RU2287341C1 - Angiogenesis inhibitor, anti-angiogenic pharmaceutical composition based on thereof and method for treatment of malignant neoplasm - Google Patents

Abstract

FIELD: chemistry of natural compounds, biotechnology, pharmacology, medicine, oncology, pharmacy.

SUBSTANCE: invention relates to a medicinal formulation of angiogenesis inhibitor comprising active compound as liposome form wherein angiostatin or endostatin are used as an active component, and their truncated peptide fragments also, and an anti-angiogenic pharmaceutical composition used for treatment. Also, invention proposes a method for inhibition of tumor growth and metastasizing of malignant neoplasms associated with disorders in angiogenesis. Method involves administration of nontoxic therapeutically effective dose of pharmaceutical composition separately or in combination with adjuvant therapy with other antitumor preparations. Invention provides reducing rate of elimination of active compound (angiostatin, endostatin or their truncated fragments) from body and enhancing effectiveness of its therapeutic effect. Invention can be used for inhibition of tumor growth and metastasizing of malignant neoplasms.

EFFECT: enhanced and valuable medicinal properties of inhibitor.

7 cl, 3 tbl, 5 ex

Description

The present invention relates to the field of chemistry of natural compounds, biotechnology, pharmacology and medicine, and can be used for the treatment of malignant neoplasms.

The invention relates to a liposome form of an antiangiogenic drug for the treatment of malignant neoplasms, namely for the treatment of primary tumors and metastases in humans, for the treatment of malignant neoplasms and other pathologies associated with impaired angiogenesis. An advantage of the invention is to enhance the antiangiogenic and antitumor activity of the active component of the drug.

Angiogenesis is the process of the formation of new blood vessels from pre-existing ones, which plays a key role in a wide range of physiological and pathological processes. Tumor progression and the course of cancer are closely related to angiogenesis [Liotta L.A. et al., Cell 64: 327-336, 1991; Fidler I.J. and Ellis L. M., Cell 79: 185-188, 1994; Folkman J., N. Engl. J.Med. 333: 1757-1763, 1995]. A developing tumor through the secretion of angiogenic factors attracts new capillaries, developing from nearby vessels. Further tumor progression is due to the tight integration of tumor cells and blood vessel endothelium. The oxygen and nutrients brought by the blood cause a rapid progression of the tumor and the initiation of the metastasis process. When using antiangiogenic agents that cause the destruction of the tumor circulatory network, in model experiments on animals with inoculated tumors, a number of authors observed a significant therapeutic effect from these drugs, expressed in suppressing tumor growth and increasing the average life expectancy of experimental animals [O'Reilly M.S. et al., Cell 79: 315-328, 1994; O'Reilly M.S. et al., Nat. Med. 2: 689-692, 1996; Holmgren L. et al., Nat. Med. 1: 149-153, 1995; Mauceri H.J. et al., Nature 394: 287-291, 1998]. Thus, the destruction of the tumor circulatory network using antiangiogenic agents is a promising approach to the treatment of malignant neoplasms.

One of the most powerful inhibitors of angiogenesis is angiostatin - a proteolytic fragment of plasminogen with mol. mass ~ 40 kDa [O'Reilly M.S. et al., Cell 79: 315-328, 1994]. By acting on endothelial cells of blood vessels, angiostatin inhibits the growth of both primary tumors and regional metastases.

At present, biologically active recombinant angiostatin expressed in various producer strains (Escherichia coli, Pichia pastoris, etc.) has been obtained by genetic engineering. Recombinant angiostatin causes apoptotic death of endothelial cells and effectively inhibits the growth of Lewis pulmonary carcinoma tumors in mice at daily doses of 1-6 mg / kg [Wu Z. et al., Biochem. Biophys. Res. Commun. 236: 651-654, 1997]. One of the important features of angiostatin is its ability to inhibit tumor growth without toxic effects [O'Reilly M.S. et al., Nat. Med. 2: 689-692, 1996; Sim B.K. et al., Cancer Res. 57: 1329-1334, 1997; Lannutti B.J. et al., Cancer Res. 57: 5277-5280, 1997]. Even with the introduction of the drug in high doses (~ 100 mg / kg weight), there are no manifestations of toxicity or the development of drug resistance of tumors to the drug [Sim B.K. et al., Cancer Res. 57: 1329-1334, 1997; Mauceri H.J. et al., Nature 394: 287-291, 1998; Drixler T.A. et al., Cancer Res. 60: 1761-1765, 2000].

Despite the obvious advantages and prospects of the therapeutic use of angiostatin, a significant drawback of the drug is its rapid elimination from the body [Drixler T.A. et al., Cancer Res. 60: 1761-1765, 2000]. In addition, to achieve the desired antitumor effect, prolonged use of the drug is necessary. Thus, the drug is administered to the patient at least twice a day in doses from 20 to 100 mg / kg of body weight. It is obvious that the therapeutic use of angiostatin will not only cause inconvenience to patients due to the need for constant injections, but will also require a huge amount of the drug. In order to avoid this, it is necessary to reduce the rate of excretion of angiostatin and increase the effectiveness of its therapeutic effect.

Another promising antiangiogenic drug that affects the survival of endothelial cells is endostatin - a 20 kDa fragment of collagen XVIII, which is a component of the basement membranes and extracellular matrix [Muragaki Y. et al., Proc. Natl. Acad. Sci. USA, 92: 8763-8767, 1995]. Endostatin specifically inhibits the proliferation and migration of capillary endothelial cells and can cause apoptosis of proliferating endothelial cells. Endostatin does not have a direct effect on tumor cells of various lines in vitro [O'Reilly M.S. et al., Cell: 88, 277-285, 1997]. In vivo, endostatin has a strong inhibitory effect on the growth of various primary tumors and their metastases. Prolonged therapy with high doses of endostatin causes an almost complete blockade of tumor angiogenesis and leads to regression of the tumor to microscopic sizes [Boehm T. et al., Nature: 390, 404-407, 1997]. Subsequently, the micro-tumors fall into a “dormant” state, in which the proliferation index of tumor cells is balanced by their apoptosis index. Even after prolonged therapy, there is no occurrence of drug resistance or any toxic reactions [Sim B.K. et al., Cancer Metastasis Rev .: 19, 181-190, 2000]. Endostatin can inhibit the cascade of intracellular signaling induced by angiogenic growth factors, as well as block the activation and catalytic activity of matrix metalloproteinases.

The closest in technical essence are angiogenesis inhibitors angiostatin, endostatin and their truncated fragments for the treatment of malignant neoplasms / Pat. RF №2240328 /. The disadvantages of these inhibitors are indicated above. In addition, the authors of the present invention propose the use of the mentioned angiogenesis inhibitors in the form of homodimeric proteins in which the inhibitor molecule is connected via a polypeptide bridge to the Fc region of the gamma immunoglobulin, which can further reduce the biological activity of the target angiogenesis inhibitor. The use of such fusion proteins also does not solve the problem of a high rate of elimination of drugs from the patient.

The purpose of the invention is to reduce the rate of excretion of the active compound (angiostatin and endostatin) from the body and increase the effectiveness of its therapeutic effect.

The goal is achieved by creating an angiogenesis inhibitor comprising the active compound in the form of liposomes. Moreover, angiostatin or endostatin, as well as their truncated peptide fragments, are used as the active compound. An anti-angiogenic pharmaceutical composition for treating malignant neoplasms and other pathologies associated with impaired angiogenesis is also provided, comprising an active compound (angiostatin, endostatin or truncated fragments thereof) and a pharmaceutical carrier, wherein the active compound is contained in an effective amount in liposome form and as a pharmaceutical carrier Acceptable injection solutions are used.

In addition, a method for the treatment of malignant neoplasms and other pathologies associated with impaired angiogenesis is proposed, which consists in administering a non-toxic therapeutically effective amount of a pharmaceutical composition, either alone or in combination with adjuvant therapy with other antitumor drugs, immunomodulators or antiangiogenic agents. As an antitumor drug for adjuvant therapy, a drug selected from the group of doxorubicin, carminomycin, mutamycin, novantrone, rubomycin, bleocin, cisplatin, taxol, etoposide, vinblastine, vincristine, gemzar, fluorouracil, methotrexate, celoda, xeloda is used carboplatin, tamoxifen. As an immunomodulator for adjuvant therapy, a drug selected from the group: interferon-α, interferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12 is used. As an antiangiogenic agent for adjuvant therapy, a drug selected from the group is used: thalidomide, vitaxin, pentosan, suramine, fumagillin, squolamine, combretastatin, prinomastat, marimastat, neovastat.

Angiostatin or endostatin as part of liposomes can be represented by various forms that differ in the length of the peptide chain — both whole proteins and their truncated peptide fragments that differ from the sequence of the whole protein by one or more terminal amino acids. Angiostatin or endostatin for liposome compositions can be obtained either from natural sources or using genetic engineering technologies, and truncated fragments of these proteins can also be obtained using genetic engineering methods or the solid-phase peptide synthesis method.

The created liposome form of the angiogenesis inhibitor is simple and manufacturable, exhibits high biological activity and is able to effectively suppress the growth of tumor vessels and tumors themselves.

The invention is based on an angiogenesis inhibitor in the form of a liposome, which in experiments both in vitro and in vivo exhibits a higher antitumor activity than the free active component. An inhibitor in the form of a liposome can be easily obtained using standard biotechnological and chemical methods; it is distinguished by its stability and manufacturability. The liposomal form of an anti-angiogenic inhibitor can be successfully used to treat a number of pathologies associated with impaired angiogenesis. Thus, the invention fully meets the criterion of "inventive step".

The invention is illustrated by the following examples.

Example 1. Obtaining liposomes loaded with angiostatin or endostatin, or their truncated fragments.

For 1 ml of liposome dispersion, 20.5 mg of egg phosphatidylcholine (JPC) and 4.5 mg of cholesterol (JPC / cholesterol 7/3 mol.) In the form of chloroform solutions are placed in a round bottom flask. The mixture of lipid solutions is evaporated on a rotary evaporator to constant weight at 38 ° C. Residual solvent is removed in an oil pump vacuum for 2 hours. The resulting lipid film is dissolved in 3 ml of ether and 1 ml of a protein solution (angiostatin, endostatin or fragments thereof) (6 mg / ml) in phosphate-buffered saline (pH 7.4) is added to the flask. ) The flask was purged with argon and the dispersion was voiced in an ultrasonic bath for 3 min at 10 ° C. Cautiously evaporate the ether on a rotary evaporator at room temperature (200 rpm). The resulting lipid dispersion is pushed 19 times through a nuclear polycarbonate filter with a pore size of 100 nm. Obtain 0.9 ml of a 100 nm liposome dispersion (25 mg / ml lipid, 6 mg / ml protein). The efficiency of protein inclusion is 14-26%. The liposomal angiogenesis inhibitor is lyophilized and stored at 4 ° C.

Example 2. Obtaining a pharmaceutical composition based on a liposomal angiogenesis inhibitor.

Injectable pharmaceutical compositions are prepared by dissolving an effective amount of a lyophilized angiogenesis inhibitor obtained as described in Example 1 in physiological saline or phosphate-saline prepared in water for injection. The pH of the solutions is about 7.4. The concentration of the active substance (protein) in the solution is 5-10%.

Example 3. Determination of the antitumor activity of liposomal angiogenesis inhibitors, including angiostatin, endostatin or their truncated fragments, in individual therapy of mice with grafted solid tumors of B16 melanoma.

, где а - короткий, b - длинный диаметр опухоли. Относительный размер опухолей (ОРО) определяли по формуле:

, где Р_оп - средний размер опухолей в опытной группе, Р_к - средний размер опухолей у контрольных животных. Увеличение продолжительности жизни леченых животных по сравнению с контролем определяли по формуле:

, где Т - средняя продолжительность жизни (СПЖ) леченых животных, дни, С - СПЖ контрольных животных, дни.B16 melanoma cells were maintained by weekly intraperitoneal inoculation in C57B1 / 6 mice. To induce solid tumors, the mice were injected subcutaneously with a cell suspension (2 × 10 ⁵ cells in 0.1 ml of physiological saline). The drugs were administered to experimental animals intravenously. Single doses of the liposome form of the angiogenesis inhibitor contained: 90 mg / kg of angiostatin, endostatin, or fragments thereof. The drugs were administered intravenously once a week, only three injections, starting from the 10th day after tumor inoculation. Each experimental group consisted of six animals, the control - of ten. The size of solid tumors was measured once every 2-3 days. Tumor volume was calculated by the formula

where a is short, b is the long diameter of the tumor. The relative size of the tumors (ORO) was determined by the formula:

where P _op - the average size of the tumors in the experimental group, P _to - the average size of the tumors in control animals. The increase in the life expectancy of the treated animals compared with the control was determined by the formula:

where T is the average life expectancy (LSS) of the treated animals, days, C - LIV of the control animals, days.

The experimental results are shown in table 1. Compared with the mild effect of the use of non-liposomal forms of angiostatin, endostatin and their truncated fragments, therapy with liposome forms led to a significant inhibition of tumor growth both during the course of treatment and after its cancellation and to increase the average life expectancy of the treated animals.

Table 1
The antitumor effect of liposome angiogenesis inhibitor against melanoma B 16 in mice compared with non-liposome forms. Group of animals ORO,% USPZH,% 24 day 35 day the control one hundred one hundred - Non-liposomal forms of drugs angiostatin 64.8 82,4 20.8 shortened angiostatin 58.3 74.0 27.1 endostatin 59.0 71.3 24.6 shortened endostatin 53.6 68.5 29.8 Liposome inhibitors angiostatin 36.1 48.9 41.8 shortened angiostatin 32.6 43.8 49.9 endostatin 31,4 42.8 47.3 shortened endostatin 28.7 37.1 52.1

Example 4. Antimetastatic activity of liposomal angiogenesis inhibitors.

To evaluate the antimetastatic activity of liposomal antiangiogenic inhibitors, an experiment was performed in mice with a subcutaneously inoculated Lewis lung carcinoma tumor (3LL) with the removal of the primary tumor node.

The primary tumor was removed on day 8 after inoculation. Hexenal was used as anesthesia. The tumor site was removed along with the adjacent area of the skin, to prevent the possibility of recurrence. The skin defect was closed by suturing, lubricated with 5% alcohol solution of iodine. The first administration of the studied drugs was carried out 12 hours before the operation. The drugs were administered intravenously at doses of 90 mg / kg once every 4 days, a total of 5 injections. Each experimental group consisted of 8 animals.

Antimetastatic effect was evaluated at the time of the onset of death of control animals. The intensity of metastasis was evaluated by comparing the average weight of the lungs affected by metastases in the control and experimental groups of animals. At the same time, conditional indicators of the intensity of metastasis were used - the number and area of metastatic nodes in the lung tissue, the frequency of tumor metastasis. Statistical processing of the results was carried out according to student criteria.

, где Л_к - средняя масса легких с метастазами в контрольной группе, Л_о - средняя масса легких с метастазами в группе леченых мышей.Inhibition of growth of metastases was calculated by the formula:

where L _to - the average weight of the lungs with metastases in the control group, L _about - the average weight of the lungs with metastases in the group of treated mice.

The experimental results are presented in table.2. Compared to control animals, in which lung metastases were detected in 100% of cases, in the groups of treated animals there were cases of the absence of metastases, and the intensity of metastasis was also reduced. Thus, the use of the proposed drugs to prevent metastasis seems very promising.

table 2
The effect of liposome angiogenesis inhibitors on the intensity of metastasis of a subcutaneous inoculated Lewis lung carcinoma tumor (3LL) against the background of removal of the primary tumor node. Group of animals TPM,% Metastasis rate Metastasis Intensity (average metastases / mouse) The control - 8/8 [5.8 ± 3.1] liposome form of angiostatin 42.7 5/8 [2.7 ± 1.3] liposome form of angiostatin (shortened) 38,2 4/8 [2.1 ± 1.9] liposome form of endostatin 40.6 5/8 [2.2 ± 0.8] liposome form of endostatin (shortened) 34.6 5/8 [1.9 ± 0.6] Notes:
1. Data are given at the time of death of the animals.
2. The column "Metastasis frequency" indicates the number of animals with metastases / number of animals in the group.
3. In the column "Metastasis intensity" (in square brackets) - the average number of metastatic colonies in the lungs per animal.

Example 5. The effectiveness of the antiangiogenic pharmaceutical compositions, including angiostatin, endostatin or their truncated fragments, in relation to mouse melanoma when they are used in combination therapy with antitumor drugs, immunomodulators and other antiangiogenic agents.

Antiangiogenic pharmaceutical compositions were administered as described in example 3, against the background of adjuvant therapy with other drugs. Thus, the animals of group 1 were additionally injected with the antitumor antibiotic doxorubicin (2 mg / kg) according to the same schedule as liposome preparations, group 2 - interferon-α daily for 3 weeks, group 3 - the antiangiogenic drug thalidomide. The experimental results are shown in table.3. As can be seen from the above data, the use of the proposed antiangiogenic pharmaceutical compositions in combination therapy with other drugs significantly increases the effectiveness of their antitumor effects.

Table 3
The efficacy of the antitumor effect of antiangiogenic pharmaceutical compositions against B16 melanoma in mice when combined with other antitumor drugs. Group of animals ORO,% (35 day) USPZH,% the control one hundred - Individual therapy Liposomal form of angiostatin 48.9 41.8 Liposomal form of angiostatin (shortened) 43.8 49.9 Liposome form of endostatin 42.8 47.3 Liposome form of endostatin (shortened) 37.1 52.1 Doxorubicin Liposomal form of angiostatin 41.6 49.8 Liposomal form of angiostatin (shortened) 37.7 56.4 Liposome form of endostatin 38,4 55,2 Liposome form of endostatin (shortened) 32.6 58.9 Interferon-α Liposomal form of angiostatin 46.1 46.2 Liposomal form of angiostatin (shortened) 39.8 54.9 Liposome form of endostatin 39.7 51,2 Liposome form of endostatin (shortened) 35.3 56.0 Thalidomide Liposomal form of angiostatin 40,2 52.9

Similar results were obtained in experiments to study the effectiveness of the claimed anti-angiogenic pharmaceutical composition in combination therapy with other antitumor drugs - carminomycin, mutamycin, novantrone, rubomycin, bleocin, cisplatin, taxol, etoposide, vinblastine, vincristine, gemzar, fluorouracil, metrotrecil, metrotrecil, methotrecl, zoladex, dacarbazine, cisplatin, carboplatin, tamoxifen; immunomodulators - interferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12; anti-angiogenic agents - vitaxin, pentosan, suramin, fumagillin, squolamine, combretastatin, prinomastat, marimastat, neovastat.

The approach proposed by the applicant is devoid of the above disadvantages of analogues and combines the simplicity and manufacturability of the liposomal form of antiangiogenic drugs with high antitumor effects due to the increase in the residence time of the drug in the blood and its selective accumulation in the area of tumor blood vessels.

Claims (7)

1. A dosage form of an angiogenesis inhibitor, comprising angiostatin or endostatin in liposomes as an active substance, characterized in that it contains a full-sized form or a shortened peptide fragment of these inhibitors as an active substance, wherein the substance is enclosed in a liposome membrane based on phosphatidylcholine and cholesterol when their molar ratio of 7: 3, the resulting liposome contains an active substance of at least 19 wt.%. 2. An anti-angiogenic pharmaceutical composition for the treatment of malignant neoplasms, containing an active substance and a pharmaceutical carrier, characterized in that as the active substance it contains the dosage form according to claim 1 and physiological saline or phosphate-saline solution, wherein the concentration of the active substance in the solution is 5-10% protein. 3. A method of inhibiting tumor growth and metastasis of malignant neoplasms associated with impaired angiogenesis, characterized in that the patient is administered a non-toxic therapeutically effective amount of the pharmaceutical composition according to claim 2. 4. The method of inhibition according to claim 3, characterized in that they additionally carry out combined adjuvant therapy with other antitumor drugs, immunomodulators or antiangiogenic agents. 5. The method according to claim 4, characterized in that as an antitumor drug for adjuvant therapy, a drug selected from the group of doxorubicin, carminomycin, mutamycin, novantrone, rubomycin, bleocin, cisplatin, taxol, etoposide, vinblastine, vincristine, gemzar, fluorouracil is used , methotrexate, xeloda, zoladex, dacarbazine, cisplatin, carboplatin, tamoxifen. 6. The method according to claim 4, characterized in that as an immunomodulator for adjuvant therapy using a drug selected from the group of interferon-α, interferon-γ, interleukin-2, interleukin-4, interleukin-6, interleukin-12. 7. The method according to claim 4, characterized in that as an antiangiogenic agent for adjuvant therapy, a drug selected from the group of thalidomide, vitaxin, pentosan, suramine, fumagillin, squolamine, combretastatin, prinomastat, marimastat, neovastat is used.