JP2012214519A - Method, composition and product for contributing to treatment of solid tumors - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, a composition and a product for contributing to the treatment of a solid cancerous tumor, capable of utilizing an endothelin B (ET) receptor agonist to enhance the delivery of a chemotherapeutic agent to a solid tumor in mammals, including humans.SOLUTION: The method for contributing to the treatment of a solid cancerous tumor includes: intravenously administering the ETreceptor agonist and the chemotherapeutic agent to a subject mammal. The ETreceptor agonist enhances the delivery of the chemotherapeutic agent to the solid tumor by selectively enhancing the blood supply to the solid tumor.

Description

  The present invention relates to methods, compositions and products for contributing to the treatment of solid tumors such as breast tumors in mammals through the administration of endothelin agonists and chemotherapeutic agents.

The incidence of breast cancer has increased substantially over the past decade and has become the single leading cause of death for women aged 40-49 in the United States. Successful treatment of solid tumors, including breast cancer, has not yet met medical goals, despite an understanding of the molecular biology of tumor cells and the availability of many potential therapeutics.

  One problem in cancer treatment is that the effective amount of a variety of possible chemotherapeutic drugs is limited due to the non-selective, highly toxic effects of these drugs on normal tissues. As a result, many patients suffer from the side effects of chemotherapy without benefiting from treatment. For example, the chemotherapeutic drug paclitaxel inhibits cell proliferation and induces apoptosis of tumor cells. However, the clinical utility of paclitaxel has been hampered by its dose limiting toxicities such as hypersensitivity, neutropenia and peripheral neuropathy. Therefore, there is a need for the development of more specific and less toxic cancer therapies.

  The ability to target delivery of chemotherapeutic drugs to tumors should provide the benefit of enhancing systemic toxicity while enhancing the benefits of chemotherapeutic drugs. Such targeted delivery would also help reduce the need for chemotherapeutic drugs and thus potentially reduce the unacceptable adverse effects of these drugs. One possible way to achieve targeted delivery of chemotherapeutic drugs is to take advantage of features unique to the tumor vasculature.

  Tumors larger than a few millimeters in size require constant nutrition and thus develop their own vascular bed and blood flow. Folkman, Cancer Res. 46: 467 (1986). Without constant feeding from these developing blood vessels, the tumor becomes hypoxic and then dies. The recruitment of new vasculature from existing blood vessels is called “angiogenesis”.

During angiogenesis, tumor blood vessels develop and differ in nature from the normal vasculature. A monolayer of epithelial cells is a tumor blood vessel that is first prepared. These newly formed tumor blood vessels do not have a smooth muscle layer or nerve distribution. Tumors also take up mature blood vessels with any self-regulating function. Mattsson et al., Tumor Blood Circuit, CRC Press, Boca Raton, pg. 129 (1979); Reinhold, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 115 (1979); Warren, Tumor Blood
Circulation, CRC Press, Boca Raton, pg. 26 (1979).
Vascular tone (the extent to which blood vessels dilate or contract) is a function of many endogenous factors, including H ⁺ , K ⁺ , Ca ²⁺ , pO ₂ , pCO ₂ and nitric oxide (NO), and endothelin (ET-1). Is governed by other regulatory substances such as: Luscher et al., The endotherium: modulator of cardiovascular function, CRC Press, Boca Raton, pg. 61 (1990). ET-1 contributes significantly to the regulation of vascular tone (Yanagisawa et al., Nature,
332: 411 (1988)), researchers have shown that expression of ET-1 and ET _B receptors solid tumors including breast cancer is increasing. Alanen et al., Histopathology, 36: 161 (2000); Nelson et al., Cancer Res, 56: 663 (1996); Kar et al., Biochem Biophys Res Commun 216: 514 (1995); Pagoto et al., J Clin Invest, 96: 2017 1995); Yamashita et al., Cancer Res, 52: 4046 (1992); Yamashita et al., Res Commun Chem Pathol Pharmacol, 74: 363 (1991). Furthermore, stimulation of ET _B receptors causes an increase in blood supply to the tumor through vasodilation of tumor blood vessels. The present invention selectively increases the blood flow to the tumor using the ET _B receptor agonist by utilizing this fact, enhance targeted delivery of chemotherapeutic agents.

Folkman, Cancer Res. 46: 467 (1986) Mattsson et al., Tumor Blood Circuit, CRC Press, Boca Raton, pg. 129 (1979) Reinhold, Tumor Blood Circulation, CRC Press, Boca Raton, pg. 115 (1979) Warren, Tumor Blood Circuit, CRC Press, Boca Raton, pg. 26 (1979) Luscher et al., The endotherium: modulator of cardiovascular function, CRC Press, Boca Raton, pg. 61 (1990) Yanagisawa et al., Nature, 332: 411 (1988). Alanen et al., Histopathology, 36: 161 (2000) Nelson et al., Cancer Res, 56: 663 (1996). Kar et al., Biochem Biophys Res Commun 216: 514 (1995). Pagotto et al., J Clin Invest, 96: 2017 (1995). Yamashita et al., Cancer Res, 52: 4046 (1992) Yamashita et al., Res Commun Chem Pathol Pharmacol, 74: 363 (1991).

The present invention is directed to the administration of endothelin agonists and chemotherapeutic agents to individuals in need thereof to contribute to the treatment of solid tumors. In particular, tumors have distinctive vasculature including an increased number of ET _B receptors (when combined cause vasodilation). Since ET _B receptors are vasodilators, ET _B receptor agonists in combination with chemotherapeutic agents are useful for the treatment of solid tumors such as those found in breast cancer. ET _B receptor agonists can deliver chemotherapeutic drugs to tumors more effectively, resulting in enhanced treatment.

Specifically, one aspect of the present invention includes a method of contributing to the treatment of solid tumors, the method comprises intravenous administration of ET _B agonist and chemotherapeutic agent need thereof a mammal. The ET _B agonist increases the delivery of chemotherapeutic agents to solid tumors by selectively increasing blood supply to the solid tumor.

In another embodiment of the process of the present invention, pharmacokinetics of chemotherapeutic agents are not affected by the ET _B receptor agonist.
In another embodiment of the process of the present invention, ET _B receptor agonists enhance the efficacy of chemotherapeutic agents.

In another embodiment of the process of the present invention, ET _B agonist, ET-1, ET-2 , ET-3, BQ3020, IRL1620 (N-suc- [Glu 9, Ala 11,15] ET-1 (8- 21)), sarafotoxin 56c, [Ala ^1,3,11,15 ] ET-1, and mixtures thereof. In another embodiment of the process of the present invention, ET _B agonist is IRL1620.

  In another embodiment of the method of the invention, the chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and the like Selected from the group consisting of: In another embodiment of the method of the present invention, the chemotherapeutic agent is paclitaxel.

  In another embodiment of the method of the present invention, the solid tumor is selected from the group consisting of ovarian tumor, colon tumor, Kaposi's sarcoma, breast tumor, melanoma, prostate tumor, meningioma, liver tumor, and breast phyllodes tumor. In another embodiment of the method of the present invention, the solid tumor is a breast tumor.

In another embodiment of the process of the present invention, ET _B agonist and the chemotherapeutic agent are administered ET _B agonist and a chemotherapeutic agent as a single composition; administering ET _B agonist and a chemotherapeutic agent as separate compositions Administer ET _B agonist and chemotherapeutic agent in turn, administer ET _B agonist prior to chemotherapeutic agent; and administer ET _B agonist and chemotherapeutic agent in order, administer chemotherapeutic agent prior to ET _B agonist Is administered according to a strategy selected from the group consisting of:

In another embodiment of the method of the present invention, the mammal is a human.
The invention also includes products. In one aspect, the product provides (a) a packaging composition comprising an ET _B agonist; and (b) an instruction providing for the intravenous administration of an ET _B agonist to contribute to the treatment of a solid tumor in a mammal. And (c) a container for (a) an ET _B agonist and (b) a package insert.

In another embodiment of the products of the present invention, the product, (d) a chemotherapeutic agent further comprises, instruction defines the administration of ET _B agonist and a chemotherapeutic agent, a container (c) is (a) ET _B agonist (B) for package inserts and (d) for chemotherapeutic drugs.

In another embodiment of the products of the present invention, if in accordance with the instruction, ET _B agonist selectively increases the blood supply to the solid tumor, thus chemotherapeutic agent delivery to solid tumors increases.
In another embodiment of the products of the present invention, pharmacokinetics of chemotherapeutic agents are not affected by the ET _B receptor agonist.

In another embodiment of the products of the present invention, ET _B receptor agonists enhance the efficacy of chemotherapeutic agents.
In another embodiment of the products of the present invention, ET _B agonist, ET-1, ET-2 , ET-3, BQ3020, IRL1620 (N-suc- [Glu 9, Ala 11,15] ET-1 (8- 21)), sarafotoxin 56c, [Ala ^1,3,11,15 ] ET-1, and mixtures thereof. ET _B agonists in another embodiment of the products of the present invention is IRL1620.
In another embodiment of the product of the invention, the chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, and the like Selected from the group consisting of: In another embodiment of the product of the present invention, the chemotherapeutic agent is paclitaxel.

FIG. 6 shows the effect of IRL1620 on changes in tumor perfusion induced by paclitaxel. FIG. 2A is a diagram showing the effect of ET-1 on systemic hemodynamics of cancer-free rats and breast tumor rats. FIG. 2B shows the effect of ET-1 on systemic hemodynamics in cancer-free and breast tumor rats. FIG. 2C is a diagram showing the effect of ET-1 on systemic hemodynamics of cancer-free rats and breast tumor rats. FIG. 2D shows the effect of ET-1 on systemic hemodynamics in cancer-free and breast tumor rats. FIG. 2E is a diagram showing the effect of ET-1 on systemic hemodynamics of cancer-free rats and breast tumor rats. FIG. 3A shows the effect of ET-1 on blood flow and local vascular resistance in breast tissue of cancer-free and breast tumor rats. FIG. 3B shows the effect of ET-1 on blood flow and local vascular resistance in breast tissue of cancer-free and breast tumor rats. FIG. 4A shows the effect of ET-1 on perfusion, mobile blood cell concentration (CMBC), and blood cell velocity in breast tissue of cancer-free and breast tumor rats. FIG. 4B shows the effect of ET-1 on perfusion, mobile blood cell concentration (CMBC), and blood cell velocity in breast tissue of cancer-free and breast tumor rats. FIG. 4C shows the effect of ET-1 on perfusion, mobile blood cell concentration (CMBC), and blood cell velocity in breast tissue of cancer-free and breast tumor rats. FIG. 5A shows the effect of BQ788 on changes in blood perfusion, CMBC, and blood cell velocity in breast tissue of cancer-free and breast tumor rats induced by ET-1. FIG. 5B shows the effect of BQ788 on changes in blood perfusion, CMBC, and blood cell velocity in breast tissue of cancer-free and breast tumor rats induced by ET-1. FIG. 5C shows the effect of BQ788 on changes in blood perfusion, CMBC, and blood cell velocity in breast tissue of cancer-free and breast tumor rats induced by ET-1. FIG. 10 shows the effect of IRL1620 on paclitaxel accumulation in tumors and other major organs of breast tumor rats. FIG. 5 shows the effect of vehicle or IRL1620 on plasma pharmacokinetic analysis of paclitaxel in normal and tumor rats as measured by HPLC. FIG. 6 shows the effect of vehicle or IRL1620 on plasma pharmacokinetics of [ ³ H] paclitaxel measured by liquid scintillation counting. FIG. 6 shows the effect of vehicle or IRL1620 on plasma pharmacokinetics of [ ³ H] paclitaxel measured by liquid scintillation counting. FIG. 10A shows the effect of IRL1620 on breast tumor perfusion measured with a laser Doppler flow meter. FIG. 10B shows the effect of IRL1620 on breast tumor perfusion measured with a laser Doppler flow meter. FIG. 5 shows the time-dependent effect of IRL1620 administration on [ ³ H] paclitaxel concentrations in tumors and major organs of breast tumor rats. It is a figure which shows the difference rate in the body weight of the breast tumor rat compared with the time of a treatment start. It is a figure which shows the influence of IRL1620 administration on the tumor volume of a breast tumor rat. And FIG. 5 shows the effect of IRL1620 administration on tumor progression, stagnation and regression.

Detailed Description Definitions Some terms used in the specification are explained as follows.
The term “treatment” or “contributes to treatment” includes prevention, slowing, reduction or elimination of solid tumor prevention, progression or growth. Thus, these terms include both medical therapeutic and / or prophylactic administration, as appropriate.

The term “container” means any receptacle and closure suitable for storage, transport, medication, and / or handling of a pharmaceutical product.
The term “package insert” refers to information attached to a pharmaceutical product that includes information on how to administer the product, along with detailed information on the use of the product by physicians, pharmacists, and patients. Provide the safety and efficacy data necessary to make decisions. Package inserts are generally considered “labels” for pharmaceutical products. The package insert can take many forms, including but not limited to paper or CD ROM.

The term “prodrug” means a compound that rapidly converts in vivo to a compound useful in the invention, for example, by hydrolysis. A detailed commentary on prodrugs can be found in Higuchi et al., Prodrugs as Novel Delivery Systems,
Vol. 14, of the A.E. C. S. D. Symposium Series, and in Roche (ed.), Bioreversible Carriers in Drug Design, American Pharmaceutical Association and Pergamon Press, 1987.

  Most chemotherapeutic drugs have cytotoxicity that is targeted to destroy cancer cells, but in the process also causes considerable damage to the body's normal physiology. Therefore, selective delivery of chemotherapeutic drugs to solid tumors has great advantages and helps to avoid these adverse effects in cancer treatment.

  The blood vessel structure of tumor blood vessels is different from that of normal blood vessels. Carmeliet & Jain, Nature, 407: 249 (2000). Therefore, the vascular reactivity of the tumor is also different from that of normal tissue. For example, administration of nitric oxide donors, nicotinamide and bradykinin agonists modulate blood flow to the tumor. Jordan et al., Int J Radiate Oncol Biol Phys, 48: 565 (2000); Fukumura et al., Am J Pathol, 150: 713 (1997); Hirst et al., Br J Radiol, 67: 795 (1994).

Endothelin is a vasoactive agent that modulates blood flow and is present in higher concentrations in breast cancer tissue compared to normal breast tissue (specifically, endothelin is about 0.12 pg / mg in normal breast tissue). In contrast, it may be present in breast cancer tissue in an amount of about 12 pg / mg). Kojima et al., Surg Oncol, 4 (6): 309 (1995); Kurbel et al., Med Hypotheses, 52 (4): 329 (1999); Patel et al., Mol Cell Endocrinol, 126 (2): 143 (1997); Yamashita Cancer Res, 52 (14): 4046 (1992); Yamashita et al., Res Commun Chem Pathol Pharmacol, 74 (3): 363 (1991). Endothelin is a family of cyclic peptides having 21 amino acids and includes three isoforms, ET-1, ET-2 and ET-3 in mammals. Inoue et al., Proc Natl Acad Sci USA 86: 2863 (1989); Yanagisawa et al., Nature, 332: 411 (1988). Endothelin acts by binding to two different cell surface receptors ET _A and ET _B. ET _B receptors bind with the same affinity as three peptide isotypes. In contrast, ET _A receptors, binds with high affinity to ET-1 than the other isoforms. Both receptors belong to the G protein coupled receptor system and mediate biological responses from various stimuli such as growth factors, vasoactive polypeptides, neurotransmitters and hormones. Masaki, J
Cardiovas Pharmacol, 35: S3 (2000); Gulati, Preface. Adv Drug Deliv Rev, 40: 129 (2000); Gulati et al., Am J Physiol, 273: H827 (1997); Levin, N Engl J Med, 333: 356 (1995). ET _B receptors are the focus of the present invention, endothelial cells (EC) and present in both the vascular smooth muscle cells (VSMC), even breast tissue (invasive breast cancer tissues of human when compared to normal breast tissue gland Tuberculosis and lobular breast cancer tissue). Wulging et al., Oncol Rep, 11: 791 (2004); Wulfing et al., Clin Cancer Res, 9: 4125 (2003); Alanen et al., Histopathology, 36 (2): 161 (2000). Endothelin cause vasodilation acts on ET _B receptors, increase blood flow to breast tumor tissue. ET _B receptors found in many over the EC, causes vasodilation by the release of factors such as prostacyclin and nitric oxide. de Nucci et al., Proc Natl Acad Sci USA, 85: 9797 (1988). Because ET-1 is causing the increased blood flow to tumors by stimulating ET _B receptors can be selectively increase blood supply to tumors Using ET _B receptor agonist. This increases the targeted delivery of chemotherapeutic drugs and the resulting efficacy.

ET _B receptors have been shown, for example, in ovarian cancer, myofibroblasts, Kaposi's sarcoma and intratumoral blood vessels, breast cancer and melanoma. Bagnat et al., Am J Pathol, 158: 841 (2001); Alanen et al., Histopathology, 36 (2): 161 (2000); Bagnat et al., Cancer Res, 59: 720 (1999); Kikuchi et al., Biochem Biophys Res. 219: 734 (1996). Thus, administration of ET _B receptor agonist in combination with a chemotherapeutic agent, ovarian cancer, colon cancer, Kaposi's sarcoma, breast cancer, and melanoma to contribute to the treatment of solid tumors including but not limited to Can be used.

Useful ET _B agonists according to the present invention, ET-1, ET-2 , ET-3, BQ3020, IRL1620 (N-suc- [Glu 9, Ala 11,15] ET-1 (8-21)), sarafotoxin 56c, [Ala ^1,3,11,15 ] ET-1, and mixtures thereof, but are not limited thereto. [Ala ^1,3,11,15 ] ET-1 is a linear analog of ET-1, with the disulfide bridge removed by replacing the Cys residue with Ala. Saeki et al., Biochem Biophys Res Commun, 179: 286 (1991). BQ3020 and IRL1620 are truncated (truncated) linear synthetic analogs of ET-1 and the most widely used selective synthetic agonists. IRL1620 is a linear ET- analog whose structure is based on the carboxy terminus of ET-1, having a 120,000 fold selectivity for ET _B receptors. Okada & Nishikibe, Cardiovasc Drug Rev, 20:53 (2002); Douglas et al., Br J Pharmacol, 114: 1529 (1995). IRL1620 is a highly selective and potent ET _B agonist, and evidence of selectivity for the ET _B1 receptor subtype over the ET _B2 subtype has been reported. Brooks et al., J Cardiovas Pharmacol, 26 Suppl 3: S322 (1995).

Chemotherapeutic agents useful in accordance with the present invention include, but are not limited to, alkylating drugs, antimetabolites, hormones and antagonists thereof, radioisotopes, antibodies, and natural products, and mixtures thereof. For example, ET _B agonists, antibiotics such as doxorubicin and other anthracycline analogs, nitrogen mustards such as cyclophosphamide, but not limited to, 5-fluorouracil (but not limited to), such as It can be administered with pyrimidine analogs, cisplatin, hydroxyurea, and natural and synthetic derivatives thereof. As another example, if the tumor is a mixed tumors, such as breast adenocarcinoma comprising gonadotropin-dependent and gonadotropin-independent cells, ET _B agonist leuprolide or goserelin (LH-RH of the synthetic peptide analogs) (these Can be administered in combination with (but not limited to). Examples of additional non-limiting chemotherapeutic agents that can be used with the present invention include adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, interferon (alpha, beta and / or gamma), interleukin 2, irinotecan, docetaxel, paclitaxel, Topotecan, and their therapeutically effective analogs and derivatives.

In one embodiment of the invention, endothelin agonists are used in combination with chemotherapeutic agents to contribute to the treatment of solid tumors. In this method, the endothelin agonist, notably ET _B agonist increases blood flow to tumors rich ET _B receptors. Therefore, ET _B agonist may provide selective targeting by for chemotherapeutic drugs, to improve the chemotherapeutic effect of the agent.

Endothelin agonists tumor vessels to expand by stimulating ET _B receptor, thereby it results in an increase of blood flow and as a result of chemotherapeutic agents delivered to the tumor is theorized, here resorting to it Absent. Increased tumor blood perfusion caused by endothelin agonists also increases tissue oxygenation. Improved oxygenation can enhance the therapeutic effects of chemotherapeutic drugs. Endothelin can also be mitogenic. The mitogenic action of endothelin can also help to increase the action of chemotherapeutic drugs when administered together. The mitogenic action of endothelin agonists can enhance the action of chemotherapeutic drugs by improving the uptake of chemotherapeutic drugs into dividing cells. Therefore, the efficacy of chemotherapeutic drugs is increased.

Chemotherapy is often indicated as an adjunct to surgery in the treatment of cancer. The goal of chemotherapy as an adjunct therapy is to reduce the risk of recurrence and increase disease-free survival when the primary tumor is controlled. Chemotherapy is often used as an adjunct treatment for cancer when the disease is metastatic. Therefore, ET _B agonists are particularly useful in combination with chemotherapy before or after surgery in the treatment of solid tumors.

Example 1. Methods of Influence of IRL1620 and Paclitaxel on Breast Tumor Perfusion The following study was conducted to investigate the systemic hemodynamic and local circulation effects of ET-1 in normal and breast tumor rats.

One widely studied breast tumor model is a chemically induced rat breast cancer development model. van Zwieten, The rat as an animal model in breast cancer research. Martinus Nijhoff Publishers, Boston, pg. 206 (1984); Dao et al., J
Natl Cancer Inst, 71: 201 (1983); Russo et al., J Natl Cancer Inst, 61: 1439 (1978). Huggins et al., Science, 137 (1962); Huggins et al., Proc Natl Acad Sci USA, 45: 1294 (1959). Chemically induced breast tumor development in rats is a model that best approximates human cancer. Russo et al., Lab Invest, 62: 244 (1990). In terms of tissue structure, the rat mammary gland resembles that of human women. It is formed by the epithelium that covers the mammary ducts and mammary vesicles, and the stroma, the connective tissue that forms the skeleton of this organ. These two compartments interact continuously throughout fetal development and maturation. Therefore, this spontaneous experimental model was chosen as the model for this study because it best approximates human cancer. Same as above.

Chemically induced rat breast cancer development is typically achieved by administration of 7,12-dimethylbenzene (a) anthracene (DMBA) or N-methylnitrosourea (MNU). Rogers et al., Chemically Induced Mammary Grand Tumors in Rats: modulation by dietary fat. Alan R. Liss, Inc. , New York 255 (1996). Tumors induced by DMBA or MNU have different morphological characteristics. In particular, tumors induced by MNU are localized by the breast and are difficult to metastasize. Macejova et al., Endocr Regul, 35:53 (2001). Therefore, MNU is often chosen as a chemical agent to specifically induce breast tumors in rats. These breast tumors can be benign or malignant with fibroadenoma and papilloma. van Zwieten, The rat as an animal model in breast cancer research. Martinus Nijhoff Publishers, Boston, pg. 206 (1984). Rats have 6 pairs of mammary glands, 1 pair in the neck, 2 pairs in the chest, 1 pair in the abdomen, and 2 pairs in the groin. Astowood et al., Am J Anat, 61 (1937). Virgin rats treated with MNU develop more tumors in the chest than in the abdomen. Russo et al., Lab Invest, 57: 112 (1987).

Methods 180-200 grams (g) of female Sprague Dawley rats (Harlan Co., Madison, Wis.) Were used. Three animals were housed in one cage under a temperature control room (23 ± 1 ° C.), humidity (50 ± 10%), and artificial light (06: 0 to 18:00). Animals were given food and water ad libitum. Experiments were performed after the animals had been habituated to the environment for at least 4 days.

  N-methylnitrosourea (MNU) is available from Ash Stevens Inc. (Detroit, Michigan). IRL1620 and endothelin-1 (ET-1) are available from American Peptide Company Inc. (Sunnyvale, Calif.). ET-1 was dissolved in 0.1% albumin.

  MNU (50 mg / kg) or saline (1 ml / kg) was administered intraperitoneally (ip) to female Sprague Dawley rats. Blood flow experiments were performed after the tumors reached 2-4 cm in diameter.

  Rats were anesthetized with urethane (1.5 g / kg, ip) (Sigma Chemicals, St. Louis, Mo.), and the left femoral vein was cannulated for drug administration (PE 50 tubes, Cley Adams, Parsippany, NJ). ).

The animals were divided into the following groups:
(I) Saline injection into normal rats, 15 minutes later paclitaxel (3 mg / kg) (N = 4);
(Ii) IRL1620 (3 nmol / kg) injection into normal rats, 15 minutes later paclitaxel (3 mg / kg) (N = 4);
(Iii) Saline injection into tumor rats, 15 minutes later paclitaxel (3 mg / kg) (
(Iv) Tumor rats were injected with IRL1620 (3 nmol / kg), 15 minutes later paclitaxel (3 mg / kg) (N = 4).

  Blood perfusion into the rat mammary gland was measured using a laser Doppler flow meter. See Song et al., Int J Radiate Oncol Biol Phys, 18: 903 (1990); Song et al., Int J Radiate Oncol Biol Phys, 17: 1041 (1989). In this process, the area around the nipple of the animal was shaved and the skin around the mammary gland was incised. A standard model fiber optic probe was fixed to the mammary artery and connected to a Periflux PF2b 4000 laser Doppler flow meter (Perimed KB, Stockholm, Sweden). The time constant was set to 1.5 seconds and the bandwidth was set to 4 KHz. Data were analyzed using analysis of variance (ANOVA) followed by Duncan's test. A level of p <0.05 was considered significant.

Results No change was observed in blood flow to the breast tissue of normal rats after administration of saline or IRL1620 and paclitaxel. Blood flow in tumor tissue was significantly different from baseline after IRL1620 injection (36.3%, p <0.05) and after paclitaxel administration (51.9%, p <0.0-5). Was observed (see FIG. 1).

Example 2 Methods of effect of ET-1 infusion on systemic hemodynamics and blood flow to breast tissue in normal and tumor rats MNU and saline treatment was performed as an intraperitoneal injection 3 months prior to the study. Rats began to palpate regularly after 4 weeks of treatment. The experiment was started when the tumor diameter reached 4-8 mm.

Rats were anesthetized with urethane (1.5 g / kg, ip) (Sigma Chemicals, St. Louis, MO). All surgical areas were shaved and wiped with alcohol sanitized cotton. The left femoral vein was cannulated for drug administration (PE 50 tube, Clay Adams, Parsippany, NJ). The left femoral artery was also cannulated (PE 50 tube) and used to collect reference blood samples for microsphere testing using a collection pump (Model 22, Harvard Apparatus, South Natick, Mass.). The right femoral artery was cannulated (PE 50 tubes) and connected to a Gold P23 ID pressure transducer through a 7PI preamplifier to record blood pressure on a Grass P7D polygraph (Grass Instrument Co., Quincy, Mass., USA). . Heart rate (HR) was recorded through a 7P4B Glass tachograph (Grass Instrument Co., Quincy, Mass.) Starting with a blood pressure signal. The right carotid artery was exposed and a PE 50 tube was introduced from the common carotid artery into the left ventricle. The presence of a cannula in the left ventricle was confirmed by recording blood pressure on a Grass polygraph using a Statham P23 DC pressure transducer (Grass Instrument Co., Quincy, Mass.). When the cannula reaches the left ventricle, diastolic blood pressure drops to zero. To keep blood pO ₂ , pCO ₂ , and pH constant, and to avoid the effects of respiration on blood pressure and HR, the animals were treated with rodent ventilators (Model 683, Harvard Apparatus Inc., Massachusetts). Constant rate ventilation was achieved by inserting an endotracheal cannula connected to South Natick.

Rats were initially divided into two groups, each receiving one of the following treatments.
(I) Rats treated with saline were treated with ET-1 (50 ng / kg / min) injection (N = 6) for 30 minutes; and (ii) MNU (50 mg / kg, ip). Rats were injected with ET-1 (50 ng / kg / min) for 30 minutes (N = 6).

Then, induced by ET-1 infusion, was tested for the following groups in order to assess the role of ET _B receptors to changes in blood flow to normal rats and mammary tumor rats systemic hemodynamics and breast tissue.

(I) BQ788 ((N-cis 2,6-dimethylpiperidinocarbonyl-L-gamma-methyl-leucyl-D-1-methoxycarbonyltryptophanyl-D-Nle); American; Peptide Company Inc. (Sunnyvale, Calif., Dissolved in physiological saline at 0.5 pmol / kg)) for 20 minutes followed by ET-1 (50 ng / kg / minute) for 30 minutes (N = 5 );
(Ii) MNU (50 mg / kg, ip) treated rats were infused with BQ788 (0.5 pmol / kg) for 20 minutes, followed by ET-1 (50 ng / kg / min) for 30 minutes (N = 5 ).

  Systemic hemodynamics and local circulation parameters were measured at baseline, 30, 60, and 120 minutes after the start of ET-1 (50 ng / kg / min) infusion. Since the injection of ET-1 is performed for 30 minutes, the 30 minute data indicates the effect of ET-1, and the 60 and 120 minute data indicate the duration of the ET-1 effect.

Systemic hemodynamics and local blood circulation were measured using procedures described in the literature. Gulati et al., J Lab Clin Med, 126: 559 (1995); Gulati et al., Life Sci. 55: 827 (1994); See Sharma et al., Artif Cells Blood Subst Immobil Biotechnol, 22: 593 (1994). For each measurement, about 100,000 microspheres (15 ± 1 μm in diameter) labeled with ⁴⁶ Sc (scandium), ¹¹³ Sn (tin), ¹⁴¹ Ce (cerium), or ⁹⁵ Nb (niobium) (New England Nuclear Corporation) , Boston, Mass.) Was injected into the left ventricle and rinsed with 0.3 ml saline for 15 seconds. To calculate blood flow, arterial blood was collected from the right femoral artery at a rate of 0.5 ml / min. Blood collection started approximately 5-10 seconds before microsphere injection and was performed for 90 seconds. At the end of the experiment, the animals were sacrificed by overdosing with sodium pentobarbital. All tissues and organs were excised, weighed and placed in a vial. Radioactivity in standards, blood samples, and tissue samples was counted with a Packard Minaxi Auto-Gamma 5000 series gamma counter (Packard Instruments Co., Downers Grove, Ill.) With a preset window that differentiates isotope energies. The following parameters were calculated: (1) cardiac output (CO) ((injected radioactivity x arterial blood collection rate) / radioactivity in collected arterial blood), (2) stroke volume (SV) (CO / HR), ( 3) Total peripheral vascular resistance (TPR) (mean arterial pressure (MAP) / CO), (4) local blood flow ((radioactivity in tissue x arterial blood recovery rate) / radioactivity in collected arterial blood), and (5) Local vascular resistance (MAP / local blood flow). Data was calculated using computer programs described in the literature. Saxena et al., Compute Programs Biomed, 12:63 (1980).

Blood perfusion into the rat mammary gland was measured using a laser Doppler flow meter. See Song et al., Int J Radiate Oncol Biol Phys, 18: 903 (1990); Song et al., Int J Radiate Oncol Biol Phys, 17: 1041 (1989). The area around the nipple of the animal was shaved. The skin around the mammary gland was dissected as a lambeau about 6 cm wide and about 4 cm long. A standard model optical fiber probe was applied to the surface of the piece and fixed to the tissue with double-sided adhesive tape. The pieces were placed in a metal holder and taped to prevent movement and then connected to a Periflux PF2b 4000 laser Doppler flow meter (Perimed KB, Stockholm, Sweden). The time constant was set to 1.5 seconds and the bandwidth was set to 4 KHz. Data were analyzed using analysis of variance followed by Duncan's test. A level of p <0.05 was considered significant.

Results Baseline systemic hemodynamic parameters in normal (saline treated) rats were: MAP: 111.1 ± 4.8 mmHg; CO: 268.6 ± 17.6 ml / min; SV: 0.87 ± 0. 06 ml; TPR: 419.6 ±. 24.37 mmHg. Min / ml; and HR: 312.5 ± 20.2 beats / min. In normal rats, a significant increase in MAP is 30 minutes after ET-1 injection (14.5%; p <0.05) and a decrease is 120 minutes (17.8%; p <0.05). Observed. TPR increased at 120 minutes (49.2%; p <0.05). CO decreased at 60 and 120 minutes after ET-1 injection (22.9% and 42.5%, respectively; p <0.05). SV decreased at 60 and 120 minutes (20.9% and 36%, respectively; p <0.05). No significant change was observed in HR (FIGS. 2A-2E).

Baseline systemic hemodynamic parameters in tumor (MNU-treated) rats were similar to those in normal rats. In tumor rats, a significant increase in MAP was observed at 30 minutes (19.1%; p <0.05) and 60 minutes (15.3%; p <0.05) after ET-1 injection. . TRF was 30 minutes after administration of ET-1 (73.9%; p <0.05), 60 minutes (39.7%; p <0.05), and 120 minutes (71.4%; p <0.05). CO decreased at 30, 60 and 120 minutes (29.4%, 16.7% and 36.1%, respectively; p <0.05). SV was significantly reduced at 30, 60 and 120 minutes (31.1%, 17.9% and 32.1%, respectively; p <0.05). No change was observed in HR (FIGS. 2A-2E).

  No change in blood flow to the breast tissue of normal saline treated rats was observed after ET-1 administration. In normal rat mammary tissue, a significant decrease (18.61%; p <0.05) in vascular resistance was observed at 60 minutes. This is 30 minutes after ET-1 injection (FIGS. 3A-3B).

  Significant differences were observed between blood flow and local vascular resistance in breast tissue of tumor (MNU treated) and normal (saline treated) rats. A significant increase (153%; p <0.05) in blood flow to the breast tissue of tumor rats was observed 60 minutes after ET-1 administration when compared to normal rats. Vascular resistance in tumor rats was significantly different at baseline (102%; p <0.05) and 60 minutes after ET-1 administration (147%; p <0.05) compared to normal rats (FIGS. 3A-3B).

  4A-4C show changes in perfusion, mobile blood cell concentration (CMBC), and red blood cell (RBC) rates in breast tissue of tumor and normal rats. Blood perfusion in the breast tissue of normal rats did not change after ET-1 administration. Perfusion in breast tissue of tumor rats at 30 minutes after ET-1 administration was significantly increased compared to normal rats (176%; p <0.05). This increase in perfusion in tumor rats returned to baseline at 60 and 120 minutes after ET-1.

  The CMBC of tumor rats was significantly increased compared to normal rats 60 minutes after ET-1 administration (54%; p <0.05). CMBC returned to baseline at 120 minutes after ET-1. The rate of RBC increased significantly compared to normal rats 30 minutes after ET-1 administration (252%; p <0.05). The rate of RBC in tumor rats returned to baseline 2 hours (120 minutes) after ET-1 administration (Figures 4A-4C).

  Figures 5A-5C show the effect of BQ788 on ET-1-induced changes in blood perfusion, CMBC, and RBC rates in tumor and normal rats, respectively. Blood perfusion in the breast tissue of normal rats did not change significantly after BQ788 administration or ET-1 infusion. However, perfusion in the breast tumor tissue of tumor rats was observed in BQ788 pretreated rats at 30 minutes (25.25. ± 5.7%; p <0.05) and 60 minutes (25 17. ± .5.7%; p <0.05). Pretreatment with BQ788 attenuated the increase in perfusion induced by ET-1 in tumor rats. In BQ788 pretreated rats, no difference was observed between perfusion in breast tissue of tumor and normal rats after ET-1 administration.

  Tumor rat baseline CMBC was significantly higher than baseline rat breast CMBC (42.2%; p <0.05). However, no difference was observed between tumor and normal rat CMBC after BQ788 injection. In addition, no difference in RBC speed was observed between the two groups (FIGS. 5A-5C).

The above studies show the effect of ET-1 on systemic hemodynamics and blood flow to breast tissue in saline and MNU treated tumor rats. ET-1 is known to stimulate angiogenesis by promoting VEGF production. Studies have shown that ET-1 is increased in many cancer tissues. For example, breast cancer (Yamashita et al., Res
Commun Chem Pathol Pharmacol, 74: 363 (1991)), breast lobe tumor (Yamashita et al., Cancer Res, 52: 4046 (1992)), prostate cancer (Nelson et al., Cancer Res, 56: 663 (1996)), liver Cancer tissues such as cancer (Kar et al., Biochem Biophys Res Commun 216: 514 (1995)), and some meningiomas (Pagotto et al., J Clin Invest, 96: 2017 (1995)). The above tests show changes in vascular response induced by ET-1 in breast tumors. The method used in these studies was a well-established radioactive microsphere technique for examining systemic hemodynamics and local blood circulation. Gulati et al., Am J Physiol, 273: H827 (1997); Gulati et al., Crit Care Med, 24: 137 (1996); Gulati et al., J Lab Clin Med, 126: 559 (1995); Gulati et al., Life Sci, 55 : 827 (1994).

  Baseline blood flow to breast tumor tissue of tumor rats was higher than that of normal animals. This has been observed in previous studies and has been theorized (not relied upon) due to the recruitment of new blood vessels in the tumor. Vaupel, Blood flow, oxygenation, tissue pH distribution, and bioenergetic status of tutors. Ernst Schering, Research Foundation Lecture 23, Berlin (1994). Blood flow to the breast tumor after ET-1 administration was significantly increased compared to that observed in the breast tissue of normal rats. Laser Doppler flow measurements showing increased blood perfusion to the breast tumor confirmed the increased blood flow observed in the breast tumor tissue after ET-1 administration. It has been theorized that the increase in blood perfusion is due to an increase in either the rate of RBC and / or CMBC, or both. At the end of the ET-1 infusion, an increase in the rate of RBC was observed, but an increase in CMBC was observed 30 minutes after the ET-1 infusion.

Furthermore, it has been theorized that the increase in blood flow observed in response to ET-1 is due to ET _B- mediated vasodilation (do not rely on it). Studies expression of ET-1 and ET _B receptors have been shown to be increased in breast cancer tissue. Alanen et al., Histopathology, 36: 161 (2000); Yamashita et al., Res.
Commun Chem Pathol Pharmacol, 74: 363 (1991). According to the present invention, it was found that administration of BQ788 prevents the increase in blood flow to tumor tissue induced by ET-1. BQ788 (i.e., N- cis-2,6-dimethyl-piperidinocarbonyl -L- gamma - methyl - leucyl -D-1-methoxycarbonyl-tryptophanyl -D-Nle) is a specific ET _B receptor antagonist It is. BQ788 inhibits binding to the ET _B receptor with an IC ₅₀ value of 1.2 nM.

BQ788 was used to determine the role of ET _B receptors in vasodilation induced ET-1 in breast tumors. This result vasodilator responses ET-1-induced is suggested to be mediated through the ET _B receptor. Expression of ET _B receptors, high endothelial cells than smooth muscle cells is regulated by various growth factors and cytokines. Smith et al., J Cardiovasc Pharmacol, 31: S158 (1998). Normal breast tissue has a high concentration of ET _B receptors than ET _A receptors (Alanen et al, Histopathology, 36: 161 (2000 )), ET B receptors during breast cancer overexpressing the tumor tissue It is theorized that it contributes to the maintenance of blood flow (do not rely on it).

In summary, the experiments described indicate that the injection of ET-1 has caused a decrease in the growth and vascular resistance of blood flow in breast tumor tissues, and this increase in blood flow can be prevented by ET _B receptor antagonist.

Example 3 Tumor blood perfusion and influence previous experiments IRL1620 on chemotherapeutic agent delivery, ET-1 administration to the breast tumor rats selectively increase blood flow to tumor tissue by stimulation of ET _B receptors It has been shown. Described in this example experiment is performed to investigate the effect on paclitaxel accumulation in affected and tumor and other major organs IRL1620 is on perfusion breast tumor and melanoma ET _B receptor agonist.

Methods Breast tumors are induced in virgin female Sprague Dawley rats (48 days old) by N-methylnitrosourea (MNU; 50 mg / kg, ip). Rats with a tumor volume of 300-500 mm ³ were anesthetized with urethane as described above (1.5 g / kg, ip), saline (0.3 ml / kg, iv) or IRL1620 (3 nmol). / Kg, iv). Tumor perfusion is measured for 10 hours using a Periflux PF2b 4000 laser Doppler flowmeter as described above. In the second study, nude mice are inoculated subcutaneously with 1 million human melanoma cells (UISO-MEL-2). Mice with a tumor volume of 300-500 mm ³ are treated with saline or IRL1620 as described above and perfusion is measured for 3 hours as described above. Experiments are also conducted to determine whether elevated perfusion increases the accumulation of paclitaxel in tumor tissue. [ ³ H] -paclitaxel (10 μCi / mouse) is administered to melanoma mice 15 minutes after IRL1620 or saline. Animals are sacrificed 3 hours after [ ³ H] -paclitaxel administration. Tumors and major organs are dissected, weighed, and dissolved in a tissue lysing agent, and radioactivity is measured by techniques well known to those skilled in the art. Specifically, the concentration of [ ³ H] -paclitaxel in the plasma sample is measured using a Beckman Coulter liquid scintillation counter (model LS 6500). Plasma is thawed and mixed with 20 mL of liquid scintillation cocktail. Count samples. The count is converted from “dpm” units to “fmol / mL” using the following equation.

fmol / mL = dpm value × decay factor × 2.2 × 10 ⁻¹² / 10 ⁻¹² × sample volume mL

Results IRL1620 significantly increases tumor perfusion in breast tumor rats and melanoma mice. Administration of IRL1620 results in a 150% and 318% increase in tumor perfusion in breast tumors and melanoma animals, respectively. Mice treated with IRL1620 show a 730% increase in paclitaxel concentration in the tumor compared to saline treated mice. However, IRL1620 does not significantly increase paclitaxel concentration in other major organs. The results are shown in FIG.

Example 4 Effect of IRL1620 on the pharmacokinetics of paclitaxel Altering blood flow dynamics in the body can significantly affect the pharmacokinetics of therapeutic agents. Paclitaxel is known to have complex pharmacokinetics. See, e.g., Spareboom et al., Cancer Res 56: 2112 (1996a); Gianni et al., J Natl Cancer Inst 87: 1169 (1995b); Sonnicsen.
& Relling, Clin Pharmacokinet 27: 256 (1994); Huizing et al., J Clin Oncol 11: 2127 (1993); Brown et al., J Clin Oncol 9: 1261 (1991); Longnecker et al., Cancer Treat 71 (1998). See Wiemik et al., Cancer Res 47: 2486 (1987b); It is therefore important to understand the effect of IRL1620 on the plasma pharmacokinetics of paclitaxel. Therefore, this study was conducted to determine whether IRL1620 selective ET _B receptor agonist changes the pharmacokinetics of paclitaxel in breast tumor rats.

Method IRL1620 was purchased from Sigma-Aldrich (St. Louis, MO). Paclitaxel (6 mg / mL solution) is available from Ben Venue Laboratories Inc. (Bedford, Ohio). Ketamine and xylazine are available from Phoenix Scientific, Inc. (St. Joseph, Missouri). [ ³ H] -paclitaxel (lmCi, 6.4 Ci / mmol, specific activity) was purchased from Moravek Biochemicals (Moravek Biochemicals, Calif.). Urethanes were purchased from Sigma Aldrich (Sigma Chemicals, St. Louis, MO).

  Virgin female Sprague Dawley rats (Harlan Co., Madison, Wis.), 48 days old (120-140 g) were used for this study. Upon arrival, all rats were housed in a cage in a controlled temperature (23 ± 1 ° C.), humidity (50 ± 10%) and artificial light (06: 0 to 18:00) room. Rats were given food and water ad libitum. Experiments were performed only after the rats had been habituated to the environment for at least 4 days.

N-methyl-n-nitrosourea (MNU) at a dose of 50 mg / kg i. p. The rats were palpated twice a week. Pharmacokinetic studies were performed when tumors reached 75-100 mm ³ .

  HPLC-UV test. Urethane (1.5 mg / kg) (Sigma Chemicals, St. Louis, MO) once i. p. Rats were anesthetized by injection. The right thigh was shaved and wiped with a surgical disinfectant and alcohol. The right femoral artery and vein were exposed and a sterile PE-50 tube was inserted. The neck was shaved and wiped with surgical disinfectant and alcohol. A midline incision was made near the neck, and tracheal intubation was performed and connected to a rodent ventilator (Model 683, Harvard Apparatus Inc., South Natick, Mass.). All surgeries were performed under aseptic conditions. Neosporine antibiotic cream (Pfizer, Mori Plains, NJ) was applied to the wound to prevent infection. A 45 minute recovery time was given before drug administration.

  Normal and tumor rats were used. Paclitaxel was administered i.v. 15 minutes after administration of IRL1620 (3 nmol / kg) or vehicle (saline, 3 mL / kg). v. Dose (3 mg / kg). Blood samples were collected before IRL1620 administration to obtain baseline values. 0.5 mL of blood was collected from rats into heparinized syringes at baseline, 5, 30 minutes after dosing with paclitaxel, and at 2, 6, and 10 hours. Samples were centrifuged and plasma was collected and stored at −80 ° C. until analysis.

Plasma samples were analyzed for paclitaxel using an HPLC system. Briefly, plasma is thawed and 50 μL of internal standard N-cyclohexylbenzamide (low standard curve 3 mM, high standard curve 30 mM) and 3 mL ethyl ether (Fisher Scientific, Chicago, Ill.) And 13 × 100 glass culture tubes. Mixed. The mixture was shaken for 5 minutes using a reciprocating shaker and then centrifuged at 3,000 rpm, 4 ° C. for 5 minutes. The resulting supernatant was transferred to a 13 × 100 borosilicate glass culture tube and evaporated in a heated water bath (37 ° C.) under a stream of nitrogen. The residue was reconstituted (returned) with 200 μL of mobile phase A (50% deionized water, 50% acetonitrile). 100 μL of the reconstituted material (low standard curve, sample collected after IV administration) was injected onto a 4 mm NovaPak 150 × 3.9 mm C18 column (Waters Associates, Milford, Mass.). In front of it is a 4 mm NovaPak 20 × 3.9 mm C18 precolumn. The apparatus used was a Waters 2695 separation module connected to a Waters 2487 absorbance detector set at 227 nm. A linear gradient was started by feeding 100% mobile phase A at a flow rate of 1 mL / min. Mobile phase A was then lowered to 70% in 10-11 minutes and mobile phase A was maintained at 70% for 11-16 minutes to remove material that slowly elutes from the column prior to the next injection. Thereafter, the mobile phase A was increased to 100% in 16 to 17 minutes, the 100% mobile phase A was maintained for 3 minutes, and the total operation time was 20 minutes. The plasma concentration of paclitaxel was calculated from the ratio of the paclitaxel peak area to the N-cyclohexylbenzamide peak area, weighted by 1 / x using linear least squares regression. The intraday and interday variability measured by the coefficient of variation was <10%. The plasma concentration profiles of normal and tumor rats were compared.

  Liquid scintillation counting test. Combination of ketamine (100 mg / kg) and xylazine (2 mg / kg) once i. p. Rats were anesthetized by injection. The neck was shaved and wiped with surgical disinfectant and alcohol. The right carotid artery was exposed and a sterile PE-50 tube was inserted. A midline incision was made near the neck and a PE50 tube was inserted into the left carotid artery for blood collection. The catheter was submerged under the neck at the base of the neck and then the incision was closed with surgical staples. Buehler et al., Free Radic Biol Med 37: 124 (2004). The open tube was plugged with fishing line. All surgeries were performed under aseptic conditions. Neosporine antibiotic cream (Pfizer, Mori Plains, NJ) was applied to the wound to prevent infection. A 45 minute recovery time was given before drug administration.

IRL1620 was administered i.v. to tumor animals at a dose of 3 nmol / kg. v. Administered. [ ³ H] -paclitaxel (160 μCi / kg) was mixed with unlabeled paclitaxel. Paclitaxel is administered i.v. 15 minutes after administration of vehicle or IRL1620. v. Administered.

  Plasma was collected prior to vehicle or IRL1620 administration to obtain baseline values. Approximately 0.2 mL of blood was collected from rats into heparinized syringes at baseline, 1, 5, 15, 30 minutes, 1, 2, 4, 6, 8, 12, and 24 hours. Samples were centrifuged and plasma was separated and stored at −80 ° C. until analysis.

The concentration of [ ³ H] -paclitaxel in plasma samples was measured using a Beckman Coulter liquid scintillation counter (model LS 6500). Briefly, plasma was thawed and mixed with 20 mL of liquid scintillation cocktail. Samples were counted, and the count was converted from “dpm” units to “fmol / mL” using the following formula.

fmol / mL = dpm value × decay factor × 2.2 × 10 ⁻¹² / 10 ⁻¹² × sample volume mL
After conversion to fmol / mL, the pharmacokinetics of all paclitaxel was calculated using the ratio of [ ³ H] -paclitaxel to unlabeled paclitaxel. Estimates of the pharmacokinetics of plasma paclitaxel were determined using both non-compartmental and compartmental analysis (run with WinNonlin Pro 4.1 (Pharsight Corp, Mountain View, Calif.)).

In non-compartmental analysis, the area under the curve (AUC0-∞) is calculated using the trapezoidal method until the final measurable concentration (Clast), and Clast is the slope (λ) of the terminal part of the logarithmic linear plasma concentration vs time curve (negative) Extrapolated to infinity by dividing by (number). The following parameters were also calculated. Mean residence time (MRTiv) was calculated as the reciprocal of systemic clearance, systemic clearance (CL) was calculated as the ratio of dose to AUC0-∞, and apparent volume distribution was calculated as the ratio of CL and λ. Plasma half-life was calculated as the product of 0.693 (natural logarithm of 2) and MRTiv.

  For compartmental analysis, a series of nonlinear compartment models were fitted to plasma concentration versus time curve data. Specifically, 1 compartment, 2 compartment and 3 compartment models were compared. Weighting based on unified and predictive data was tested. Final model selection was based on diagnostic plots (observed vs predicted and residual plots), Akaike Information Criteria (AIC) and Schwartz Criteria (SC). Models with low AIC and SC criteria were considered final models.

  Data were analyzed by one-way ANOVA for HPLC-UV followed by Duncan assay and by t-test for liquid scintillation testing. p <0.05 was considered significant. The main result measured in these pharmacological response studies was the difference in plasma paclitaxel concentration.

Results The pharmacokinetic profile of paclitaxel was not affected by IRL1620 administration in both normal and tumor rats (FIGS. 7 and 8). HPLC analysis of the plasma pharmacokinetic profile is similar to the broader kinetic profile of radiopaclitaxel. FIG. 8 depicts the pharmacokinetic profile of radioactivity of paclitaxel in vehicle-treated and IRL1620-treated tumor rats. Pharmacokinetic profiles were analyzed by non-compartment and compartment methods.

In a non-compartmental analysis, the AUC calculated for the vehicle + paclitaxel group was 9433.53 ± 1465.00 ng ^* h / mL, similar to that of IRL1620 treated tumor rats (p> 0.05). The elimination half-life was calculated to be 0.14 ± 0.08 hours. Clearance calculated as dose / AUC is 0.56 ± 0.07 L / h / k
g. The distribution volume calculated as clearance / Kel is 10.11 ± 4.
It was found to be 17 L / kg. In general, as can be seen from the table below, IRL1620 did not affect the pharmacokinetic profile of paclitaxel.

The plasma concentration of paclitaxel was calculated from the dpm count of the plasma sample. A three-compartment model best described the pharmacokinetics of paclitaxel. FIG. 9 depicts a plot of observed vs predicted pharmacokinetics for both vehicle-treated and IRL1620-treated rats. The AUC of paclitaxel in vehicle-treated rats was 9.42 ± 3.18 μg-h / mL. The distribution volume (Vss) at steady state was 10.31 ± 4.54 L / Kg. The clearance was estimated to be 0.69 ± 0.17 L / h / Kg. α t1 / 2,
β t1 / 2 and γ t1 / 2 were 0.03 ± 0.01 h, 1.0 ± 0.32 h, and 25.87 + 17.81 h, respectively. The average residence time was 27.92 ± 19.84h. As can be seen from the table below, these parameters estimated in the IRL1620 treatment group were not significantly different from those in the vehicle treatment group.

  In this study, a three-compartment model best described the plasma pharmacokinetics of paclitaxel. This model suggests that paclitaxel is distributed in various organs regardless of whether the blood perfusion in the organ is high, medium or low. Administration of IRL1620 did not change the distribution of paclitaxel. Plasma pharmacokinetic parameters obtained by the three-compartment model showed similar clearance, volume of distribution and absorption, distribution and elimination half-life for the vehicle and IRL1620 treatment groups. However, IRL1620 increases tumor blood perfusion and tumor paclitaxel concentration. Rai et al., American Association of Pharmaceutical Scientists, Pharmaceuticals and Drug Delivery Conference. Philadelphia, PA (2004); Rai & Gulati, Cancer Chemother Pharmacol, 51:21 (2003). Thus, IRL1620 selectively increases tumor perfusion without significantly altering the pharmacokinetic profile of paclitaxel.

  The use of IRL1620 had no effect on the pharmacokinetics of paclitaxel. Pharmacokinetics can often be viewed as a surrogate for compound safety. Therefore, these results also suggest that the safety of paclitaxel is not altered by administration of IRL1620. As a result, IRL1620 could be used to improve the efficacy of paclitaxel and allow appropriate dose escalation to minimize its severe toxicity. These results indicate that the pharmacokinetic profile of paclitaxel in normal rats is similar to that of tumor rats, indicating that paclitaxel kinetics do not change with tumor model systems.

Example 5 FIG. The dose-response effect of IRL1620, the effect of IRL1620 on the biodistribution of [ ³ H] paclitaxel in major organs and tumor tissues, and the effect of IRL1620 on the efficacy of paclitaxel on tumor status The experiments described in this example include (a ) ET _B receptor agonist IRL1620 dose response effect on breast perfusion of normal and tumor rat, effect of IRL1620 on the biodistribution of ^[3 H] paclitaxel in (b) major organs and tumor tissues, and (3) MNU Designed to investigate the effect of IRL1620 on the efficacy of paclitaxel on tumor status in induced breast tumor rats.

Method IRL1620 is available from Sigma Chemical Co. (St. Louis, Missouri). [ ³ H] -paclitaxel was purchased from Moravek Biochemicals (Blair, CA). Paclitaxel (6 mg / ml solution) is available from Ben Venue Laboratories Inc. (Bedford, Ohio). Ketamine and xylazine are available from Phoenix Scientific, Inc. (St. Joseph, Missouri). Tissue lysing agent (TS-2) is available from RPI Corp. (Chicago, Illinois).

Virgin female Sprague Dawley rats (Harlan Company, Madison, Wis.) Were purchased at 40 days of age and placed in cages two by two in a temperature-controlled room at 23 ± 1 ° C., under a 12 hour light / 12 hour dark schedule Maintained. Rats were given water and standard rodent food ad libitum. At 48 days of age, each animal was injected once with N-methylnitrosourea (MNU, Ash Stevens, Detroit, MI) at a dose of 50 mg / kg. MNU is dissolved in 3% acetic acid and diluted in 0.9% NaCl (final concentration 12.5 mg / ml) which is i. p. Administered by injection. This treatment induces breast adenocarcinoma with an incidence of nearly 100% approximately 100 days after the carcinogenesis treatment. Mehta Eur J Cancer, 36: 1275 (2000). Tumor appearance and location were monitored by palpation of the mammary gland by hand, and the tumor surface was measured with a digital caliper. Rats with a tumor volume of 500-800 mm ³ were selected and tested.

  Perfusion test. Rat perfusion to breast tissue and tumor was measured using a Periflux PF2b 4000 laser Doppler flowmeter (Perimed, Stockholm, Sweden) as described above. Briefly, ketamine (100 mg / kg) and xylazine (2 mg / kg) were combined once i. p. The rats were anesthetized by injection. The area near the nipple was shaved and the animals were placed on a heating pad (37 ° C.) to minimize body temperature fluctuations. The skin around the mammary gland was incised about 6 mm wide and 4 mm long. A standard model optical fiber probe (MP3 flow probe, Moors Instruments, Devon, UK) was applied to the surface of the exposed tissue. It was connected to a Periflux PF2b 4000 laser Doppler flow meter. The time constant was set to 1.5 seconds and the bandwidth was set to 4 KHz. This method measures the Doppler shift in the laser beam (flux). This depends on the number and velocity of red blood cells and is proportional to the total blood flow in a given volume of tissue. Flux values were obtained using Polyview software. Saline or IRL1620 was administered after obtaining a baseline of 15 minutes stable recording. Animals received 1, 3 or 9 nmol / kg of IRL1620 and perfusion was recorded for 3 hours. Each dose was administered to at least 4 animals.

Biodistribution test. Rats were combined with ketamine (100 mg / kg) and xylazine (2 mg / kg) once i. p. Anesthetized by injection. Rat body weight, tumor location and tumor volume were recorded. The animals were randomly grouped and administered with saline or IRL1620 (3 nmol / kg) from the tail vein to a final volume of 0.2 ml. Each group of rats received [ ³ H] paclitaxel (50:50 Cremophor EL and 40 μCi / rat in ethanol) at a final volume of 1.0 ml at 15, 120 and 240 minutes after IRL1620 administration. Since six rats were tested at each time point, a total of 36 rats were used. The animals were sacrificed 3 hours after [ ³ H] paclitaxel administration. [ ³ H] paclitaxel concentrations in tumor tissue, kidney, liver, lung and spleen were measured. Tumors and organs were sliced into small pieces. Approximately 500 mg of tissue or tumor was placed in a separate vial with tissue lysing agent (6 ml) and incubated in a 50 ° C. water bath. When the tissue or tumor has dissolved, remove the vial from the water bath and add 1.2 ml of 10%
Glacial acetic acid was added. The contents of the vial were aliquoted into 3 vials and 15 ml of liquid scintillation cocktail (Safety Solve, RPI Corp, Chicago, Ill.) Was added to each vial and kept overnight for equilibration. Tube radioactivity was counted using a liquid scintillation counter (Beckman Coulter, LS 6500).

Efficacy test. Tumor rats were randomly divided into 7 groups (12 rats / group). Group I—Saline; Group II—IRL1620 (3 nmol / kg); Group III—Cremophor EL
: Ethanol; group IV-vehicle (saline) + paclitaxel (1 mg / kg); group V-vehicle (saline) + paclitaxel (5 mg / kg); group VI-IRL1620 (3 nmol / kg) + paclitaxel (1 mg) / Kg); and Group VII-IRL1620 (
3 nmol / kg) + paclitaxel (5 mg / kg). The administration schedule was once every 3 days for a total of 5 times. Body weight, tumor size and location were monitored every third day for a total of 30 days after the last dose. The following categories were used for evaluation. Progression: tumor has grown more than 40% in area compared to the start of treatment; stagnation: tumor has not changed more than 40% from its initial area throughout the course of treatment; partial regression: tumor has its Regressed more than 40% from the initial area; complete remission: when the tumor can no longer be palpated and measured. Tumor multiplicity (appearance of new tumors) was also recorded during treatment and during the 30 day observation period. The animals were sacrificed 30 days after the last (5th) dose. Data were analyzed using analysis of variance followed by Duncan's test. A level of p <0.05 was considered significant.

Results Dose response in breast perfusion of IRL1620. The effect of IRL1620 administration on tumor perfusion was found to be transient and dose dependent (FIG. 10A). A maximum increase from baseline of 244.0% (p <0.001) in tumor perfusion was observed 15 minutes after administration of 3 nmol / kg IRL1620. Increased perfusion was found to be significant at 15, 30 and 60 minutes as compared to baseline and saline treated rats (FIG. 10B). Administration of 1 and 9 nmol / kg of IRL1620 produced only a slight increase in breast tumor perfusion compared to baseline perfusion and saline treated rats. Maximum increases in perfusion (60.9 and 63.3%) were recorded at 90 and 30 minutes after 1 and 9 nmol / kg IRL1620, respectively. However, the increase in perfusion in animals treated with 9 nmol / kg IRL1620 was found to be significant at 15, 30 and 60 minutes as compared to saline treated rats (FIG. 10A). Administration of saline to tumor rats did not cause any significant changes in blood perfusion compared to baseline (FIG. 10A). Administration of saline, 1, 3 or 9 nmol / kg IRL1620 did not cause any significant changes in mammary perfusion in normal female rats (data not shown).

Biodistribution test. The concentration of [ ³ H] paclitaxel in the tumor was significantly increased in IRL1620 (3 nmol / kg) treated rats compared to saline treated rats. The greatest effect was seen in the group of animals that received paclitaxel 15 minutes after IRL1620. A 277.1, 151.9 and 34.7% increase in paclitaxel concentration in the tumor was observed when paclitaxel was administered 15, 120 and 240 minutes after IRL1620 administration, respectively (FIG. 11). It was found that even when IRL1620 was administered, the accumulation of paclitaxel in the liver, lung, kidney and spleen was unchanged compared to control animals (FIG. 11).

  Efficacy test: body weight. FIG. 12 shows the difference in body weight from the baseline (before the start of treatment) to 30 days after the final administration. The rate of increase in body weight monitored at the end of the experiment in saline treated control rats was 7.2 ± 1.7% compared to baseline body weight (FIG. 12). In animals treated with vehicle + paclitaxel (1 mg / kg), vehicle + paclitaxel (5 mg / kg), IRL1620 + paclitaxel 1 mg / kg and IRL1620 + paclitaxel 5 mg / kg, body weights were 5.1 ± 3.6, 9.4 ±, respectively. There was an increase of 2.4, 14.3 ± 3.1 and 13.1 ± 1.8% (FIG. 12). Cremophor EL: The rate of increase in body weight of animals receiving ethanol and IRL1620 compared to baseline was found to be <10% (data not shown).

Efficacy test: tumor volume. At the start of treatment, the tumor sizes of the various groups were similar and not significantly different from each other (Figure 13). The tumor volume of control rats increased at a rapid and varying rate. The great variability in tumor growth may be due to the random growth pattern of spontaneously growing tumors. At the end of the 30 day observation period, the control tumor had a tumor volume of 2693.4 ± 790.9 mm ³ . IRL1620-treated rats also had a similar development pattern with a final tumor volume of 2560.5 ± 844.4 mm ³ . Cremophor EL: Ethanol treatment had a similar growth pattern with a final tumor volume of 2338 ± 1329 mm ³ . Thus, IRL1620 and cremophor El: ethanol did not significantly affect the growth of MNU-induced breast tumors. Vehicle + paclitaxel 1 mg / kg treated rats showed slightly reduced tumor size growth when compared to control rats (1960.8 ± 611.9 mm ³ ), whereas in vehicle + paclitaxel 5 mg / kg group Was more prominent (1682.7 ± 497.3 mm ³ ). IRL1620 + paclitaxel 1 mg / kg treated rats showed reduced tumor size (1707.2 ± 621.1 mm ³ ). However, the lowest mean tumor size (730.1 ± 219.4 mm ³ ) was observed in the group of animals treated with IRL1620 + paclitaxel 5 mg / kg (FIG. 13). Administration of paclitaxel 5 mg / kg 5 times every 3 days following IRL1620 significantly (p <0.05) reduced tumor volume compared to saline + paclitaxel (5 mg / kg) administered rats (FIG. 13). Tumor volume was found to be lower in the IRL1620 + paclitaxel 5 mg / kg group compared to rats treated with either IRL1620 or cremophor EL: ethanol (data not shown).

  Efficacy test: Tumor multiplicity. All animals in all treatment groups had developed additional tumors by the end of the 30 day observation period. Animals treated with saline, cremophor EL: ethanol and IRL1620 showed an increase of 58.4, 57.1 and 60.8% in additional tumor appearance, respectively. It was found that the incidence of new tumors was 78.3% and 41% in animals dosed with vehicle + paclitaxel 1 and 5 mg / kg, respectively. However, with IRL1620 + paclitaxel 1 and 5 mg / kg, the percentage of additional tumors was found to be 69.2 and 44.8%, respectively.

  Efficacy test: tumor progression. The percentage of tumors that progressed, stagnated, regressed or disappeared were calculated as described above. In the saline treatment group, 73.5% of tumors progressed over 40% of the initial tumor size. The IRL1620 (82.7%) and cremophor EL: ethanol (80.4%) treated groups also showed similar percent tumor progression over 40% of the initial tumor size. Tumor progression was low percent in the vehicle + paclitaxel (5 mg / kg) group (71.4%), vehicle + paclitaxel (1 mg / kg) group (61.1%) and IRL1620 + paclitaxel (1 mg / kg) Group (69%). However, the lowest percentage (40%) was found in the IRL1620 + paclitaxel (5 mg / kg) group (FIG. 14).

  Efficacy test: tumor stagnation. 16.9% of the rats treated with saline were stagnant. That is, it did not grow beyond the range of 40% by the end of 30 days. IRL1620 (13.7%), cremophor EL: ethanol (15.2%), vehicle + paclitaxel (1 mg / kg) (22.2%) and vehicle + paclitaxel (5 mg / kg) (19.5%) treated rats The percentage of stagnant tumors was low. IRL1620 + paclitaxel (1 mg / kg) (23.8%) and IRL1620 + paclitaxel (5 mg / kg) treated rats had a higher percentage of stagnant tumors (26.6%) (FIG. 14).

  Efficacy test: tumor regression. Administration of IRL1620 prior to paclitaxel 5 mg / kg treatment significantly reduced tumor progression compared to control animals. Saline-treated rats showed regression of 9.2% of the tumor from the initial tumor volume. Cremophor EL: Ethanol (4.3%), IRL1620 (3.4%) and vehicle + paclitaxel (5 mg / kg) (9.5%) treated rats were significantly larger in size than the control group There was no difference. At the end of the fifth dose, tumors were 76.1 ± 10.5 and 45.9 ± 11.5% compared to control rats in IRL1620 + paclitaxel 5 mg / kg treated vehicle and vehicle + paclitaxel 5 mg / kg treated rats, respectively. It was regressing. Animals in groups treated with IRL1620 + paclitaxel 5 mg / kg and vehicle + paclitaxel 5 mg / kg had 80.2 ± 6.9% (p <0.05) and 33.8 ± 19.4% regression ( FIG. 14). Tumor regression rates in the IRL1620 + paclitaxel 1 mg / kg and vehicle + paclitaxel (1 mg / kg) groups were found to be 47.1 ± 15.4 and 37.7 ± 16.2%, respectively. Administration of paclitaxel (5 mg / kg) 15 minutes after IRL1620 resulted in significantly greater tumor regression compared to administration of paclitaxel (5 mg / kg) 15 minutes after saline. Cremophor El: ethanol and IRL1620 treated groups were not significantly different in tumor regression compared to control rats at any time point (data not shown).

Efficacy test: tumor remission. Complete remission with complete disappearance of the tumor was observed in only two groups. IRL1620 + paclitaxel (1 mg / kg) (2.3%) and IRL1620 + paclitaxel (5 mg / kg) (15%) treated rats (FIG. 14).
The results obtained in this study indicate that administration of a 3 nmol / kg dose of IRL1620 results in a significant increase in tumor perfusion compared to baseline and vehicle treated rats. 1, 3 and 9 nmol / kg of IRL1620 also increased tumor perfusion, while a dose of 3 nmol / kg of IRL1620 produced the greatest effect. High dose (9 nmol / kg), it is also possible to stimulate the ET _B receptor is involved in vasoconstriction. Brooks et al., J Cardiovas Pharmacol, 26 Suppl 3: S322 (1995).

Therefore, vasodilation mixed with vasoconstriction by ET _B receptor stimulation with high doses results in reduced vasodilator effect. A low dose (1 nmol / kg) may not be sufficient to produce maximal vasodilation. Therefore, a 3 nmol / kg dose of IRL1620 was selected for the next study.

The effect of IRL1620 on paclitaxel concentration in tumor tissue was found to be time dependent. When [ ³ H] paclitaxel was administered at 15 minutes, 2 hours, and 4 hours after IRL1620 administration, the concentration of [ ³ H] paclitaxel in the tumor was 277.1, 151.9, and 34, respectively, compared to vehicle-treated rats. There was a difference of 7%. Studies have shown that administration of IRL1620 does not result in a significant increase in paclitaxel accumulation in liver, lung, kidney and spleen. Efficacy test results show that administration of IRL1620 significantly increased paclitaxel-induced reduction in tumor volume compared to saline-treated rats receiving paclitaxel. The enhanced therapeutic benefit seen with the 5 mg / kg dose of paclitaxel was maintained until 30 days after the last dose. This means that the tumor volume did not rebound and the effect of IRL1620 in enhancing the efficacy of paclitaxel was unchanged until the end of the study. However, paclitaxel (1 and 5 mg / kg) after saline treatment did not cause such a significant change in tumor growth. Furthermore, tumor multiplicity was reduced in the group of rats treated with 5 mg / kg of paclitaxel after IRL1620. Therefore, administering IRL1620 before paclitaxel 5 mg / kg has a significant effect on the efficacy of paclitaxel. This is indicated by a decrease in tumor burden, percent tumor regression, unaffected body weight and multiplicity. In addition, the group of animals treated with IRL1620 after paclitaxel 1 and 5 mg / kg had 2.3 and 15% initial complete tumor remission, respectively, compared to all other groups.

Experiments described in this example clearly show that ET _B is a receptor agonist IRL1620 can significantly increase the blood flow of the tumor. Administration of IRL1620 caused an increase in tumor blood flow but did not change perfusion in healthy control tissues. The increase in tumor perfusion lasted for 3 hours. Administration of [ ³ H] paclitaxel during periods of increased perfusion significantly increased the concentration of [ ³ H] paclitaxel only in tumor tissue but not in other organs. Furthermore, the results of the experiments described in this example provide evidence that administration of IRL1620 stimulates the antitumor effect of paclitaxel. Tumor volume of rats treated with paclitaxel 5 mg / kg every third day for a total of 5 times was reduced by 60.0% compared to control rats. However, paclitaxel administration 15 minutes after IRL1620 administration reduced tumor volume to 268.9% compared to control rats when recorded one month after final paclitaxel administration. There was a 130.4% reduction in tumor volume in IRL1620 treated rats compared to rats treated with paclitaxel alone. It is possible that increased tumor perfusion may increase the supply of nutrients that promote tumor growth. Our results show that there was no significant increase in tumor volume and multiplicity of IRL1620-treated rats compared to saline-treated rats, and IRL1620 alone had no effect on tumor volume and multiplicity. It suggests that there was not. In conclusion, IRL1620 can be used as a tumor selective vasodilator and can be used to selectively increase the efficacy of chemotherapy. This study clearly demonstrates that many times higher drug concentrations can be achieved in tumor tissue by adopting this treatment strategy. Finally, ET _A receptor antagonists have also been proposed for the improvement of tumor blood flow (Sonveaux et al., Cancer Res, 64: 3209 ( 2004)), enhance delivery of anticancer drugs to the tumor in accordance with the present invention Can be used to do.

  A pharmaceutical composition containing the active ingredient is suitable for administration to a human or other mammal. Typically, pharmaceutical compositions are sterile and do not contain toxic, carcinogenic, or mutagenic compounds that cause an adverse reaction when administered. Administration of the pharmaceutical composition can be performed before, during or after the onset of solid tumor growth.

  The methods of the present invention can be achieved using the active ingredients described above or as physiologically acceptable salts, derivatives, prodrugs or solvates thereof. The active ingredient can be administered as it is or as a pharmaceutical composition containing either or both entities.

  Pharmaceutical compositions include those in which the active ingredients are administered in an amount effective to achieve their intended purpose. More specifically, “therapeutically effective amount” means an amount effective to prevent, eliminate, delay progression or reduce the size of a solid tumor. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

A “therapeutically effective dose” is the amount of active ingredient that achieves the desired effect. The toxicity and therapeutic efficacy of such active ingredients can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. For example, determine the LD ₅₀ (dose lethal for 50% of the population) and ED ₅₀ (dose that is therapeutically effective for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio between LD ₅₀ and ED ₅₀ . A high therapeutic index is preferred. The data obtained can be used to design dose ranges for use in humans. The dosage of the active ingredient is preferably within a range of circulating concentrations that include the ED ₅₀ with little to no toxicity. The dose can vary within this range depending on the dosage form used and the route of administration utilized.

The exact formulation and dosage will be determined by the physician in view of the patient's condition. Dosage amount and interval can be adjusted individually to provide concentrations of the active ingredients that are sufficient to maintain therapeutic or prophylactic effects.
The amount of pharmaceutical composition administered can depend on the patient being treated, the patient's weight, the severity of the disease, the mode of administration, and the judgment of the prescribing physician.

  The active ingredients can be administered alone or mixed with a pharmaceutical carrier selected with regard to the intended route of administration and standard pharmaceutical practice. Thus, the pharmaceutical composition used according to the present invention comprises one or more physiologically acceptable carriers comprising excipients and adjuvants that facilitate the processing of the active ingredient into a pharmaceutically usable formulation. Can be formulated in a conventional manner.

  When a therapeutically effective amount of the active ingredient is administered, the composition may be in the form of a parenterally acceptable aqueous solution that is free of pyrogens. The preparation of such parenterally acceptable solutions with due consideration of pH, isotonicity, stability, etc. is within the skill of the art. Suitable compositions for intravenous injection typically contain isotonic vehicles, but this feature is not required.

  For veterinary use, the active ingredient is administered as a suitably acceptable formulation in accordance with normal veterinary practice standards. The veterinarian can readily determine the dosing regimen that is most appropriate for a particular animal.

  Various adaptations and modifications of the embodiments can be made and used without departing from the scope and spirit of the invention. The invention may be practiced other than as specifically described herein. The above description is for illustrative purposes and is not intended to be limiting. The scope of the invention is determined only by the claims.

  The terms and expressions used herein are used for purposes of description, not limitation. The use of such terms and expressions is not intended to exclude equivalents or portions of features shown and described. It will be appreciated that various modifications are possible within the scope of the claimed invention. Furthermore, any one or more features of any aspect of the invention may be combined with any one or more features of any other aspect of the invention without departing from the scope of the invention. .

  Unless stated otherwise, all numbers used in the specification and claims to indicate the amount of a component, such as molecular weight, reaction conditions, etc., are changed by the term “about” in all examples. It should be understood that Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that can vary depending on the desired properties sought to be obtained by the present invention. At a minimum, and not trying to limit the application of equivalent doctrine to the claims, each numeric parameter is at least in terms of the number of significant figures reported and by applying conventional rounding techniques. Should be interpreted. Although the numerical ranges and parameters representing the broad scope of the present invention are approximate, the numerical values shown in the specific examples are reported as accurately as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

As used herein in the context of describing the present invention (especially in the context of the following claims), the terms “a (an)” and “the” and similar indications are used herein. Unless specifically stated otherwise or unless clearly contradicted by context, it should be construed as covering both singular and plural. The recitation of a range of values herein is only a shorthand for referring individually to each other number that falls within that range. Unless stated otherwise specifically in the specification, each individual numerical value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or example languages (e.g., "such as") provided herein are merely intended to better illuminate the invention and are claimed separately. It is not intended to limit the scope of the invention being described. No language in the specification should be construed as referring to any unclaimed element essential to the practice of the invention.

  Groupings of alternative elements or aspects of the invention disclosed herein are not to be construed as limitations. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements found herein. It is anticipated that one or more members of a group may be included or removed from the group for convenience and / or for patentability. If any such inclusion or deletion occurs, the specification is considered herein to contain the modified group and thus satisfies all Markush group descriptions used in the appended claims.

  Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Of course, variations on those preferred embodiments will become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect experts to use such variations as necessary, and the inventors intend that the invention be practiced other than as specified herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Furthermore, numerous references have been made to patents and publications throughout this specification. Each of the references and publications cited above are individually incorporated herein by reference in their entirety.
Finally, it should be understood that the embodiments of the invention disclosed herein are intended to illustrate the principles of the invention. Other variations that can be used are also within the scope of the present invention. Thus, for example (but not limited to), alternative configurations of the present invention can be utilized in accordance with the teachings herein. Accordingly, the present invention is not limited to that precisely as shown and described.

Claims (26)

Use of a composition comprising ET _B receptor agonist in the manufacture of a medicament for the treatment of breast tumors, the pharmaceutical is intended to be used in conjunction with a chemotherapeutic agent, the ET _B receptor agonist is an IRL1620 the composition is intended to be administered intravenously to a mammal in need of said treatment, the ET _B receptor agonist selectively increases the blood supply to the breast tumor, thus the to the breast tumor increasing the delivery of chemotherapeutic agents, and the ET _B receptor agonists enhance the effect of the chemotherapeutic agent, the use. The pharmacokinetics of chemotherapeutic agent is not affected by the ET _B receptor agonists Use according to claim 1. Wherein the chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, or a mixture thereof, to claim 1 Use of description.   4. Use according to claim 3, wherein the chemotherapeutic agent is paclitaxel. The chemotherapeutic agent and the ET _B receptor agonist,
Administering the chemotherapeutic agent with the ET _B receptor agonist as a single composition;
Administering the chemotherapeutic agent with the ET _B receptor agonists as separate compositions;
Administered or the ET _B receptor agonist and said chemotherapeutic agent in order; the administration of ET _B receptor agonist and said chemotherapeutic agent are sequentially, the ET _B receptor agonist is administered prior to the chemotherapeutic agent administering the chemotherapeutic agent prior to the ET _B receptor agonist;
The use according to claim 1, wherein For the treatment of breast tumors in a mammal in need of treatment, a use of a therapeutically effective amount of ET _B receptor agonist and a therapeutically effective amount of a chemotherapeutic agent, the ET _B receptor agonist is The use, which is IRL1620. Wherein the chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, or a mixture thereof, to claim 1 Use of description.   Use according to claim 1, wherein the chemotherapeutic agent is paclitaxel. The ET _B receptor agonist and said chemotherapeutic agent are administered simultaneously, use according to claim 1. The ET _B receptor agonist and said chemotherapeutic agent are administered as a single composition, Use according to claim 9. The ET _B receptor agonist and said chemotherapeutic agent are administered as separate compositions, use according to claim 9. The ET _B receptor agonist and said chemotherapeutic agent are administered sequentially, Use according to claim 1. The ET _B receptor agonist is administered prior to the chemotherapeutic agent Use according to claim 12. (A) packaging composition comprising ET _B receptor agonist is IRL1620;
(B) a package insert that provides instruction for the intravenous administration of to contribute to the treatment of breast tumors in a mammal (a), when according to the instruction, the ET _B receptor agonist the selectively increase blood supply to the breast tumor, thus increasing the chemotherapeutic drug delivery to the breast tumor, and the ET _B receptor agonists enhance the effect of the chemotherapeutic agent, the package insert; And (c) a product comprising a container for (a) and (b). The product further comprises (d) a chemotherapeutic agent, the instructions provide for the administration of (a) and (d), and the container (c) is for (a), (b) and (d) It is of, when in accordance with the instruction, the ET _B receptor agonist selectively increases the blood supply to the breast tumor, and thus increase the delivery of the chemotherapeutic agent to the breast tumor, Then the ET _B receptor agonists enhance the effect of the chemotherapeutic agent, product of claim 14.   The chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, or a mixture thereof. Product listed.   15. The product of claim 14, wherein the chemotherapeutic agent is paclitaxel. (A) packaging composition comprising ET _B receptor agonist is IRL1620;
(B) a package insert providing instructions for administering (a) with a chemotherapeutic agent to treat a breast tumor in a mammal; and (c) a breast tumor comprising a container for (a) and (b) A product that can be treated. (A) packaging composition comprising ET _B receptor agonist is IRL1620;
(B) a packaging composition comprising a chemotherapeutic agent;
(C) a package insert providing instructions for the simultaneous or sequential administration of (a) and (b) with a chemotherapeutic agent to treat a breast tumor in a mammal; and (d) (a), (b ) And a product capable of treating a breast tumor comprising a container for (c). (A) IRL1620 packaging composition comprising ET _B receptor agonist and the chemotherapeutic agent is;
(B) a package insert providing instructions for administering (a) with a chemotherapeutic agent to treat a breast tumor in a mammal; and (c) a breast tumor comprising a container for (a) and (b) A product that can be treated.   19. The chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, or mixtures thereof, The product according to 19 or 20.   21. A product according to claim 18, 19 or 20 wherein the chemotherapeutic agent is paclitaxel. A pharmaceutical composition for the treatment of breast tumors including ET _B receptor agonist, said composition is intended to be used in conjunction with a chemotherapeutic agent, the ET _B receptor agonist is IRL1620, the composition to a mammal have a need of the treatment is intended to be administered intravenously, the ET _B receptor agonist selectively increases the blood supply to the breast tumor, thus of the chemotherapeutic agent to the breast tumor delivery increase, and the ET _B receptor agonists enhance the effect of the chemotherapeutic agent, the pharmaceutical composition. Pharmacokinetics of the chemotherapeutic agent is not affected by the ET _B receptor agonist, a pharmaceutical composition of claim 23.   The chemotherapeutic agent is adriamycin, camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin, alpha interferon, beta interferon, gamma interferon, interleukin 2, irinotecan, docetaxel, paclitaxel, topotecan, or mixtures thereof. The pharmaceutical composition as described.   26. The pharmaceutical composition of claim 25, wherein the chemotherapeutic agent is paclitaxel.

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