RU2789607C1 - Use of polymerized hemoglobin for increasing efficiency of malignant tumor chemotherapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: present invention relates to the field of medicine, namely to the use of polymerized hemoglobin in the form of lyophilizate for the preparation of a solution for infusions as an agent increasing an effect of chemotherapeutic drugs in the treatment of brain tumors.

EFFECT: present invention provides expansion of the arsenal of remedies simultaneously having oxygen-transport, phase-modulating, and chemo-modifying properties at low concentrations and doses of administration of polymerized hemoglobin.

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Description

The invention relates to medicine and can be used in oncology as a means to increase the effectiveness of chemotherapy with simultaneous administration.

The invention also relates to veterinary medicine and can be used as an adjuvant in chemotherapy for oncological diseases in domestic animals.

As a rule, analogs of the drug - polymerized hemoglobin are used as drugs with the function of oxygen transfer. These are drugs such as "Gelenpol", "Biopyur" and the drug "Gelenpol" /RU 2132687, A61K 35/18, publ. 10.07.1999/ obtained from donated blood. The drug "Gelenpol" is a mixture of hemoglobin tetramer and oligomers with different chain lengths. Oligomers are obtained by cross-linking hemoglobin tetramers with glutaraldehyde and modified with glutamic acid. Biopure preparation obtained from bovine erythrocyte hemoglobin (patents US 5084558, C07K14/805, publ. 01.28.1992 and US 6506725, C07K14/805, publ. 14.01.2003).

Known polymerized hemoglobin with the function of oxygen transfer [RF Patent No. 2340354, A61K 38/42, publ. 12/10/2008 and RF Patent No. 2361608, A61K 38/42, publ. 20.07.2009]. Said hemoglobin polymerized with glutaraldehyde is obtained from animal blood, is a lyophilisate and contains at least 90% polymerized hemoglobin with a molecular weight in the range of 192,000-320,000 Da, and the content of methemoglobin is not more than 5%. For all the above drugs, the function of increasing the sensitivity of tumor cells to the effects of chemotherapy has not been shown.

Known agent for protecting blood cells and restoring the hematopoietic system and pharmacological combination for the treatment of cancer patients [Patent No. 2328304, A61K 38/42, publ. 07/10/2008], which is characterized by the fact that it includes polyhemoglobin modified with glutamic acid. The present invention also relates to a pharmacological combination comprising polyhemoglobin modified with glutamic acid and a cytostatic, wherein the components of the combination are intended for separate or sequential administration to a patient. In this combination, polyhemoglobin provides a pool of red blood cells to replace those damaged during therapy.

Known tool proposed in US patent 5631219, C12N5/0641, publ. 05/20/1997, where it is proposed to improve the quality of life of cancer patients and increase its duration by stimulating erythropoiesis by introducing highly pure hemoglobin obtained by recombinant technology, extracted from E. Coli, in which the hemoglobin gene is expressed, and a mutation is introduced into this gene that provides crosslinking of two alpha or two beta or several alpha and several beta subunits into a hemoglobin tetramer via a peptide bridge.

Polymerized hemoglobin is a mixture of hemoglobin tetramer and oligomers with various chain lengths and is an oxygen carrier, but so far they have not been considered as adjuvants of chemotherapy drugs coming from the bloodstream in the treatment of tumors.

Thus, there are currently no direct analogues - products based on mammalian hemoglobin that can be used to increase the effectiveness of chemotherapy for brain tumors.

The drug already described above (US Patent 5631219, C12N5/0641, publ. 05/20/1997) describes the mechanism of stimulation of erythropoiesis when introduced into the bloodstream. This property of hemoglobin, due to the reaction of the hematopoietic system by the mechanism of positive feedback to the presence of extra-erythrocyte hemoglobin, complements the overall picture of the positive effect of the introduction of polymerized hemoglobin to cancer patients. As a probable mechanism, one can consider the induction of the release of mature erythrocytes from the depot, followed by the induction of the maturation of immature proforms of blood cells. These data allow us to consider polymerized hemoglobin as a three-component drug: an inducer of endogenous CO formation, an inducer of erythropoiesis and an increase in the efficiency of cellular respiration, in addition to the oxygen transfer effect that polymerized hemoglobin has.

In addition, the drug is a source of free iron, which, entering a significant amount in the tumor, induces ferroptosis. Ferroptosis is an oxidative form of necrotic cell death that is highly relevant to many degenerative diseases and cancers. Ferroptosis is characterized by iron-dependent lipid peroxidation and is distinct from other modes of cell death. The key factor in ferroptosis is the enzyme glutathione peroxidase-4, which provides effective control of lipid peroxidation. The development of new inhibitors and inducers of ferroptosis opens up great potential in the development of innovative drugs for the treatment of diseases associated with ferroptosis. It is known that iron ions are able to overcome the blood-brain barrier, and the brain cells contain the largest amount of erythrocyte iron.

An important characteristic of gliobastoma multiforme is the production of a significant amount of VEGF with an increase in the number of vessels, as a rule, having an imperfect stage of neoangiogenesis. The possibility of increasing the amount of chemotherapy entering the tumor can be provided by increasing the permeability of the blood-brain barrier and expanding the diameter of the vessels that make up the developed vascular network of the tumor.

The technical problem to be solved by the claimed invention is the expansion of the arsenal of agents that simultaneously have oxygen transport, vasomodulatory and chemomodifying properties at low concentrations and doses of polymerized hemoglobin.

The claimed technical problem is solved by using polymerized hemoglobin as an adjuvant that enhances the effectiveness of chemotherapy.

What is new in the proposed invention is that polymerized hemoglobin (a lyophilisate for preparing a solution for infusion) containing at least 90% polymerized hemoglobin with a molecular weight in the range of 192000-320000 Da and methemoglobin in its composition is not more than 5%.

The essence of the invention is illustrated by examples of a specific implementation, which uses polymerized hemoglobin (lyophilizate for preparing a solution for infusion in a 100 ml vial), obtained in a standard way / RF Patent No. 2361608, A61K 38/42, publ. 07/20/2009 and RF Patent No. 2340354, A61K 38/42, publ. 12/10/2008.

Exogenous hemoglobin acts as an activator of inducible heme oxygenase-1, which leads to an increase in endogenous production of the most important low molecular weight mediator - carbon monoxide (CO), which modulates vascular tone, including providing vasodilation of blood vessels leading to the tumor.

As a result of our experimental studies, a hitherto unknown property of polymerized hemoglobin (a lyophilizate for preparing a solution for infusions) was found to stimulate the formation of endogenous carbon monoxide, which, in turn, induces an expansion of the diameter of blood vessels, including arterioles and capillaries.

The presence of the blood-brain barrier in the brain creates a big problem for the delivery of chemotherapeutic drugs in the development of various types of malignant tumors, including astrocytoma, glioblastoma, and other neuroectodermal tumors. Unlike tumors of the meninges (meningiomas), these tumors are diagnosed at late stages, grow into both the white and gray matter of the brain without the development of pain, and are difficult to surgically treat due to difficult access and, as a rule, multiple metastases. different size. In addition, in addition to primary tumors, it is in the brain that metastases of uterine cancer, breast cancer, melanoma and other tumors are found. According to the US Central Brain Tumor Registry [http://www.CBTRUS.org], about 13,000 deaths and 18,000 new cases of primary malignant tumors of the brain and central nervous system (CNS) occur annually in the United States. Gliomas account for approximately 77-81% of primary malignant brain tumors. It is the most common primary intracranial tumor. Glioblastoma, the most common type of glioma (~45% of all gliomas), has a 5-year relative survival of ~5% [Ostrom QT, Bauchet L, Davis FG, Deltour I, Fisher JL, Langer CE, Pekmezci M, Schwartzbaum JA, Turner MC , Walsh KM, Wrensch MR, Barnholtz-Sloan JS. The epidemiology of glioma in adults: a "state of the science" review. Neuro Oncol. 2014 Jul; 16(7):896-913. doi: 10.1093/neuonc/nou087. PMID: 24842956; PMCID: PMC4057143]. They are localized both in the cerebral hemispheres and in the cerebellum; they can be single or multiple. Currently, it is customary to combine surgical treatment, radiotherapy (gamma knife, proton therapy) and chemotherapy treatment.

The property of polymerized hemoglobin to modulate vasodilation and vasoconstriction during chemotherapy makes it possible to increase its effectiveness, providing an increase in the permeability of the blood-brain barrier (BBB), while it is an unobvious fact for oncologists and does not follow from the state of the art in this field, it is not found in the patent and scientific and medical literature .

The above property of polymerized hemoglobin can be used in practical public health and veterinary medicine to improve the quality of treatment. Thus, the proposed technical solution meets the criteria for patentability of the invention, namely "novelty", "inventive step" and "industrial applicability".

The significant effect of the introduction of polymerized hemoglobin on blood hemodynamics and vasodilation of microvessels, found in the course of a study on large laboratory animals, allows us to consider it as an exogenous inducer of endogenous production of the most important low molecular weight mediator - CO by activating the enzyme systems of the body.

The claimed invention is illustrated in Fig. 1, which shows the Kaplan-Meier curve, which allows you to evaluate the effect of the therapy; and figure 2 - total indicator of the severity of tumor growth.

Statistical processing of the research results was carried out using parametric and non-parametric methods. Differences between groups were considered statistically significant at P≤0.05.

Example 1. Hemoglobin Polymerized cattle as a means that increases the sensitivity of tumor cells of multiform glitorotoma to chemotherapy, was evaluated on the model of the redeemed glyoblastoma of the rat 101/8 (Bank of the cellular crops of human morphology. Moscow) on adult sexually mature rags Wistar, with body weight 150-180 g.

List of abbreviations Key words: LS - life expectancy, LS - increase in life expectancy, ALE - average life expectancy, DOX - doxorubicin hydrochloride, BBB - blood-brain barrier.

Rat transplantable glioblastoma 101/8 (developed at the Research Institute of Human Morphology, where it is stored, maintained and used) Animals were injected with the drug (hereinafter IP) on days 2, 5 and 8 after tumor implantation. The drugs were injected into the tail vein (left or right). In the combination therapy group, drugs were administered sequentially into one vein.

The rats were divided into four groups:

Group 1. Control. Animals were implanted with glioblastoma 101/8 and observed for lifespan. Therapy was not carried out.

Group 2. Doxorubicin. Animals with implanted glioblastoma were treated with an aqueous solution of doxorubicin (Teva, Israel) at a dose of 1.5 mg/kg.

Group 3. Polymerized bovine hemoglobin. Animals with implanted glioblastoma were treated with a solution of the composition in PBS at a dose of 228 mg/kg.

Group 4. "Polymerized hemoglobin + doxorubicin". Animals received combined therapy: doxorubicin at a dose of 1.5 mg/kg and hemoglobin polymerized at a dose of 228 mg/kg.

Glioma 101/8 was implanted into the brain of a rat under ketamine-xylazine (80 mg/kg, 20 mg/kg) anesthesia with cryopreserved or freshly ground tumor tissue in the amount of 800,000–1 million cells. To do this, a hole was made in the right parietal bone of the rat skull with a dental cutter 2 mm in diameter at a distance of 2 mm from the sagittal suture and 2 mm caudally from the coronal suture. Using a trocar with a diameter of 1.8 mm, the tumor tissue was injected to a depth of 4 mm from the surface of the skull bone, which corresponded to the bottom of the right lateral ventricle. If necessary, bleeding was stopped with hydrogen peroxide. The wound was covered with a powder of sulfa drugs.

The studied drugs were administered to immobilized rats using a disposable syringe into the lateral tail vein. Animals were immobilized in a Kogan immobilization plexiglass chamber. Before the administration of the drug, the animal's tail was placed in warm (+40°C) water for 2–3 min, dried with a gauze cloth, and wiped with a 70% ethanol solution. Holding the base of the tail with the left hand, a filled syringe was taken in the right hand. A thin needle first pierced the skin on the lateral surface of the tail, and then the vessel wall. When the needle entered the lumen of the vein, blood appeared in the syringe, and when the piston moved, a free flow of liquid was then felt.

Efficacy criteria: Lifespan and lifespan of rats treated with intracerebral tumors compared with untreated controls and the doxorubicin-treated group.

On histological sections of the brain of the Wistar rat, glioblastoma 101/8 is represented by a malignant glial tumor with high cellularity, numerous mitoses, focal microvascular proliferation, extensive foci of necrosis and hemorrhage. Vessels in the tumor had different diameters and shapes; as a rule, they were tortuous, lacuna-like dilated. The endothelial lining of the vessels was focally disturbed. Tumor cells had a high nuclear-cytoplasmic ratio. The nuclei are pleomorphic, small or medium in size, with an atypical arrangement of heterochromatin. The boundaries of the tumor are indistinct, with a pronounced infiltration of the surrounding brain tissue by tumor cells. The capsule was missing. According to the morphological picture and by analogy with human tumors, this tumor met the criteria of the World Health Organization (WHO) for glioblastoma multiforme/astrocytoma grade IV.

The death of animals after glioma transplantation in the group without treatment occurred on days 14-21 as a result of tumor spread in a limited space of the cranial cavity. All animals after therapy felt satisfactory. With the development of the tumor process, the following clinical signs were noted: the animals lost activity and appetite, the coat became dull and disheveled, other signs of intoxication were noted. Animals in groups 1 and 3 steadily gained weight until the onset of symptoms of glioblastoma (weight loss 2-3 days before death, lethargy, tremor, refusal to eat). Animals in groups 2 and 4 had a slight delay in weight gain (no gain or slight weight loss) during therapy, which is typical with doxorubicin therapy at this dose. Subsequently (starting from day 10), weight gain persisted until the symptoms of glioblastoma worsened.

Opening. In one animal No. 3 from gr. 4, euthanized on the 46th day after tumor implantation, due to the onset of paralysis of the hind limbs, no tumor node was found. The probable cause of death is the destruction of the spinal cord or the blockage of the spinal canal by tumor cells. All other animals that died or euthanized as a result of an increase in the symptoms of glioblastoma development had significant tumor nodes in the region of the transplant site (right hemisphere) with significant hemorrhages. At autopsy, all typical signs of death from hemorrhage as a result of the development of glioblastoma were found. Animals No. 2 and 4 from group 4, despite the end of the experiment, were not killed, however, no signs of tumor development were found in them. These animals were considered cured of glioblastoma, since this strain is characterized by 100% grafting.

Animal lifespan analysis. Based on the results of the studies, an analysis of the survival of animals in groups was carried out. On FIG. 1 shows the Kaplan-Meier curve, which allows you to evaluate the effect of the therapy.

As a result of combined therapy in group 4, a significant increase in the life expectancy of animals compared to other groups was shown. Life expectancy in 4 increased by 231% (Table 1), with 2 out of 10 animals (20%) surviving the observation threshold of 100 days without showing signs of tumor growth. UPZh in gr. 4 compared with gr.2 was 160% (not shown in the table). The life span of animals in gr. 4 significantly differed from similar indicators in groups 1, 2 and 3.

The life expectancy of animals from group 3 was comparable in terms to those from group 1, which suggested that the Composition does not have its own antitumor effect against glioblastoma 101/8. Some numerical decrease in life expectancy in group 3 compared to group 1 (-8%) is not statistically significant and is caused more by the heterogeneity of tumor growth in animals than by the hypothetical antitumor effect of the composition, since these differences are not statistically significant.

Despite the fact that doxorubicin does not penetrate the BBB into gr. 2, a small LLS was found - 18%. This effect is typical under given conditions, and is probably due to the fact that the tumor BBB becomes partially permeable by the 3rd injection (day 8 of tumor growth) and doxorubicin penetrates the brain and has a small cytotoxic effect.

The table shows the life expectancy, life expectancy and median (Me) life expectancy of animals in four groups.

Table 1 - Average life expectancy and increase in life expectancy of animals after the therapy.

Number of animals n ALE (M±SD) Me LLS to control, % Significance of difference according to Mann-Wittney To gr.1 (Control) To gr. 2. (Doxorubicin) Gr. 1 Control 12 16.3±2.2 16 - P≤0.05 Gr.2 Doxorubicin 9 20.7±7.1 18 27 P≤0.05 - Gr.3 Polymerized hemoglobin 6 15±4.2 13.5 -8 Not reliable P≤0.05 Gr.4 Dox + Hemoglobin polymerized 10 54±31 48.5 231 P≤0.01 P≤0.01

Designations: M - mean, SD - standard deviation, Me - median, LL - increase in life expectancy.

Example 2. Hemoglobin polymerized cattle was used as a means that has adjuvant properties in the chemotherapy of multiform glioblastoma. The effectiveness of the use was evaluated on the model of the digestible glitor of the rat 101/8 on adult sexually mature roofs Wistar, males with body weight 150-180 g.

Rat transplantable glioblastoma 101/8. Animals underwent IP on days 2, 5, and 8 after tumor implantation. The drugs were injected into the tail vein. In the combination therapy group, drugs were administered sequentially into one vein.

The rats were divided into four groups:

Group 1. Control. Animals were implanted with glioblastoma 101/8 and observed for lifespan. Therapy was not carried out.

Group 2. Doxorubicin. Animals with implanted glioblastoma were treated with an aqueous solution of doxorubicin (Teva, Israel) at a dose of 1.5 mg/kg.

Group 3. Polymerized bovine hemoglobin. Animals with implanted glioblastoma were treated with a solution of hemoglobin polymerized in PBS at a dose of 456 mg/kg.

Group 4. "Polymerized hemoglobin + doxorubicin". The animals received combined therapy: doxorubicin at a dose of 1.5 mg/kg and polymerized hemoglobin at a dose of 456 mg/kg.

Animals were dissected on the 14th day after tumor inoculation, the skull was opened, the tumor was isolated from the brain. Macroscopic (visual) examination of the brain was performed according to the following parameters:

• changes in the appearance of the brain (color, shape);

• the presence of visually determined cerebral edema;

• the presence of visually determined foci of hemorrhages and their localization.

Changes were assessed semi-quantitatively from 0 to 2 points for each parameter, where 0 - no changes, 2 - moderately pronounced changes, 2 - pronounced changes. The total indicator of tumor growth severity (TTS) for individual animals ranged from 0 to 9.

The isolated brain was fixed in buffered formalin for 48 hours, sections were made, and histological examination was performed according to the standard method.

The results of macromorphological studies: in all groups, a tumor was found at the site of inoculation (right hemisphere, around the lateral ventricle with invasion into the lateral ventricle), the severity of hemorrhages in the surrounding brain tissues, visually determined edema and necrosis in the tumor tissue were the same for all groups.

In groups 2 and 4, on the 14th day of tumor development, the total tumor growth rate was less than that in the control group without treatment. At the same time, in group 4 (polymerized hemoglobin + doxorubicin), this indicator was lower than in group 2, which received only doxorubicin.

According to the results of the assessment of macromorphological changes in the brain, the introduction of polymerized hemoglobin increased the effectiveness of doxorubicin hydrochloride. The total indicator of the severity of tumor growth in group 4 was statistically significantly different from this parameter for groups 1-3 (Figure 2) .

Table 2 - Total indicator of tumor growth

Number of animals, n Me (25-75%) Significance of difference according to Mann-Wittney To gr.1 (Control) To gr. 2. (Doxorubicin) Gr. 1. Control 8 8.50 (8-9) - - Gr. 2. Doxorubicin 6 8.00 (7-8) P≤0.05 - Gr. 3. Polymerized hemoglobin 6 8.50 (7-9) - P≤0.05 Gr. 4. Dox + Hemoglobin polymerized 6 7.00 (6-7) P≤0.05 P≤0.05

The introduction of only polymerized hemoglobin did not cause a significant decrease in tumor volume. Significant heterogeneity of the results was found, both in the control and experimental groups, due to the individual genetic polymorphism of Wistar rats. However, in the value of the total tumor growth rate for different groups, statistically significant differences were revealed, showing the adjuvant activity of polymerized hemoglobin, which increases the effect of doxorubicin, compared with the administration of doxorubicin alone.

According to the results of the histological evaluation of tumor sections, all the main characteristics of the growth of glioblastoma 101/8, which determine the course of the tumor formation process, were noted. Compared with untreated animals, the intensity of cellular renewal of glioblastoma 101/8 was higher in animals treated with doxorubicin, the effect was more pronounced due to an increase in the intensity of cell death. There were no differences in the number of mitoses between the groups.

As a result of the studies, it was found that the proposed adjuvant agent based on polymerized hemoglobin has the function of a modifier of the effectiveness of chemotherapy for malignant tumors. Intravenous administration of polymerized hemoglobin may have an adjuvant effect during chemotherapeutic treatment of glioma/glioblastoma.

Thus, the modifying chemotherapeutic effect was achieved by introducing the following doses into the body of experimental animals:

• 228 mg/kg body weight polymerized hemoglobin,

• 456 mg/kg body weight polymerized hemoglobin.

The dose of polymerized hemoglobin prescribed for administration varies depending on many factors, such as age, gender, body weight, symptoms and severity of the disease, the route of administration of the chemotherapy drug in which it is prescribed.

Usually, the total prescribed dose is from 114 mg/kg to 456 mg/kg per day. The total drip dose of the active substance can be divided into several doses, for example, for administration from 1 to 4 times. An effective therapeutic dose of the drug can be selected by the attending physician.

Claims (1)

The use of polymerized hemoglobin in the form of a lyophilizate for the preparation of a solution for infusion as a means of enhancing the effect of chemotherapeutic drugs in the treatment of brain tumors.

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