RO128486B1 - Polyphenol extract for veterinary use, to be used as adjuvant in anti-cancer chemotherapy - Google Patents

Abstract

The invention relates to a product with antioxidant properties, used as adjuvant in cytostatic treatment in animals. According to the invention, the product consists of a polyphenolic concentrate extracted from a vegetal material consisting of fruits of blueberry (Vaccinium mytillus), sea buckthorn (Hippophae rhamnoides), hawthorn (Crataegus monogyna), aerial parts of tutsan (Hypericum perforatum) and celandine (Chelidonium majus), in a mass ratio of 2 : 1 : 1 : 0.5 : 0.5 having a polyphenol concentration of 5 g gallic acid equivalent/100 ml and a saccharose content of 40%.

Description

The invention relates to a polyphenolic extract for veterinary use, used as an adjuvant in anti-cancer chemotherapy.

From the in vitro studies presented in the literature, it is known that polyphenols have the ability to annihilate reactive oxygen species (hydroxyl radical, superoxide anion, hydrogen peroxide, singlet oxygen, hypochlorite antonus) and reactive nitrogen species (nitric oxide). , inhibits the oxidation process of unsaturated poly fatty acids, acts as chelators of transition metal ions, especially Fe ²⁺ ion, considered a hydroxyl radical generator in the Fenton reaction.

In vivo Zn studies, namely, experiments in rats have shown that, at the liver level, polyphenols enhance the activity of antioxidant enzymes catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), increase the level of reduced glutathione and decrease the concentration of compounds that reacts with thiobarbituric acid (malondialdehyde MDA).

It is known that the resistance of tumors to cytostatic action is the main cause of failure and inefficiency of cytostatic therapy with chemotherapy or concomitant with several anticancer drugs. This resistance, congenital or acquired, is known in the specialty literature as multidrug resistance (Many Drug Resistence - MDR).

Currently, strategies are being sought to avoid, prevent or overcome the installation of multidrug resistance. Among the strategies addressed we mention the prevention of MDR during therapy, by using several cytostatic, alternatively and at maximum tolerated doses, as well as the administration of adjuvants, including natural antioxidants.

Thus, known from US patent application 2011/0136751 A1, polyphenolic compounds and their use for the treatment or prevention of cancer in a subject, as well as methods for administering an effective amount of polyphenolic compounds. Said polyphenolic compounds include petrostylbenzene, resveratrol, catechin, gallic acid, caffeic acid, anthocyanin, quercetin and any combination thereof.

Also known from patent application WO 03057201 A2, a pharmaceutical nutrient formulation and its use for cancer treatment, more specifically are disclosed in pharmaceutical formulations based on polyphenols selected from the group consisting of epigalocatechin gallate, epicatechin gallate and catechin.

The known solutions of the prior art have the disadvantages below.

Data from the literature, regarding the effect of polyphenols on the antioxidant status of the animals undergoing chemotherapy, on the multidrug resistance, as well as on the reduction of the toxicity of the chemotherapies, are insufficient. The researchers studied either the antioxidant character of some plant extracts, the effect of multidrug resistance or the toxicity of cytostatic chemotherapies. The 3 major problems in anticancer therapy have not been studied in interaction.

The technical problem that the invention solves is to improve the antioxidant status of the animals undergoing chemotherapy, with the help of a phytotherapeutic composition with adjuvant effect.

Thus, the object of the invention consists in obtaining and using a natural product, consisting mainly of polyphenols, with a strong antioxidant effect.

This product has two major actions:

1. The annihilation of the free radicals arising from the action of oxidative stress, instituted by the mental state, the drug treatment, the processes of multiplication of the malignant cells, as well as the chelation of the ions of the transition metals (antioxidant action),

RO 128486 Β1

In patients with different forms of cancer, it was found that the free radicals resulting from 1 different oxidative processes, as well as from the action of the cancerous antibodies, induce oxidative stress, which generates secondary processes, such as the change of the permeability of 3 cell membranes. These oxidative processes affect the health of the patients, causing it to worsen, or, in the case of chemotherapy, they may contribute to the installation of resistance to the administered drug.

Studies have shown that antineoplastic agents, due to their toxicity, 7 reduce the body's ability to annihilate free radicals and, as a result, promote lipid peroxidation and fatty acid levels in cell membranes. The 9 peroxidation processes in the phosphoJipids of the cytomembrane constitution affect the cytotransmembrane transport functions, affecting their permeability. 11

2. Modulation of the activity of some proteins and enzymes involved in the processes of transport and metabolism of the cytostats. 13

The invention consists of a polyphenolic extract for veterinary use, used as an adjuvant in the treatment with cytostatic chemotherapy in animals, which consists of a poly-phenolic concentrate extracted from a plant material consisting of blueberry fruit (Vaccinium myrtillus), fruit of heather (Hippophae rhamnoides), hawthorn fruit (Crataegus monogyna), aerial parts of Stingrays (Hypericumperforatum) and aerial parts of rattlesnake (Chelidonium majus), in mass ratio 2: 1: 1: 0: 0.5: 0.5, with a polyphenol concentration of 5 g equivalent gallic acid / iOO ml and a sucrose content of 40%.

The polyphenolic extract according to the invention is conditioned as a natural product in 21 syrup form, having a polyphenol content of 5 g gallic acid equivalent (EAG) / 100 ml.

The polyphenolic extract according to the invention has the following advantages: 23

- it has antioxidant activity (it annihilates the free radicals generated by cancer disease and chemotherapy) and regulates the activity of antioxidant enzymes; 25

- reduces cytostatic toxicity;

- reduces the chemoresistance of tumors; 27

- has hepatoprotective effect;

- does not interfere with the metabolic line of other drugs; 29

- is non-toxic and has no adverse effects.

The chosen form of presentation of the preparation, respectively, alcoholic syrup, has as an advantage 31 the efficient absorption in the digestive tract of the active substances contained, ensuring an increased bioavailability of the active principles associated in this product, as well as the easy administration in animals 33, taking into account the fact that the preparation is for veterinary use.

The selection of the mentioned plants is supported by the existing data in the 35 specialized literature. Thus, it is known that blueberries are rich in polyphenols (flavonoids and anthocyanins) with a strong antioxidant effect, which provides protection to liposomal membranes against oxidative processes. Anthocyanins from blueberries have a hypoglycemic, cardioprotective, neuroprotective, anti-inflammatory, genoprotective and anticancer effect. Studies have shown that polyphenols extracted from seafood have a strong antioxidant, anti-inflammatory and anti-proliferative effect in colon and liver cancer. Polyphenols extracted from hawthorn fruits 41 have antioxidant, hepatoprotective, gastroprotective, diuretic, radioprotective, immunostimulatory, anticancer and antidepressant effect. The aerial parts of the ringworm are rich in flavonoids, 43 routinely, quercitin, kaempferol and hypericin. These polyphenolic compounds have antioxidant, antidepressant and anticancer activity. The aerial portions of the aftertaste contain 45 polyphenols and alkaloids with therapeutic properties. Apigenin, polyphenol extracted from rostopască, exhibits anti-carcinogenic, antioxidant activity and modulates multidrug resistance. 47

RO 128486 Β1

The natural product made according to the invention does not interfere with the metabolic line of other medicines, has no side effects and does not cause adverse reactions. The recommended dose is between 5 and 10 mg EAG poJifenol / kg.

The following is an example of an embodiment of the invention.

Example of product realization

The vegetal material used consists of blueberries (Vaccinium myrtillus), seafood (Hippophae rharnnoides), hawthorn fruit (Crataegus monogyna '), aerial parts of hummingbirds (Hypericum perfo / atum) and aerial parts of redcurrant (Chel), chelpea (Chel), mixed in a ratio of 2: 1: 1: 0.5: 0.5 (m / m / m / m / m) - mass ratio, in the dry and ground state. The plant material is subjected to extraction by stirring at reflux for 3 hours, with 60% ethyl alcohol. After cooling, the extraction solution is separated by filtration at normal pressure. Once obtained, the extraction solution is concentrated under low pressure (150 mm col. Hg), until the ethanol is completely evaporated. The residue obtained is dissolved in a 40% sucrose solution, prepared in 60% ethanol, up to a concentration in polyphenols of 5 g equivalent gallic acid (EAG) / 100 ml.

An example of the use of the product is given below.

Example of using the product

The use of the natural polyphenolic extract product according to the invention is made in chemotherapy treatment regimens in dogs with breast tumors.

Pharmaco-clinical tests. The experimental protocol that was applied in the case of puppies with mammary tumors caught in the stages of evolution and T ₂ is presented in table 1.

Table 1

Scheme of treatment with cytokines and polyphenolic extract, according to the invention, in dogs with mammary tumors caught in stages TI and T2

the weekly hand Treatment with chemotherapies and polyphenolic extract according to the invention Chemotherapy treatment chemotherapy Polyphenolic extract according to the invention (mg EAG / kg) chemotherapy Polyphenolic extract according to the invention (mg EAG / kg) 1 Cyclophosphamide - 50 mg / m ² / day, 4 consecutive days Covinan (proligeston) -1 ml (100 mg) / 10 kg, first day administration 5 mg / kg daily Cyclophosphamide - 50 mg / m ² / day, 4 days in a row Covinan (proligeston) -1 ml (100 mg) / 10 kg, first day administration 2 Pause 5 mg / kg daily Pause - Surgery (mastectomy) 3 Pause 5 mg / kg daily Pause - 4 Pause 5 mg / kg daily Pause - 5 Holoxane (ifosfamide) - 200 mg / m ² / day, 2 days in a row 5 mg / kg daily Holoxane (ifosfamide) - 200 mg / m ² / day, 2 days in a row -

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Table 1 (continued) 1

the weekly hand Treatment with chemotherapies and polyphenolic extract according to the invention Chemotherapy treatment chemotherapy polyphenolic extract according to the invention (mg EAG / kgj chemotherapy extract polyphenolic conformable of the invention (mg EAG / kg) 6 Gemcitabine - 200 mg / m ² one dose 5 mg / kg daily Gemcitabine - 200 mg / m ² one dose 7 Pause 5 mg / kg daily Pause - 8 Pause 5 mg / kg daily Pause - 9 Pause 5 mg / kg daily Pause - 10 Holoxan (ifosfamide) - 200 mg / nT / day, 2 days in a row 5 mg / kg daily Holoxane (ifosfamide) 200 mg / m ² / day, 2 days in a row 11 Gemcitabine - 200 mg / m ² one dose 5 mg / kg daily Gemcitabine - 200 mg / m ² one dose

improving the antioxidant status under the influence of the natural product based on 23 polyphenolic extracts according to the invention

The administration of the polyphenolic preparation in dogs with mammary tumors, treated with cytostatics 25 and subjected to mastectomy (according to table 1), resulted in increased levels of erythrocyte antioxidant enzymes superoxide dismutase (r-SOD), catalase (r-CAT), glutathione peroxidase (r-27).

GPx), decreased erythrocyte malondialdehyde concentration (r-MDA) and increased glutathione concentration (r-GSH), In dogs that did not benefit from polyphenolic extract 29 preparation, according to the invention, the activity of erythrocyte antioxidant enzymes (r-SOD, r-CAT, r-GPx) decreased, and the level of malondiaidide increased. A significant decrease was also recorded in the level of reduced glutathione from erythrocytes (Table 2). These results demonstrate a decrease in the level of lipid oxidation process at the level of the erythrocyte membrane, 33 and the improvement of antioxidant status in the animals in which the polyphenolic extract was administered. 35

RO 128486 Β1

Table 2

Activity of redox superoxide dismutase (r-SOD), erythrocyte catalase (r-CAT), glutathione erythrocyte peroxidase (r-GPx), concentration of red cell malondialdehyde and reduced glutathione (r-GSH)

Custom mammary dogs, treated with cytostatics and polyphenolic extract according to the invention Dogs with mammary tumors, treated with cytostatics r Sod (U / g Hb) γ- ΟΑΤ (U / g Hb) r GPx (U / g Hb) r MDA (pmol / g Hb) r GSH (Mg / g Hb) r SOD (U / g Hb) Γ- ΟΑΤ (U / g Hb) r GPx (U / g Hb) r MDA (prnol / g Hb) r GSH (Mg / g Hb Control 125.3 400.2 39.3 0.31 3.51 125.3 400.2 39.3 0.31 3.51 Day 0 (before treatment) 99.5 378.2 30.9 0.47 2.14 88.5 380.2 27.1 0.55 2.11 week 2 99.4 377.3 31.0 0.47 2.01 89.1 380.3 27.5 0.57 1.92 week 6 101.3 387.1 31.9 0.41 1.97 89.1 380.2 27.1 0.46 1.91 week 7 101.2 388.9 32.1 0.39 2.28 87.3 372.5 26.7 0.48 1.78 week 11 109.7 390.0 34.1 0.35 2.43 84.2 364.5 26.0 0.65 1.65

“The values presented are the average of 5 determinations

The hepatoprotective effect of the natural product based on polyphenolic extract according to the invention

The introduction in the treatment scheme of the polyphenolic extract product according to the invention, according to table 1, has led to the reduction of the toxic effects of cytostatic effects on the liver. Due to the hepatoprotecfor effect exerted by the polyphenolic extract product according to the invention, the activity of serum enzymes alkaline phosphatase (ALP), aspartate-aminotransferase (AST), alanyl-aminotransferase (ALT) and lactate-dehydrogenase (LDH) has been improved. In dogs that did not benefit from the polyphenolic extract according to the invention, the activity of the mentioned serum enzymes increased, which demonstrates the intensification of the hepatic cytolysis process (Table 3).

Thus, it is observed that the values of the activity of these enzymes are increased 3 ... 4 times compared to the normal ones, in the case of patients with mammary tumors treated only with cytostatics, as opposed to the patients who were administered, besides cytostatics, and plant polyphenolic extracts, at which the activity values of these enzymes are above the normal limits, reaching only up to double the normal values, and this only for certain parameters. These observations indicate that polyphenols extracted from plants, by their antioxidant action, reduce the hepatic toxicity of cytostatic chemotherapies.

RO 128486 Β1

Table 3 1

Activity of hepatic cytolysis marker enzymes: alkaline phosphatase (ALP), aspartate transaminase (AST), alanyl transaminase (ALT) and lactate dehydrogenase (LDH) 3 in breast cancer patients

Dogs with mammary tumors, treated with cytostatics and polyphenolic extract according to the invention Dogs with mammary tumors, treated with cytostatics ALP (U / l) AST (U / l) OTHER (U / l) LDH (U / l) ALP (U / l) AST (U / l) OTHER (U / l) LDH (U / l) Control 88.5 21.0 27.9 148.3 88.5 21.0 27.9 148.3 day 0 (before treatment) 93.2 24.2 34.2 238.5 96.9 29.6 42.7 265.2 week 2 107.2 28.9 47.5 245.2 115.6 39.2 61.3 297.6 week 6 142.6 34.5 76.5 294.2 137.5 53.2 96.2 324.7 week 7 140.2 40.1 90.7 307.2 179.2 65.4 120.5 370.5 week 11 135.4 41.2 72.5 320.5 210.2 95.6 131.2 440.7

'The values presented are the average of 5 determinations 17

Reducing the multidrug resistance under the influence of the natural polyphenolic extract 19 according to the invention

As an important result obtained following the therapeutic strategy addressed, a regression of the tumor was observed clinically 21, respectively, a clinical remission from stage T ₄ or T ₃ to stage T ₂ or T _v After treatment with cytostatics associated with the poiifenolic mix, in one of tumor cases 23 involved from the T ₄ N ₂ M ₁ stage to the T ₂ N ₁ M _{X stage} , the blood tests revealing a slight remission of the hematological constants and the enzymes studied. 25

Claims (4)

1 Claim 3 Polyphenolic extract for veterinary use, used as an adjuvant in the treatment with cytostatic chemotherapy in animals, characterized in that it consists of a polyol concentrate5 nolic extracted from a plant material consisting of berries (Vacciniummyrtillus'), seafood (Hippophae) rhamnoides), hawthorn (Crataegus monogyna), aerial parts 7 Stingrays (Hypericum perforatum) and aerial parts of rattlesnake (Chelidonlum majus), in mass ratio 2: 1: 1: 0.5: 0.5, with a concentration in polyphenols of 5 g equivalent acid 9 gallic / 100 ml, and a sucrose content of 40%.