RU2721771C1 - Use of composition of copper oxide nanoparticles and n-acetylcysteine for death induction of cells of chronic myeloid leukaemia - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: present invention relates to use of a pharmaceutical composition containing nanoparticles of copper (II) oxide (CuO) and N-acetylcysteine in an effective amount for inducing cell death of chronic myeloid leukaemia.EFFECT: present invention provides efficacy of the composition with respect to inducing cell death of chronic myeloid leukaemia of various types, regardless of their resistance to other anti-tumour preparations, for example, BCR-ABL inhibitors, cooperative resistance.4 cl, 4 dwg, 3 ex

Description

The present invention relates to the field of pharmaceuticals and medicine, and more particularly to a composition intended for inducing cell death of chronic myeloid leukemia, including CuO (divalent copper oxide) nanoparticles and N-acetylcysteine.

Chronic myeloid leukemia (CML) is a malignant tumor of white blood cell cells. In the pathogenesis of CML, the most important role is played by a genetic event - translocation of the sites of the 9th and 22nd chromosomes. The confluent (chimeric) protein BCR-ABL resulting from genetic rearrangement is a tyrosine kinase that phosphorylates substrate proteins; Intracellular signaling pathways are activated. BCR-ABL stimulates accelerated cell cycle phase change, cell proliferation and survival. In myeloid differentiation cells, signaling by BCR-ABL leads to malignancy.

Initially, busulfan, hydroxyurea, and interferon, non-specific cytostatics, were used to treat CML, but such treatment was accompanied by pronounced general resorption toxicity and low five-year survival.

Currently, more selective agents have been developed and used for CML treatment: BCR-ABL inhibitors. Therapy has acquired a directed character (targeted therapy). The first drug to change the approach to treatment was imatinib (STI571, Gleevec®). Its use in the early phases of the disease led to complete remission in 98% of cases (Druker B, Tamura S, Buchdunger E, et al. Effects of a selective inhibitor of the Ab1 tyrosine kinase on the growth of Bcr-Ab1 positive cells. Nature Medicine 1996 ; 2 (5): 561-566).

However, the effectiveness of imatinib in the later phases of the disease dropped to 34% and 31% for the acceleration phase and blast crisis, respectively (Kerr D. J. et al. (Ed.). Oxford textbook of oncology. - Oxford University Press, 2016.).

A frequent occurrence with the use of imatinib was a relapse of the disease after 10-12 months. In these situations, the tumor cells become imatinib resistant. Two mechanisms of such resistance were revealed: 1) mutations in the ABL kinase domain, leading to a change in the structure of the polypeptide, which prevents the successful binding of the inhibitor to the target; 2) activation of other signaling mechanisms for maintaining the tumor phenotype.

To combat the mutational resistance of tumor cells, the following generations of inhibitors were created: dasatinib (BMS-354825, Spryce®) and nilotinib (AMN107; Tasigna®), which are active for most mutant forms of BCR-Ab1 with the exception of replacing the threonine residue with isoleucine at position 315 ( T315I). Only with the advent of ponatinib (AP24534; Iclusig ^® ), a 3rd generation drug, was it possible to obtain a drug that works for a tumor with T315I.

These drugs are effective in case of drug resistance caused by genetic mutations of the Ab1 tyrosine kinase. However, if the resistance of tumor cells is due to an epigenetic event, activation of other intracellular signaling pathways, in addition to Bcr-Ab1, is not sufficient to use Bcr-Ab1 inhibitors. Such resistance can be caused, in particular, by the cooperation of tumor cells and signals from the microenvironment that support the malignant potential.

Thus, the problem of creating compounds or their combinations capable of overcoming cooperative resistance in chronic myeloid leukemia remains unresolved due to insufficient knowledge of its mechanism.

They tried to solve this problem in various ways. The use of busulfan and hydroxycarbamide is known. These drugs were used before the invention of tyrosine kinase inhibitors, but caused significant side effects - pulmonary fibrosis, prolonged bone marrow aplasia. Currently, such therapy is used in very rare cases as a inhibiting factor (induction) for the tumor before the introduction of inhibitors.

Interferon-alpha (IFNα) was also used in clinical practice, but later gave way to imatinib as a drug with minimal general resorption toxicity. The importance of IFN is considered for combination treatment, however, no significant results have been obtained so far.

The authors of the present invention associate the solution of the above problem with the use of metal nanoparticles in combination with N-acetylcysteine.

The use of metal nanoparticles in the treatment of tumors is known in the art. For example, the authors (Jin Y. et al. Hyaluronic acid modified tantalum oxide nanoparticles conjugating doxorubicin for targeted cancer theranostics // Bioconjugate chemistry. - 2015. - T. 26. - No. 12. - P. 2530-2541; Podsiadlo P. et al. Gold nanoparticles enhance the anti-leukemia action of a 6-mercaptopurine chemotherapeutic agent // Langmuir. - 2008. - T. 24. - No. 2. - C. 568-574.) it is shown that the use of nanoparticles as transporters increases the effectiveness of chemotherapeutic agents. However, in the case of drug resistance, this may not be enough, since an antitumor agent proper is required that can overcome the molecular mechanism of resistance.

There are also works in which the introduction of metal nanoparticles (copper, zinc, iron or gold) leads to a slowdown in tumor growth and its regression (RF Patent No. 2417942 from 09/25/2009; RF Patent No. 2392668 from 12/15/2008). The difficulty lies in the fact that the toxic effect is manifested in rather high concentrations, which inevitably affect the functioning and viability of non-tumor cells (general resorption toxicity).

The present invention is directed to solving this problem and eliminating the disadvantages inherent in previously known solutions.

The claimed composition is intended for the induction of CML cell death, including CuO nanoparticles and N-acetylcysteine (NAC) as active agents in effective amounts.

Moreover, this composition is also effective against cells of chronic myeloid leukemia with drug resistance. Such resistance may be due to mutations in the ABL kinase domain. Drug resistance can also be caused by the activation of other signaling mechanisms for maintaining the tumor phenotype, in particular, by the cooperation of tumor cells and signals from the microenvironment that support the malignant potential.

The effectiveness of the composition with respect to various types of CML is due to the fact that copper oxide (CuO) nanoparticles exhibit a cytotoxic effect due to the generation of oxidative stress, which is significantly enhanced in the presence of NAC and leads to the death of tumor cells regardless of individual genetic events (mutations) or intercellular interactions.

N-acetylcysteine (NAC) is a derivative of the amino acid cysteine with an acetyl group at the nitrogen atom. In domestic pharmacology, NAC is used for the treatment and prevention of inflammatory diseases of the respiratory tract. Antioxidant properties are realized due to the presence of a thiol (-SH) group.

The technical result of the invention is the effectiveness of the composition with respect to the induction of the death of CML cells of various types, regardless of whether they have resistance to other antitumor drugs, for example, to BCR-ABL inhibitors, cooperative resistance. Drug resistance may be due to mutations in the ABL kinase domain or activation of other signaling mechanisms for maintaining the tumor phenotype, in particular, cooperation of tumor cells and signals from the microenvironment that support the malignant potential.

The combined (combination) use of CuO and NAC nanoparticles leads to a synergistic effect: NAC does not cause death of CML cells at reasonable concentrations, and the use of CuO nanoparticles as an independent antitumor agent is ineffective, since they act in a high concentration range, having a negative effect on non-tumor cells. The proposed composition is effective in the low concentration range of each component.

The sizes of the CuO nanoparticles used in the present invention preferably range from 20 to 110 nm.

For example, the average nanoparticle size may be 80 ± 20 nm.

The concentration of nanoparticles in the composition may vary from 0.1 to 50 μM, preferably from 0.625 to 5 μM.

The NAC used in the present invention may be N-acetyl-L-cysteine, N-acetyl-D-cysteine, or a mixture thereof.

The concentration of NAC in the composition may vary from 0.1 to 50 mM, preferably from 0.156 to 5 mM.

The composition may contain at least one pharmaceutically acceptable carrier.

Such a carrier can be water, an isotonic solution or a buffer solution (phosphate, acetate-ammonia, etc.), or a solution of glycerol in water up to 30%. Image List:

Fig. 1. The survival of cell culture K-562 (chronic myeloid leukemia) of various densities during incubation with different concentrations of the tyrosine kinase inhibitor BCR-ABL PF-114.

Fig. 2. Cell survival (%) upon addition of CuO nanoparticles in mono-exposure (A), NAC 2.5 mM (B) and CuO + NAC combination (C). The cell culture density is 25 thousand / ml.

Fig. 3. Comparison of IC ₅₀ for CuO and CuO + NAC at different cell culture densities.

Fig. 4. Annexin-PI staining for cells supplemented with CuO + NAC:

A: After 4 hours. B: 6 hours. At: 24 hours.

Examples:

1. Obtaining nanoparticles

CuO nanoparticles were synthesized by precipitation using CuSO ₄ * 3H ₂ O as a precursor. It was dissolved to a concentration of 0.02 M in 50 ml of distilled water, heated to 80-90 ° C with constant vigorous stirring, and 2 ml of 0.1 M NaOH was added. A sign of a successful reaction was a change in the color of the solution from bright blue to black. Then the precipitate was washed in ethanol and water. Particle size (80 ± 23 nm) is determined by dynamic light scattering measurements using a particle size analyzer.

2. Preparation of the composition

The preparation of the composition was carried out immediately before use directly in the culture medium: RPMI with the addition of 5% fetal calf serum and 0.5% gentamicin. 0.2 ml of a solution of CuO nanoparticles at a concentration of 0.14% (1.4 μg / ml) and 5 μl of N-acetylcysteine - 500 mM were added to 2 ml of the medium containing cancer cells.

3. Induction of CML cell death

CML resistance to BCR-ABL inhibitors is modeled by culturing CML cells (K562 line) in an increased density (4 × 10 ⁵ cells in 1 ml of culture medium), since the possibility of paracrine intercellular interactions and the emergence of “cooperative” resistance are increased in a dense culture (Fig. 1 )

In mono-exposure, CuO nanoparticles exhibited a moderate toxic effect with respect to CML cells (line K562). The addition of NAC increased the toxicity of copper oxide nanoparticles by 2–3 orders of magnitude (Fig. 2).

The cytotoxicity of CuO, NAC, and combinations thereof were evaluated in an MTT assay. K562 cells were scattered into 96-well plates of 5 × 10 ³ or 80 * 10 ³ in 180 μl of RPMI medium supplemented with 5% fetal calf serum and 0.5% gentamicin. NAC and CuO at various concentrations were added to the wells. The cultures were incubated for 72 hours at 37 ° C, 5% CO ₂ in a humidified atmosphere. At the end of the incubation, 20 μl (100 μg) of an aqueous solution of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT; 5 mg / ml) was added to the wells, incubated for 2 hours in the same conditions, the medium was removed, the cells were resuspended in 100 μl of dimethyl sulfoxide, and the optical density of the solution was measured at a wavelength of 570 nm. The percentage of cells that survived under the action of each dose of the test compound was calculated as the quotient of the division of the optical density in the wells (average value of 3 measurements) after incubation with this concentration to the average optical density of the control wells (the values of the latter were taken as 100%).

As a result, it turned out that at a cell culture density of 25,000 pcs / ml, the IC50 (the concentration of a substance at which half of the cell population dies) for CuO nanoparticles is about 67 μM, and when NAC is added at a concentration of 2.5 mm it decreases to 0.1125 μM, those. toxicity increases 520 times. In a dense culture (400 thousand / ml), these values increase somewhat: for CuO nanoparticles in a single action, up to 126 μM; combinations up to 1 μM at the same NAC concentration (Fig. 3). However, firstly, the difference in concentrations remains high compared with mono-exposure, and secondly, BCR-Ab1 inhibitors do not cause tumor cell death under similar conditions, unlike the combination of CuO + NAC.

To elucidate the mechanism of tumor cell death, staining with annexin V conjugated with fluorescein isothiocyanate (FITC) and propidium iodide (PI) fluorescent in the phycoerythrin channel (PE) was used. Apoptosis (fluorescence in the FITC channel), necrosis (fluorescence in the PE channel), or a combination of death mechanisms (luminescence in both channels) are judged by the luminescence of cells in one or another channel. 4 hours after the addition of the combination (0.7 μg / ml CuO; 2.5 mM NAC) to the cell culture, an increase in the luminous intensity was observed in the FITC channel, but not in the FE channel. After another 2 hours, most of the cells fluoresced in the FITC channel, and part of the population in the FE channel. After 24 hours, almost all cells fluoresced in both channels (Fig. 4). This means that the initial apoptosis of the cells is supplemented by a necrotic component - damage to the integrity of the plasma membrane; cells get unrepairable damage.

In addition, the addition of a caspase inhibitor Z-VAD did not affect survival, which indicates independence of death from caspase activity and makes the composition effective against various types of CML.

Claims (4)

1. The use of a pharmaceutical composition comprising nanoparticles of copper oxide (II) (CuO) and N-acetylcysteine in an effective amount for the induction of cell death of chronic myeloid leukemia. 2. The use of the pharmaceutical composition according to claim 1, wherein N-acetylcysteine is N-acetyl-L-cysteine, N-acetyl-D-cysteine, or a mixture thereof. 3. The use of the pharmaceutical composition according to claim 1, containing at least one pharmaceutically acceptable carrier. 4. The use of the pharmaceutical composition according to claim 3, wherein the carrier is water, an isotonic solution, a buffer solution or a solution of glycerol in water up to 30%.

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