Combinations of albumin-based drug delivery systems

Abstract

The present invention relates to a composition comprising at least two different albumin-based drug delivery systems, as well as to a pharmaceutical composition comprising said composition.

Claims

1. A composition comprising a combination of at least two different albumin-binding prodrugs, wherein one of said at least two different albumin-binding prodrugs is the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH), and wherein one of the at least two different albumin-binding prodrugs is the methotrexate derivative EMC-D-Ala-Phe-Lys-Lys(γ-MTX)-OH, wherein EMC=6-maleimidocaproic acid, (AW054).

2. The composition according to claim 1, wherein the at least two different albumin-based prodrugs are each present in separate containers to be sequentially administered.

3. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier and/or a pharmaceutically acceptable adjuvant and/or diluent.

Description

(1) The Figures show:

(2) FIGS. 1A AND 1B: Chemical structure of the albumin-binding prodrug DOXO-EMCH (FIG. 1A) which is the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin, and of the albumin-binding prodrug AW054 (FIG. 1B) which is a methotrexate derivative.

(3) FIG. 2: Therapeutic efficacy of DOXO-EMCH (3×24 mg/kg), AW054 (3×20 mg/kg), and a combination of DOXO-EMCH (3×12 mg/kg) and AW054 (3×10 mg/kg) or of doxorubicin (3×6 mg/kg), of methotrexate (3×125 mg/kg) or a combination of doxorubicin (3×3 mg/kg doxorubicin) and methotrexate (3×62.5 mg/kg) against the MiaPaCa-2 pancreatic carcinoma xenograft model (7 animals per group); tumor volumes are depicted on a logarithmic scale.

(4) FIG. 3: Body weight curves under therapy with DOXO-EMCH (3×24 mg/kg), AW054 (3×20 mg/kg), and a combination of DOXO-EMCH (3×12 mg/kg) and AW054 (3×10 mg/kg) or of doxorubicin (3×6 mg/kg), of methotrexate (3×125 mg/kg) or a combination of doxorubicin (3×3 mg/kg doxorubicin) and methotrexate (3×62.5 mg/kg) in the MiaPaCa-2 pancreatic carcinoma xenograft model (7 animals per group) are depicted.

(5) The compositions of the present invention surprisingly and advantageously produce an improved efficacy and in vivo tolerability over the use of the drugs as such, and further provide an effective treatment of diseases which have previously been refractory to the therapeutics available in the prior art. Accordingly, it is advantageously possible to efficiently treat a great variety of diseases by combining at least two albumin-based drug delivery systems, such as albumin-binding prodrugs, whereby the advantageous properties of said drugs can be improved beyond the properties of the individual drugs alone.

(6) The present invention will now be further illustrated in the following example without being limited thereto. Many further combinations according to the present invention can be realized as outlined above.

EXAMPLE

Example: Evaluation of the Antitumor-Efficacy of a Combination of Two Albumin-Binding Prodrugs

(7) The antitumor efficacy of a combination of the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH) and an albumin-binding prodrug of methotrexate, AW054 (see FIGS. 1A and B), that is cleaved by two proteases that are over-expressed in solid tumors, cathepsin B and plasmin was evaluated in the MiaPaCa-2 pancreatic carcinoma xenograft model.

(8) The 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH) (FIG. 1A) is an albumin-binding prodrug of doxorubicin that binds rapidly to the cysteine-34 position of circulating albumin after administration and is taken up by solid tumors. Following tumor uptake, doxorubicin is cleaved in the acidic environment of tumor tissue, either extra- or intracellularly. DOXO-EMCH has an maximum tolerated dose (MTD) of 3×24 mg/kg doxorubicin equivalents in nude mice and has shown superior efficacy over free doxorubicin in several xenograft and orthotopic tumor models (F. Kratz et al. (2002): Probing the Cysteine-34 Position of Endogenous Serum Albumin with Thiol-binding Doxorubicin Derivatives: Improved Efficacy of an Acid-sensitive Doxorubicin Derivative with Specific Albumin-binding Properties Compared to the Parent Compound, J. Med. Chem. 45, 5523-5533; R. Graeser et al. (2009): INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin); and R. Graeser, N. Esser, H. Unger, I. Fichtner, A. Zhu, C. Unger, F. Kratz (2010): INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model, Investigational New Drugs 28, 14-19).

(9) AW054, i.e. EMC-D-Ala-Phe-Lys-Lys(γ-MTX)-OH (EMC=6-maleimidocaproic acid), rapidly binds to the cysteine-34 position of circulating albumin after administration to form a conjugate which is stable in human plasma. Due to two lysine residues it can be cleaved by cathepsin B and plasmin, two enzymes which are found in high levels in solid tumors. After binding to endogenous albumin, AW054 accumulates in tumor tissue and tumor cells where a methotrexate-lysine derivative is released either extra- or intracellularly by plasmin and cathepsin B, respectively (A. Warnecke, I. Fichtner, G. Sass, F. Kratz (2007): Synthesis, cleavage profile, and antitumor efficacy of an albumin-binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B., Arch. Pharm., Pharm. Med. Chem. 340, 389-395). AW054 has a MTD of 3×20 mg/kg (methotrexate equivalents) and is superior compared to methotrexate (MTD 3×125 mg/kg) in an ovarian carcinoma xenograft model (OVCAR3) (A. Warnecke, I. Fichtner, G. Sass, F. Kratz (2007): Synthesis, cleavage profile, and antitumor efficacy of an albumin-binding prodrug of methotrexate that is cleaved by plasmin and cathepsin B., Arch. Pharm., Pharm. Med. Chem. 340, 389-395).

(10) The combination of DOXO-EMCH with AW054 was evaluated in comparison to the respective albumin-binding prodrugs alone as well as to the conventional drugs doxorubicin and methotrexate and to a combination of doxorubicin and methotrexate in the MiaPaCa-2 pancreatic carcinoma xenograft model.

(11) The experiment in the ovarian carcinoma MiaPaCa-2 xenograft model was carried out as follows:

(12) In vivo efficacy studies in the MiaPaCa-2 xenograft model were carried out with doxorubicin, the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH), the albumin-binding prodrug of methotrexate AW054, a combination of the 6-maleimidocaproyl(hydrazone) derivative of doxorubicin (DOXO-EMCH) and the albumin-binding prodrug of methotrexate AW054, doxorubicin, methotrexate and a combination of doxorubicin and methotrexate.

(13) DOXO-EMCH was dissolved in sterile 10 mM sodium phosphate, 5% D-(+)-glucose (pH 5.8) and the respective dose administered intravenously within 30 minutes after dissolution. All doses of DOXO-EMCH and AW054 administered in the studies are stated in doxorubicin and methotrexate equivalents, respectively. Adrimedac® from medac, Germany, was used as the doxorubicin reference (c=2 mg/mL). Methotrexate (Sodium salt from medac) was used as a stock solution (12.5 mg methotrexate/mL in 5% D-Glucose from Braun, sterile). The stock solution AW054 for intravenous administration was 2.0 mg AW054/mL in 5% D-Glucose from Braun, sterile.

(14) For in vivo testing in the MiaPaCa-2 xenograft model, female NMRI: nu/nu mice (Taconic, Denmark) were used. The mice were held in individually ventilaged cages (IVC) under sterile and standardized environmental conditions (25±2° C. room temperature, 50±10% relative humidity, 12 hour light-dark-rhythm). They received autoclaved food and bedding (ssniff, Soest, Germany) and acidified (pH 4.0) drinking water ad libitum. All animal experiments were performed under the auspices of the German Animal Protection Law.

(15) MiaPaCa-2 cells (5×10.sup.6 cells/mouse) were transplanted subcutaneously (s.c.) into the left flank region of each mouse on day zero. Mice were randomly distributed to the experimental groups (7 mice per group). Treatment was initiated at day 10 when the tumors were grown to a size of ˜60 mm.sup.3. Mice were treated on a weekly schedule with 3×6 mg/kg doxorubicin (MTD), 2×125 mg/kg methotrexate (MTD), 3×24 mg/kg DOXO-EMCH (doxorubicin equivalents) (MTD), 3×20 mg/kg AW054 (methotrexate equivalents) (MTD), and a combination of 3×12 mg/kg DOXO-EMCH and 3×10 mg/kg AW054 or a combination of 3×3 mg/kg doxorubicin and 3×62.5 mg/kg methotrexate. All compounds were injected once a week for three weeks (days 10, 17, 24). The injection volume was 0.2 mU20 g body weight. Tumor size was measured twice weekly with a caliper-like instrument in two dimensions. Individual tumor volumes (V) were calculated by the formula V=(length×[width].sup.2)/2 and related to the values on the first day of treatment (relative tumor volume, RTV). The experiment was ended on day 43. Statistical analysis was performed with the U-test (Mann and Whitney) with p<0.05. The body weight of mice was determined every 3 to 4 days.

(16) Pancreatic cancer has a poor prognosis: For locally advanced and for metastatic disease, which collectively represent over 80% of individuals, median survival is approximately 10 and 6 months, respectively, when treated with either 5-fluorouracil or gemcitabine.

(17) 3×6 mg/kg doxorubicin is the maximum tolerated dose (MTD) in nude mice models. The MTD of methotrexate in nude mice models is 3×125 mg/kg (i.v.). Combination therapy with doxorubicin and methotrexate was carried out at in a three weekly schedule (i.v.) at half of their maximum tolerated dose (MTD), i.e. 3×3 mg/kg for doxorubicin and 3×62.5 mg/kg methotrexate. Both DOXO-EMCH and AW054 were administered simultaneously (i.v.) in a three weekly schedule at half of their maximum tolerated dose (MTD), i.e. 3×12 mg/kg for DOXO-EMCH (doxorubicin equivalents) and 3×10 mg/kg (methotrexate equivalents) and at their respective MTDs, i.e. 3×24 mg/kg for DOXO-EMCH (doxorubicin equivalents) and 3×20 mg/kg for AW054 (methotrexate equivalents) (see the following Table 1).

(18) TABLE-US-00001 TABLE 1 Comparison of doses, mortality, and final tumor volume of doxorubicin, DOXO-EMCH, and the combination of DOXO- EMCH and doxorubicin against human Mia-Paca-2 cancer xenografts (median initial tumor volume on day 1 was 0.062 cm.sup.3) Dose (i.v.) Tumor volume Schedule [mg/kg/ (day 43) Mice Compounds [days] inject.] toxic death cm.sup.3/d 43 7 Glucose- 10, 17, 24 0 .sup. 0.992 ± 0.362 - Phosphate Puffer 7 Doxorubicin 10, 17, 24  6 0 0.190 ± 0.125* 7 Methotrexate 10, 17, 24 125  0 0.396 ± 0.307* Doxorubicin + 10, 17, 24   3 + 62.5 0 0.199 ± 0.071* Methotrexate 7 DOXO-EMCH 10, 17, 24 24 1 (d 39) 0.034 ± 0.064* 7 AW054 10, 17, 24 20 0 0.264 ± 0.226* 7 DOXO-EMCH + 10, 17, 24 12 + 10 0   0.001 ± 0.001* ** AW054 5 × 10.sup.6 MiaPaca-2 cells/mouse, s.c. day 0 *Statistically significant to control (Glucose phosphate buffer treated group) ** Statistically significant to all groups Mann-Whitney test, p < 0.05 NMRI: nu/nu mice, female

(19) The efficacy results of the drugs alone and their combination are shown in FIG. 2

(20) Compared to the combination of DOXO-EMCH or AW054 alone, where DOXO-EMCH showed a partial remission (one of the seven mice died on day 39) and AW054 only showed moderate antitumor effects over control animals treated with 5% glucose phosphate buffer (see FIG. 2), the combination of the two albumin-binding prodrugs produced complete remissions and an acceptable body weight loss of −13% at the end of the experiment compared to the starting tumor volume (see FIG. 3).

(21) With respect to antitumor efficacy, the free drugs doxorubicin and methotrexate at their optimal and maximum tolerated doses or as combination of the two drugs only showed moderate antitumor efficacy up to the end of the experiment (day 43) (see FIG. 2).

(22) In summary, treatment with a combination of the two albumin-binding prodrugs DOXO-EMCH at 3×12 mg/kg and AW054 at 3×10 mg/kg was the only therapy schedule which produced complete remissions against subcutaneously growing MiaPaCa-2 pancreatic carcinoma xenografts, this effect being statistically significant (p<0.05, Mann-Whitney test) compared to all other groups. Thus, the treatment combining DOXO-EMCH with AW054 has a distinct advantage over treatment with either DOXO-EMCH or AW054 alone or doxorubicin, methotrexate or a combination thereof with respect to antitumor efficacy.