtered through 0.2 micron filters prior to use.
Animal studies were approved and conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee. A cannulated rat model was used to study the intravenous (iv) and oral absorption 2-ME2 and 2-ME2-PD.
Sprague Dawley rats (male, 250-300 gram, Charles River Laboratories) were implanted with carotid artery, and jugular and/or femoral vein catheters. These studies were automated with the animals connected to the Culex Automated Pharmacology System allowing for direct comparison of pharmacokinetic behavior between the orally and iv administered compounds in the same animal. Following surgery, the animals were connected to the Culex and allowed to recover and acclimate. Fasted animals were dosed intravenously and orally with solutions of 2-ME2-PD in 0.1 M Captisol® (CyDex Pharmaceuticals, Inc) and 2-ME2 parent in 0.2 M HP-13-CD (Sigma-Aldrich). The dose levels are provided in Table I (below).
Oral doses were given via gavage to the animal under light anesthesia. Blood was sampled at times ranging from five to 1440 minutes into heparinized vials stored on the chilled fraction collector, and remained there until sampling was complete. Blood samples were processed to plasma via centrifugation and stored at −80° C. until analysis for 2-ME2-PD, 2-ME2 and associated metabolite concentration by liquid chromatography-mass spectrometry (LC-MS/MS). Bioanalytical methods for the 2-ME2-PD and 2-ME2 analysis were modified from those of Lakhani et. al. Quantitation was made relative to deuterated internal standards.
Pharmacokinetic analysis of the resulting plasma concentration time data was performed using PK Solution software (Summit PK). Results for the studies conducted are illustrated in
The data suggest that the 2-ME2 prodrug (2-ME2-PD) strategy employed enables the delivery of 2-ME2 to the systemic circulation. The approach provides absolute bioavailabilities from oral administration of the 2-ME2 prodrug in the 4-5% range (0-480 minutes) in these limited studies.
1Relative to 2ME2 iv and corrected for dose; AUC through 480 minutes
2Average of two animals
After demonstrating the in vitro antitumor properties of 2-ME2 against OE33 growth and invasion, we determined the in vivo effects of 2-ME2 on OE33-generated xenografts. We injected ˜2.0×106 OE33 cells subcutaneously into the left hind leg flank of each nude mice (n=2) for the development of tumor. The mice were divided into two groups (two mice per group) with a control group and Pro-2-ME2 treatment group. After forming palpable tumors, the nude mice bearing xenografts of OE33 cells were given daily pro-2-ME2 doses (75 mg/kg/day) by orogastric feeding or vehicle (control). Pro-2-ME2 was dissolved in 500 μl of saline water. We used 500 μl of saline water as a vehicle control. Tumor growth was monitored for 8 days by measuring two perpendicular diameters twice weekly. Tumor volume was calculated according to the formula V=a×b2/2, where a and b are the largest and smallest diameters, respectively.
As illustrated in
The results obtained indicate that the growth of OE33 xenografts was significantly inhibited in the pro-2-ME2 group, than in animals treated with vehicle (control).
While this invention has been described as having preferred sequences, ranges, steps, materials, structures, components, features, and/or designs, it is understood that it is capable of further modifications, uses, and/or adaptations of the invention following in general the principle of the invention, and including such departures from the present disclosure as those come within the known or customary practice in the art to which the invention pertains, and as may be applied to the central features herein before set forth, and fall within the scope of the invention and of the limits of the appended claims.
The following references, and those cited in the disclosure herein, are hereby incorporated herein in their entirety by reference.
The present application claims priority on prior U.S. Provisional Application Ser. No. 61/457,327, filed Mar. 1, 2011, which is hereby incorporated herein in its entirety by reference.
The work leading to the present invention was supported by one or more grants from the U.S. Government, and specifically NIH, Centers of Biomedical Research Excellence (COBRE Grant Number P20 RR015563), and Department of Veteran Affairs Merit Review Grant. The U.S. Government therefore has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 61457327 | Mar 2011 | US |
