WO2022175858A1 - Dicarboximide derivative for use in cancer treatment - Google Patents
Dicarboximide derivative for use in cancer treatment Download PDFInfo
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- A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
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- A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
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Definitions
- the present invention relates to derivative of dicarboximide for use in cancer treatment, in particular in the treatment of leukemias (e.g. chronic myeloid leukemia (CML) or acute myeloid leukemia (AML)) and other diseases characterized by clonal hyperplasia and impaired proliferation of white blood cells, such as leukemias.
- leukemias e.g. chronic myeloid leukemia (CML) or acute myeloid leukemia (AML)
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- pharmaceutically acceptable formulations have been developed enabling the use of the active ingredient in liquid compositions, their sterilization and stable storage.
- the compound to be used according to the invention can be used as monotherapy or in combination treatment, e.g. together with a tyrosine kinase inhibitor, such as imatinib.
- Leukemias remain an unsolved problem of clinical medicine and pharmacology in the 21 st century. Even though a number of clinically useful anti-cancer compounds are available on the market, many problems remain without an effective solution.
- the main challenges in leukemia therapies are the shortage of drugs that specifically act on cancer cells and do not damage normal cells, and that have no negative side effects.
- a big problem is development of multidrug resistance which desensitizes cancer cells to current chemotherapeutics, and shortage of drugs effective against cancer-initiating stem cells, which can be an origin of cancer recurrence.
- anticancer drugs with a better pharmacological profile in relation to those currently used.
- CML chronic myeloid leukemia
- the effect of translocation is the transfer of the ABL oncogene (located on chromosome 9) to a strictly defined breaking site (breakpoint cluster region-BCR) on chromosome 22 and the formation of the fusion BCR-ABL gene.
- This gene encodes a protein that exhibits abnormal, increased activity of receptor tyrosine kinase (TK).
- BCR-ABL TK expression is the primary driving factor of neoplastic transformation in CML (3 Kurzrock 2003, 4 Colicelli 2010, 30 Thompson 2016, 31 Jabbour E 2018).
- BCR-ABL can transform myeloid progenitor cells into cancer cells and drives development in 95% of CML cases.
- BCR-ABL promotes leukemogenesis by activating further signaling proteins that increase survival and proliferation of cancerously altered cells [4 Colicelli 2010]
- These pathways include, among others, RAS/mitogen-activated protein kinase (RAF/M EK/ERK), phosphatidylinositol/AKT 3-kinase cascades (PI3K/AKT), and JAK/STAT signaling cascades [5 Steelman 2004]
- the first-line drugs approved for use in humans in CML therapy are hydroxyurea, available as Hydroxyurea Medac, among others, and Imatinib (IM), available as Glivec®, among others.
- IM binds to the kinase domain of ABL kinase and inhibits the phosphorylation of its substrates.
- IM therapy there is a significant improvement in patient survivability, especially when used at an early stage.
- CML treatment has been optimized by using therapy with compounds from the group of new generation tyrosine kinase inhibitors, having different activity against leukemic cells.
- IM and other tyrosine kinase inhibitors do not show satisfactory efficacy in the advanced stage of the disease, which is the so-called blast crisis, as well as resistance leading to relapse may develop during therapy with these compounds [6 An 2010, 7 Bubnoff 2003]. Cancer recurrence caused by clonal growth of cancer-initiating stem cells in patients after treatment also remains a problem.
- IM is a low-molecular inhibitor of protein tyrosine kinases that strongly inhibits the activity of BCR-ABL tyrosine kinase (TK) and many other receptor tyrosine kinases, e.g., Kit, a stem cell growth factor (SCF) receptor encoded by c-Kit proto-oncogene, colony-stimulating factor receptors (CSF-1 R), and 17 alpha and beta receptors for platelet-derived growth factor (PDGFR-alpha and PDGFR-beta).
- TK BCR-ABL tyrosine kinase
- Kit a stem cell growth factor (SCF) receptor encoded by c-Kit proto-oncogene, colony-stimulating factor receptors (CSF-1 R), and 17 alpha and beta receptors for platelet-derived growth factor (PDGFR-alpha and PDGFR-beta).
- SCF stem cell growth factor
- this drug does not eliminate cancer-initiating stem cells from the patient's body (11 Hamilton 2012, 12 Andersson 1979). Moreover, in about 3% of patients lack of response to IM treatment is observed, which is associated with primary drug resistance. In addition, during the IM treatment, an increase in acquired (secondary) resistance is observed, which can be observed in about 20% of patients in the chronic phase of CML.
- Threonine at position 315 forms a hydrogen bond with IM that stabilizes the enzyme-drug complex.
- Replacement of threonine by isoleucine prevents formation of the binding and consequently greatly reduces the potency of the drug (14 Apperley 2007 a, 15 Apperley 2007 b, 20 Nardi 2004, 29. Tamai 2016).
- Important mechanisms of resistance to IM also include: gene amplification within BCR- ABL, clonal evolution consisting in arising of genetic changes leading to the activation of new signal conduction pathways, the presence of a population of stem cells insensitive to the drug, the emergence of mechanisms leading to the reduction of intracellular concentration of IM (binding of the drug by alpha-1 -acid glycoprotein, reduction of hOCT 1 - human organic cation transporter - expression, increase in expression of MDR1 which is responsible for the active ejection of the drug from cells) (17 Lahaye 2005, 18 Hochhaus 2002).
- resistance may be the result of one or more mechanisms, including molecular resistance caused by a mutation in the BCR-ABL gene, or independent BCR-ABL resistance, e.g. associated with stem cell-specific survival factors, e.g. increased signaling of the RAF/MEK/ERK pathway, which is not dependent on RAS but is initiated by PKC.
- CML patients are considered resistant to IM when the response is lost or not observed after a daily dose of >400 mg imatinib (Valenti, 2007; Kantarjian etal., Blood. 101 (2): 473, 2003; Baccarani et al., Blood 108 (6): 1809-20, 2006).
- CD34(+) CML stem cells are also a reservoir of cells with mutations that confer resistance to IM (24. Sorel 2004).
- the present invention relates to the use of 4-[2-hydroxy-3-(propan-2-ylamino)propylj- 1 ,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.0 2 ' 6 ] dec-8-en-3,5,10-trione, also described as BK124.1 , in therapy of CML or AML and other leukemias.
- BK124.1 is a derivative of compounds from the group of dicarboximides.
- the synthesis of the compound was disclosed and described in the patent applications EP2687509, EP2687509, PL400000.
- the synthesis and cytotoxic activity against selected cell lines is described in the publication (23, Kuran 2016).
- the BK124.1 compound is defined by the Formula I:
- a pharmaceutically acceptable salt of the compound of formula I is: Studies performed on normal cell lines (HUVEC) as well as leukemic cell lines (e.g. HL-
- BK124.1 human leukemia cell lines: CML leukemia line (K562 cells) and AML leukemia line (HL-60 cells), and yet low cytotoxicity against other cancer lines and non-cancer cells (23 Kuran).
- the inventors of the present solution have also developed a system of solvents neutral to an organism to enable administration of BK124.1 in vivo.
- Pharmacokinetic parameters and therapeutic window parameters for BK124.1 were determined, including the route of administration of the compound to the body and the dose of the compound that conditions activity in reducing growth of cancer cells in the absence of toxicity (on the example of a mouse model of human chronic myeloid leukemia CML - immunodefective mice after xenotransplantation of human CML leukemia cells).
- BK124.1 has been shown to be highly cytotoxic against hematopoietic blood stem cells (cells that initiate cancerogenesis) obtained directly from patients with leukaemia.
- BK124.1 High cytotoxic activity of BK124.1 against multidrug resistant leukemic cells was also demonstrated. Also the molecular mechanism of cytotoxicity of the compound in human CML cells has been described (described mechanism of action of BK124.1 is therapeutically justified and clinically interesting, as confirmed in the present application by demonstrating the efficacy of BK124.1 in monotherapy and in IM-combination therapy in a mouse model).
- the BK124.1 substance is characterized by very untoward rheological properties from the technological point of view of producing oral solid as well as liquid forms of the compound. As disclosed in the patent applications EP2687509, EP2687509, PL400000, the BK124.1 substance is in the form of white non-hygroscopic powder having electrostatic properties. In addition, this compound is characterized by poor solubility in aqueous solutions. The solubility of the compound at the synthesis stage was found only for 100% DMSO (23 Kuran).
- the form of BK124.1 as a concentrate is suitable for administration, e.g. once or twice a day, and can be administered at different doses to help the patient comply with the recommendations for use as well as to facilitate the doctor to select an appropriate therapeutic dose.
- the preparation gives the possibility of controlled administration of the drug into the circulatory system and achieving the therapeutic concentration of the compound in blood during treatment.
- Another problem with a long-term therapy is the need to determine the optimal dose that the patient can tolerate. If such a dose is not established, this may lead to reduction in the effectiveness of the drug being administered.
- An aim of the present invention is therefore also providing preparations suitable for long-term administration of an active compound of formula I.
- the compound of formula I can be administered, for example, by intraperitoneal or oral or intravenous routes.
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- BK124.1 monotherapy a new type of therapy for a patient with IM-resistant CML or AML were disclosed.
- the invention provides a new method of treatment chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) or other leukemia in a mammal subject, that involves administering a combination of a BCR-ABL inhibitor and BK124.1 .
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- BK124.1 BCR-ABL inhibitor
- the invention provides an additional type of therapy for a patient with chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) or another disease characterized by a clonal increase in the number of white blood cells that is treated with a BCR-ABL inhibitor or other drug, e.g. a MEK inhibitor.
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- another disease characterized by a clonal increase in the number of white blood cells that is treated with a BCR-ABL inhibitor or other drug, e.g. a MEK inhibitor.
- the subject of the invention is therefore a compound of formula I: or its pharmaceutically acceptable salt for use in the treatment of cancer, wherein the cancer is leukemia or another cancer characterized by a clonal increase in the number of white blood cells, wherein the cancer shows resistance to previous treatments, in particular multidrug resistance.
- Resistance to previous treatment includes both primary and secondary (acquired) resistance, developing after exposure of the tumor to a given therapy.
- the cancer responds to therapy in an unsatisfactorily way or not at all, or the efficiency of therapy significantly weakens over time, which can lead to recurrence of the disease.
- Multidrug resistance is defined as the acquisition by cancer cells of simultaneous insensitivity to several groups of different therapeutic agents, which develops in response to the use of even a single anticancer agent.
- the cancer is myeloid leukemia, in particular, chronic myeloid leukemia or acute myeloid leukemia.
- the individual to be treated suffers from chronic myeloid leukemia or acute myeloid leukemia and is in the phase of blast crisis.
- leukemia is chronic myeloid leukemia or acute myeloid leukemia with multidrug resistance or resistance to imatinib.
- a compound of formula I prior to administration to the individual to be treated, is dissolved in a mixture containing at least one pharmaceutically acceptable water- soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer, and the concentrate thus obtained is then used to dilute and prepare the final form for administration, which is administered to the individual to be treated.
- the pharmaceutically acceptable water-soluble polar compound comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
- the pharmaceutically acceptable lipophilic polymer comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
- the compound of formula I prior to administration to the individual to be treated, is dissolved in a mixture containing ethanol and macrogol-15- hydroxy stearate (e.g., Solutol® HS 15), the resultant concentrate is then used to dilution and preparation of the final formulation which is administered to the individual to be treated.
- a mixture containing ethanol and macrogol-15- hydroxy stearate e.g., Solutol® HS 15
- the prepared concentrate is diluted in an aqueous solution, and then sterilized, the resultant diluted form of the drug is administered to the individual to be treated.
- a compound of formula I is administered orally or intraperitoneally in a mixture containing ethanol 10% : macrogol-15-hydroxystearate 10%.
- ethanol 10% macrogol-15-hydroxystearate 10% refer to a mixture obtained by diluting the concentrate of dissolved compound of formula I, e.g. in water (such as water for injection) or in physiological salt solution.
- the resulting concentrate diluted in an aqueous solution is then re-diluted, e.g. in a 5% glucose solution or a 0.9% sodium chloride solution, and the resulting formulation is administered intravenously.
- the compound of formula I is administered to an individual at least once a day, at a daily dose in the range of 10-30 mg/kg body weight.
- the individual to be treated has previously undergone therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
- another BCR-ABL kinase inhibitor was imatinib.
- a compound of formula I is used as monotherapy.
- the compound of formula I is used in combination with therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
- another BCR-ABL kinase inhibitor is imatinib.
- the present inventors have shown that it is also effective to use the compound of formula I together with imatinib. This implies that for patients who have previously been treated with this compound and who develop or may develop resistance to treatment, it is possible to introduce treatment with the compound of formula I without withdrawing imatinib therapy.
- the compound of formula I can be used in an individual in need thereof, preferably in mammals, while particularly preferably, an individual is a human.
- the subject of this disclosure is also a new formulation of the compound of formula I, which allows not only its sufficiently effective dissolution, in a solvent system that is pharmaceutically acceptable, but to obtain a solution that can be sterilized by filtration at the same time.
- the compound of formula I is a substance that is hardly soluble.
- solubility was observed only in 100% solvent.
- the solubility obtained was already unacceptable in the case of a small dilution of DMSO with water.
- a compound of formula I before administration to the individual to be treated, is dissolved in a mixture containing at least one pharmaceutically acceptable water-soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer, preferably wherein the pharmaceutically acceptable water- soluble polar compound is ethanol and the pharmaceutically acceptable lipophilic polymer is macrogol-15-hydroxystearate, and the concentrate thus obtained is then used to dilute and prepare the final form for administration, which is administered to the individual to be treated.
- BK 124.1 a method of formulating BK 124.1 into a concentrate which can be then diluted and administered, e.g., intraperitoneally or orally, or after further dilution also, e.g., intravenously, as by infusion, or it can be used to make another pharmaceutical form.
- the first step of formulating according to the preferred embodiment of the invention consists in dissolving BK124.1.
- a dose thereof preferably in the range of up to 3 mg (optimally 1 mg) is measured into a transparent, glass or plastic vial, then a mixture of a water-soluble polar compound and a lipophilic polymer is added, with the water- soluble polar compound used in the amount of 40-60% and the lipophilic polymer used in the amount of 40-60%.
- a water-soluble polar compound and a lipophilic polymer can be in a ratio of 50%/50%.
- a mixture used to dissolve a compound of formula I is obtained by mixing Solutol HS 15 and anhydrous ethanol 99.8%, e.g. so as to obtain a solution of 50% Solutol HS 15/ 50% ethanol.
- the mixture is gently shaken for completely wetting the drug product.
- the vial is rotated/shaken for a period of time (e.g. several minutes or e.g. 1-2 hours) to obtain a transparent solution.
- the vial can be heated on a water bath at 30-40°C.
- the solution prepared is carefully inspected to ensure that the product has been dissolved and that the solution does not contain particulate matter. It is preferable to use clear and colorless solutions, without particulate matter.
- BK124.1 concentrate thus prepared is colorless.
- the second step involves dilution of the BK124.1 concentrate obtained in the first step using a compatible solvent, such as, e.g. water for injection, aqueous solution, saline, physiological multi-electrolyte solution, 5% glucose solution, saline mixture with 5% glucose solution in a ratio of 2:1 , lactated and non-lactated Ringer's solution, and their modifications, etc.
- a compatible solvent such as, e.g. water for injection, aqueous solution, saline, physiological multi-electrolyte solution, 5% glucose solution, saline mixture with 5% glucose solution in a ratio of 2:1 , lactated and non-lactated Ringer's solution, and their modifications, etc.
- the concentrate is diluted so that in the resulting solution the content of the water-soluble polar compound and the lipophilic polymer is 10% or less, respectively.
- the concentrate solution thus obtained can be sterilized, for example, by filtering on a 0.2 pm filter.
- stability studies of the prepared concentrate were carried out and it was found that the form prepared in this way allows the concentrate to be stored for at least 30 days without affecting its parameters.
- An additional advantage of the concentrate is the ease of selecting its doses, which is important for therapeutic applications.
- the solution thus obtained may be used for administration to the individual to be treated, e.g. intraperitoneally or orally.
- an additional dilution step is preferred, e.g. in 5% glucose solution or 0.9% sodium chloride solution, preferably in polyethylene, polypropylene or glassware, but not in PVC vessels. Only a clear and colorless solution should be administered.
- concentration of the compound of formula I in the solution for intravenous infusion may preferably be in the range from 0.004 mg/mL to 0.1 mg/mL.
- Dissolution of the therapeutic compound should be performed under aseptic conditions. If the prepared concentrate does not contain preservatives, it is used for single use, the unused solution should be discarded.
- preservatives such as e.g. benzalkonium chloride, thiomersal, chlorocresol, benzyl alcohol, extends its shelf life. It is also recommended to sterilize said solution, e.g. by filtering on a 0.2 pm filter.
- a pharmaceutically acceptable lipophilic polymer should constitute from 40% to 60% by weight of the entire solution, optimally 50%. At least one pharmaceutically acceptable polar compound should be present in the mixture, in a proportion of 40% to 60% by weight respectively, based on the weight of the solution, optimally 50%.
- Examples of a pharmaceutically acceptable lipophilic polymer are compounds including: N-vinyllactam homopolymer, N-vinyllactam copolymer, cellulose ester, cellulose ether, polyalkylene oxide, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl alcohol, vinyl acetate polymer, oligosaccharide and polysaccharide Solutol HS 15 (macrogol-15- hydroxystearate; Polyethylene glycol (15)-hydroxystearate); CAS No: 70142-34-6).
- the soluble form of BK124.1 thus obtained can be further dissolved in aqueous solutions such as saline. Once dissolving, these solutions can then be administered to the body of the individual to be treated, e.g. by intravenous route, or can be used to prepare another pharmaceutical form of the compound (e.g. granules, tablets, capsules).
- aqueous solutions such as saline.
- the invention encompasses development of a pharmaceutical formulation containing BK124.1 for administration once or twice a day, wherein the formulation contains the active compound BK124.1 in a liquid form.
- the developed formulation allows dosing BK124.1 , for example, at intervals of 12 or 24 hours, which is sufficient to obtain therapeutic levels of the compound in blood within 12-24 hours after administration.
- BK124.1 was the only one of the group of dicarboximide derivatives (see EP2687509, EP2687509, PL400000) for which it was possible to develop a formulation that provides not only the possibility of easy administration of the compound, but also to maintain stability and effectiveness.
- pharmaceutically acceptable refers to substances, compounds, mixtures, solutions, preparations, etc. which are generally not toxic or cause no damage to the individual to whom they are administered.
- pharmaceutically acceptable salt means the salts of the compound according to the invention which are safe and effective for topical use in mammals and which continue to retain its desired biological activity.
- Pharmaceutically acceptable salts include salts of acidic or alkaline groups.
- salts are, for example, but not limited to, hydrochloride, chloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulfate, phosphate, hydrogen phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (that is, 1 ,1'-methylene- bis-(2-hydroxy-3-naphthoate).
- FIG. 1 depicts a representative immunoblot illustrating the change in the level of selected proteins in human leukemia K562 cells treated for 4, 8, 16, 24 and 48 hours with 0.1% DMSO solution or BK124.1 at a concentration of 5 mM in 0.1% DMSO.
- FIG. 2 depicts a representative immunoblot documenting the level of FOX03A and P21 proteins in the nuclear and cytoplasmic fractions of K562 cells treated for 8 or 24 hours with BK124.1 at a concentration of 5 mM in 0.1% DMSO.
- BK124.1 a concentration of 5 mM in 0.1% DMSO.
- the purity of separated fractions documents the level of control proteins: ACTB (beta-actin) - marker of cytoplasmic fraction, LMNB1 (laminin B1 ) - marker of nuclear fraction.
- FIG. 3 shows the effect of BK 124.1 on the cell cycle analyzed by flow cytometry after staining with propidium iodide (PI).
- PI propidium iodide
- FIG. 4 presents the results of the MTT viability test for cells with multidrug resistance MDR1 of the K562-MDR1 line and for primary K562 cells (without multidrug resistance) treated with BK124.1 in a concentration range of 0.1-10 mM in 0.1% DMSO.
- the results show the mean ⁇ SD absorbance in wells with BK124.1 relative to absorbance in control wells with 0.1% DMSO for 3 independent experiments.
- FIG. 5 illustrates the effect of BK124.1 as compared to the reference compounds: taxol and vincristine on cells with K562-MDR1 multidrug resistance after 24 h incubation.
- Treatment of K562-MDR1 cells with BK124.1 results in an increase of the percentage of MDR1 -positive cells (left panel) while maintaining high cytotoxicity of this compound (right panel).
- K562-MDR1 cells were treated with 0.1% DMSO or compounds such as BK124.1 , taxol and vincristine dissolved in 0.1% DMSO at a concentration of 2xlC 5 o, respectively. The cells were stained and analyzed by flow cytometry.
- A The plot shows the average percentage of MDR1 -positive cells ⁇ SEM.
- FIG. 6 shows that BK124.1 has high cytotoxic activity and causes cell death via apoptosis regardless of the presence of MDR1 multidrug resistance in cells.
- B The plot shows the average percentage of living K562-MDR1 cells ⁇ SEM.
- C The plot shows the average SEM ⁇ for the percentage of K562-MDR1 cells in the early and late stages of apoptosis.
- FIG. 7 shows the results of the MTT viability test for CD34(+) stem cells isolated from the blood mononuclear cell fraction (PBMC) of patients with chronic myeloid leukemia (CML).
- PBMC blood mononuclear cell fraction
- CML chronic myeloid leukemia
- Cell viability was calculated on the basis of absorbance in wells with the compound relative to absorbance in control wells with 0.1% DMSO. Normal distribution was verified using the Shapiro-Wilktest, statistical analysis was performed using one-way ANOVA with post-hoc Dunnett’s test * P ⁇ 0.05, ** 0.001 ⁇ P ⁇ 0.05, *** 0.0001 ⁇ P ⁇ 0.001 , **** P ⁇ 0.0001.
- Fig. 8 shows that BK124.1 induces death of CML patient’s CD34+ stem cells via apoptosis after 24 h of incubation with 2.5 mM or 5 mM BK124.1.
- CD34+ cells isolated from a CML patient were treated for 24 hours either with a 0.1% DMSO solution or BK124.1 dissolved in 0.1% DMSO at a concentration of 2.5 mM or 5 mM, and then the number of cells in early and late apoptosis (dead cells) was determined using FITC Annexin V labeling and flow cytometry.
- the graph shows the average percentage of CD34(+) cells ⁇ SEM (A) alive and (B) in early apoptosis and (C) in late apoptosis (dead cells).
- the experiment was performed in at least 3 independent repeats, each in a technical duplicate.
- Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P ⁇ 0.05, ** 0.001 ⁇ P ⁇ 0.05, *** 0.0001 ⁇ P ⁇ 0.001 , **** P ⁇ 0.0001 .
- FIG. 9 presents the results of a pharmacokinetic study of BK124.1 in rats (Wistar breed) after 5 min, 15 min, 30 min, 1 h, 2h, 6h and 24h from a single intravenous administration of the compound at a concentration of 5 or 10 mg/kg body weight.
- the upper panel shows the average concentrations of the compound in blood ⁇ SD, the lower panel shows individual results for each rat.
- FIG. 10 illustrates no effect of the carrier used in the administration of BK124.1 (10% Solutol FIS 15 / 10% ethanol) on tumor growth in a mouse xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells).
- BK124.1 10% Solutol FIS 15 / 10% ethanol
- Experimental conditions were given in Table 2.
- Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P ⁇ 0.05, ** 0.001 ⁇ P ⁇ 0.05, *** 0.0001 ⁇ P ⁇ 0.001 , **** P ⁇ 0.0001.
- FIG. 11 shows antileukemic activity of BK124.1 in a mouse xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells) as determined by measuring tumor size at the end of the experiment. Administration of the compounds took place on days 3 - 17 after implantation of K562 cells. The experiment was completed on day 21. The diagram of the experiment was given in Table 2, and the results are summarized in Table 7. Each of the variants used was statistically significant compared to the control group, which was not treated with any compound. However, there were no statistically significant differences in tumor size for each of the compounds tested. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P ⁇ 0.05, ** 0.001 ⁇ P ⁇ 0.05, *** 0.0001 ⁇ P ⁇ 0.001 , **** P ⁇ 0.0001.
- FIG. 12 shows mean theoretical mass of the tumor observed during the experiment with BK124.1 administration in a xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells).
- the formula for the theoretical mass of the tumor and the exact diagram of the experiment are presented in Table 2.
- Administration of the compounds was carried on days 3 - 17 after implantation of K562 cells.
- the experiment was completed on day 21.
- FIG. 13a shows the results compared to the control not treated with any compound
- Fig. 13b provides a more detailed comparison of the effects of the individual compounds tested.
- FIG. 13 provides an exemplary plot of the change in average weight of animals during the experiments presented in Fig. 13 testing the effect of compounds on tumor growth.
- the weight of animals receiving the test compound - BK124.1 at a dose of 20 mg/kg (IP) was found to be similar to that of animals receiving imatinib.
- the weight of animals not treated with compounds was increased due to the unlimited growth of tumor mass.
- FIG. 14 shows selected representative images of isolated tumors in the experiment depicted on Fig. 12 - 14.
- the tumors were isolated after 21 days from the K562 cell implantation.
- Fig. 15 shows representative flow cytometry images (A) and percentages of cells (B) in subpopulations identified based on CD34/CD38 antigens in cells from PBMCs of CML and AML patients before any treatment (purity) and after treatment for 24 h with BK124.1 at a concentration of 2.5 microM and 5 microM. Data from individual patients is presented.
- Fig. 15C illustrates results of apoptosis assessment with Annexin V-FITC/Propidium iodide flow cytometry assay in CD34+/CD38+ and CD34+/CD38- cell subpopulations from CML or AML patients after treatment for 24 h with 0.1% DMSO or BK124.1 at the concentration of 2.5 mM or 5 mM.
- IM imatinib
- BK124.1 Described is a method for formulating and preparing a BK124.1 concentrate allowing administration of different doses.
- the preparation of BK124.1 thus prepared can be used in the treatment of cancer, in a preferred variant, e.g. leukemias, including CML.
- BK124.1 in monotherapy and in combination therapy for the CML treatment have been described. Any method known in the art can be used to diagnose CML, including detecting the presence of Philadelphia chromosome (Ph) translocation in the patient's leukemia cells. Other diagnostic methods can also be used.
- the CML stem cells described herein are characterized as bone marrow cells of the CD34(+) phenotype with the Ph chromosome present. Routine methods can be used to detect expression levels as well as to determine the phenotype of the patient's cells.
- BCR-ABL+ IM-resistant leukemia e.g. Ph+ acute lymphocytic leukemia (-20% in adults, 5% in children), Ph+ acute myeloid leukemia (-2%) and potentially KIT+ gastrointestinal stromal neoplasms (GIST, as IM can also inhibit KIT kinase).
- BK124.1 can be used in a combination therapy with other BCR-ABL inhibitors.
- GLEEVEC imatinib
- Nilotinib AN107, Tasigna
- Dasatinib BMS-345825, Sprycel
- Bosutinib SKI-606, Bosulif
- Ponatinib AP24534 lclusig
- Prominent in therapy are molecules belonging to the new generation of BCR- ABL inhibitors, which have not yet entered common clinical practice for CML and ALL therapy. They can also be used in a combination therapy together with BK124.1. These compounds are Bafetinib (INNO-406), Rebastinib (DCC-2036), Tozasertib (VX-680, MK-0457), Danusertib (PHA-739358), HG-7-85-01 , GNF-2 and -5, and 1 ,3,4 thiadiazole derivatives.
- BK124.1 affects the constitutive signaling of BCR-ABL1 kinase, known as the main cause of CML. This signaling includes pathways such as PI3K/AKT and JAK/STAT.
- the activity of BK124.1 is associated with induction of the apoptosis process in target cells.
- this compound causes a decrease in STAT5, AKT, mTOR and P65 protein level.
- an increase in F0X03A and P21 protein level is observed.
- F0X03A protein level which initiates an increase in P21 protein synthesis.
- these proteins are transported from the cytoplasm to the cell nucleus. Increasing the level of nuclear P21 triggers apoptosis, a natural process of programmed cell death, decreasing the cell population without the occurrence of an inflammatory process.
- the mechanism of action of this compound is therefore significantly different from that of kinase inhibitors such as imatinib, allowing for use thereof when resistance to treatment with these inhibitors develops.
- compositions that include a dicarboxyimide derivative, e.g. BK124.1 , and optionally also a BCR-ABL inhibitor, e.g. IM, as active ingredients.
- a dicarboxyimide derivative e.g. BK124.1
- a BCR-ABL inhibitor e.g. IM
- compositions usually comprise a pharmaceutically acceptable carrier.
- “Pharmaceutically acceptable carrier” as used herein includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, compatible with pharmaceutical administration.
- compositions are typically formulated to be compatible with the intended route of administration.
- routes of administration include parenteral administration, e.g. intravenous, intradermal, subcutaneous, oral (e.g. inhalation), percutaneous (topical), mucosal, intraperitoneal and rectal.
- Suitable pharmaceutical compositions are known in the art and are described in the Polish Pharmacopoeia or in the European Pharmacopoeia, for example, solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulphite; chelating compounds such as versenic acid; buffers such as acetates, citrates or phosphates and tonicity-adjusting agents such as sodium chloride or dextrose.
- a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents
- antibacterial agents such as benzyl alcohol or methylparabens
- the pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.
- the parenteral preparation may be closed in ampoules, disposable syringes or multidose vials made of glass or plastic.
- Pharmaceutical compositions suitable for use for injection may include sterile aqueous solutions when the active ingredients are water-soluble, or dispersions and sterile powders for the preparation ex-tempore of sterile solutions or dispersions for injection.
- suitable carriers include saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be liquid to such an extent that there is an ease of injection.
- the carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof.
- Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the event of dispersion and by using surfactants.
- Preventing the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, timerosal and the like.
- isotonic agents for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride.
- Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, for example, aluminum monostearate and gelatin and corresponding mixtures thereof.
- Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the event of dispersion and by using surfactants.
- Sterile injectable solutions can be made by introducing the active compound in the required amount into a suitable solvent together with one or a combination of the ingredients listed above, as required, and then sterilization by filtration.
- Dispersions are generally produced by incorporating the active compound into a sterile carrier that contains the primary dispersion medium and optionally other components from those listed above.
- a sterile carrier that contains the primary dispersion medium and optionally other components from those listed above.
- the advantageous ways of manufacturing are vacuum drying and freeze drying, which give the active ingredient powder plus optionally any additional desirable component from its previously sterile filtered solution.
- Oral compositions in general contain an inert diluent or edible carrier.
- the active compound can be combined with excipients, and used in the form of tablets, lozenges or capsules, e.g. gelatin capsules.
- Oral compositions can also be made using a liquid carrier for use as a mouthwash.
- Pharmaceutically compatible binders and/or adjuvant materials can be included as part of the composition.
- Tablets, pills, capsules, lozenges and the like may contain any of the following ingredients or compounds having similar nature: a binder such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel or maize starch; a lubricant such as magnesium stearate or Sterotes; a lubricant, such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin or a flavoring agent such as peppermint, methyl salicylate or orange flavor.
- a binder such as microcrystalline cellulose, tragacanth gum or gelatin
- an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel or maize starch
- a lubricant such as magnesium stearate or Sterotes
- a lubricant such as colloidal silicon dioxide
- a sweetener such as sucrose or
- the compounds can be provided as an aerosol from a pressurized container or dispenser that contains a suitable propellant, e.g. a gas such as carbon dioxide, or from a nebulizer.
- a suitable propellant e.g. a gas such as carbon dioxide, or from a nebulizer.
- Systemic administration of the therapeutic compound, as described herein, can also be carried out through the mucous membranes or transdermally.
- penetrating agents suitable for the barrier to be penetrated are used in the preparation.
- penetrants are generally known in the art and include, for example, for administration through mucous membranes, detergents, bile salts and fusidic acid derivatives.
- Administration through the mucous membranes can be achieved by using nasal sprays or suppositories.
- active compounds are formulated in ointments, lotions, gels or creams generally known in the art.
- compositions can also be produced in the form of suppositories (e.g. with conventional vehicles for suppositories such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
- suppositories e.g. with conventional vehicles for suppositories such as cocoa butter and other glycerides
- retention enemas for rectal delivery.
- the active compound(s) is (are) produced with carriers that will protect therapeutic compounds from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems.
- a controlled-release formulation including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers such as [poly(ethylene-co-vinyl acetate)], polyanhydrides, Polyglycolide, collagen, poly(ortho-esters) and Polylactide [poly(lactic acid)] can be used.
- Such preparations can be manufactured using standard techniques or can be obtained commercially, e.g. from Alza Corporation and Nova Pharmaceuticals, Inc. They can be produced according to the methods known to persons skilled in the art, according to the requirements and specifications described in the European Pharmacopoeia.
- compositions can be contained in ampoules, container, package or dispenser along with instructions for administration.
- the therapeutic dose is the amount of compound sufficient to obtain beneficial or desirable therapeutic results. This dose may be the same or different from the prophylactic dose, which is the amount necessary to prevent the onset of symptoms of the disease or onset of the disease.
- the therapeutic dose may be administered in one or more administrations, applications or doses.
- the therapeutic dose is characteristic of each compound, it can be administered once or more times a day, in addition, therapy can be continuous or can be used once or more times a week.
- the choice of therapeutic dose depends on the severity of the disease or disorder, previous treatment, general health and factors such as body weight, age of the patient and other accompanying diseases. Therefore, the therapeutic dose is selected by a person skilled in the art, who determines the dosage and time required to effectively achieve therapeutic effects.
- Dosages, toxicity and therapeutic efficacy of compounds can be determined by standard procedures, which are in vitro experiments in cell cultures and in vivo experiments in animals.
- LD50 lethal dose for 50% of the population
- ED50 therapeutically effective dose in 50% of the population
- therapeutic index is determined, which is expressed as the LD50/ED50 ratio.
- Compounds are considered therapeutically beneficial when their therapeutic indicators are high.
- Another parameter used may be the IC 5 o (the concentration at which proliferation/viability of tumor cells is inhibited by 50%, relative to the control).
- Toxic compounds may be used in therapy, but at the design stage, appropriate drug transport systems are created, which direct the molecules to the affected area. Such systems are created to minimize potential damage to normal cells and thus reduce side effects.
- Data from cell culture and animal studies can be used to create a dosage range for humans.
- Such dosage is in the range of concentrations of compounds in the circulation, for which low toxicity or no toxicity is observed.
- the dosage may vary within that range depending on the dosage form and the route of administration used.
- the therapeutically effective dose has been initially estimated based on tests on cell cultures. Then the dose was tested in animal models, in which the IC 5 o value (i.e., the concentration of the test compound that inhibited symptoms at 50%) was determined. In addition, the activity of the compound tested was compared with the activity of the preparations used in therapy. Such information was used to determine therapeutic doses for humans.
- FDA-approved doses can be used, e.g., up to a maximum of 800 mg a day of IM and for BK124.1 based on in vitro, in vivo (mouse model), and ex vivo (patient blood model) experiments performed and described below, in which the optimal dosage without adverse effects is administration of BK124.1 twice a day at a daily dose of 20 to 30 mg/kg body weight.
- the overall object of the study was to understand the mechanisms underlying the activity of BK124.1 and to investigate its potential independent anti-cancer effects and synergistic effects on the increase in cytotoxicity of imatinib used in the treatment of CML and other leukemias, e.g. AML.
- the study was divided into an in vitro stage conducted on cell lines and an in vivo stage in which the compound was tested in a mouse model.
- These studies used, among others, the human CML cell line K562 (BCR-ABL+), a xenogeneic mouse model of human CML leukemia (NSG mouse with K562 cells administered), human cells isolated from the blood of individuals diagnosed with CML and AML.
- the study consisted of a series of controlled laboratory experiments in which a number of parameters such as cell viability, apoptosis, cell signaling pathway activity and leukemia progression were analyzed.
- a number of parameters such as cell viability, apoptosis, cell signaling pathway activity and leukemia progression were analyzed.
- K562 BCR-ABL+ cells
- mice were randomly assigned to each group, and the results were analyzed in an unblinded manner. Animal sample sizes were chosen based on the procedure established in previous publications. To achieve statistical significance, the animal groups studied consisted of at least 4 individuals. K562 (BCR-ABL+) cells were used for mouse experiments.
- CML cell line K562 (BCR-ABL+) was purchased from an ATCC supplier.
- Cells were cultured in RPMI 1640 medium containing HEPES (Biiest) with 2 mM GlutaMAX (Gibco), supplemented with 10% fetal bovine serum (Sigma) and 100 pg/mL penicillin and 100 U/mL streptomycin (Gibco).
- Cell viability was monitored using the Muse Cell Analyzer (Millipore).
- K562 cells with multidrug resistance type 1 were selected in the Laboratory for Preclinical Research of Elevated Standard (Marceli Nencki Institute of Experimental Biology, Polish Academy of Sciences) according to the protocol published by Tsuruo (26 Tsuruo 1983).
- K562 cells were cultured in increasing concentrations of vincristine starting at the IC50 determined by MTT assay (Sigma). The final concentration of DMSO in the culture was 0.1%. Vincristine dose was doubled every 2-3 weeks after the cells reached at least two confluences. Cell viability was above 90%, which was confirmed using the Muse Cell Analyzer (Millipore).
- K562 or K562-MDR1 cells were seeded into 50 pi 96-well dishes in the amount of 7x10 3 cells per well.
- the test compounds dissolved in 0.1% DMSO were added to the cells.
- 50 pi of medium with appropriate drug concentration was added to each well.
- the final DMSO concentration per well was 1% for K562 and K562-MDR1 , and 0.1% for CD34(+).
- 20 mI of Tetrazolium Bromide (Thiazolyl Blue) at a concentration of 5 mg/mL was added to each well.
- K562 or K562-MDR1 cells were seeded in a 6-well plate at a concentration of 0.1 - 0.2 x10 6 cells/mL.
- CD34(+) cells were seeded at a concentration of 0.25 x10 6 cells/mL in a 24-well plate. After 24 hours of pre-incubation, the test compound dissolved in DMSO was added to achive a final concentration of DMSO of 0.1%. After 24 hours of incubation, cells were harvested and stained according to the manufacturer's protocol using FITC Annexin V apoptosis detection kit (BD Pharmingen). Stained cells were immediately analyzed on a BD FACSCalibur flow cytometer, (USA) using CellQuest software (BD Biosciences, USA).
- Cells were seeded at a concentration of 0.1 - 0.2 x10 6 cells/mL in a 6-well plate. After 24 hours of pre-incubation, the test compound - BK124.1 in 0.1% DMSO was added to the cells, and then the cells were further incubated for 24 or 48 hours. After incubation, cells were washed twice with ice-cold PBS solution (without Mg and Ca), resuspended in ice-cold 70% ethanol solution, and placed at -20°C for 24 hours.
- K562 cells were seeded at a concentration of 0.1 - 0.2 x10 6 cells/mL in a 6-well plate. After 24 hours of pre-incubation, the test compound - BK124.1 dissolved in 0.1% DMSO at the analyzed concentration was added and incubated for a specified time. In all experiments using this technique, the final DMSO concentration was 0.1%. After incubation, the cells were collected and lysed using a RIPA (Sigma) lysis buffer supplemented with complete Protease Inhibitor (Roche) and PhosSTOP (Roche). Protein concentration in cell lysates was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific).
- Lysates prepared for immunoblotting (Western blotting) analysis contained the same amount of protein (20 pg) in equal final volume. Protein lysates were then incubated for 5 min in Laemmli buffer (Biorad) at 95°C. SDS PAGE electrophoresis was performed using TGX StainFree FastCast (Biorad) in TGS Running Buffer (Biorad). After electrophoretic separation, transfer of proteins to membranes (AppliChem) in Towbin buffer was performed at 4°C for 1 h at 100 V. Both electrophoresis and transfer used BioRad apparatus.
- Membranes (AppliChem) were blocked with 5% non-fat milk powder and then incubated with primary antibodies (Cell Signaling Technology) overnight at 4°C with gentle shaking. HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies) were used to visualize the labeling results. Blots were developed and analyzed densitometrically using Clarity Western ECL Substrate from ChemiDoc XRS+ (Biorad). Protein levels normalized to controls were determined using ImageJ software.
- K562 cells were plated at a concentration of 1.5 c 10 5 cells/mL in a 6-well plate. After 24 h of pre-incubation, test compound - BK124.1 , dissolved in DMSO, was added at the analyzed concentration and incubated for the specified time. In all experiments using this technique, the final DMSO concentration was 0.1%. Cells were harvested and washed with ice cold PBS.
- hypotonic buffer (10 mM HEPES pH 7.9; 10 mM KCI, 0.1 mM EDTA pH 8; 0.1 EGTA pH 8) supplemented with complete Protease Inhibitor and PhosSTOP (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated on ice for 15 minutes, followed by addition of 25 pL of 5% NP-40 (Sigma- Aldrich, St. Louis, MO, USA) to each lysate and the tube was intensively vortexed for 10 seconds. Tubes were then centrifuged at 4 °C, 1,000 c g for 15 minutes.
- Cytosol fraction was collected as supernatant.
- the pellet was washed twice with hypotonic buffer and resuspended in hypertonic buffer (20 mM HEPES pH 7.9; 0.4 M NaCI, 1 mM EDTA pH 8; 1 mM EGTA pH 8) supplemented with complete Protease Inhibitor and PhosSTOP. Lysate was incubated for 30 minutes on ice, followed by centrifugation at 4 °C, 10,000 c g for 15 minutes. Nuclear fraction was collected as the supernatant. Protein concentration in cell lysates was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA).
- Lysates contained the same amount of protein (at least 15 pg) in the same final volume.
- the purity of the separated fractions is documented by the level of control proteins: ACTB (beta- actin) - cytoplasmic fraction marker (Cell Signaling Technology, Danvers, MA, USA), LMNB1 (laminin B1 ) - nuclear fraction marker (Santa-Cruz Biotechnology, Dallas, TX, USA).
- ACTB beta- actin
- LMNB1 laminin B1
- SDS- PAGE Western Blot procedure was performed as described for whole cell lysates.
- Table 1 Administration scheme in an assay of in vivo model toxicity.
- red blood cells [erythrocytes, RBC] monocytes [MONO], white blood cells [leukocytes WBC], lymphocytes [LYMPH], hematocrit [HCT], platelets [thrombocytes PLT], red blood cell volume distribution [RDW], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC]
- the obtained parameter values were compared with the values observed in the control group of animals. Body weight was measured daily during the experiment.
- a xenogeneic NSG mouse model was generated as previously described (21 Huang R 2018, 22 Zhao WH 2017, 25 Vlahovic G 2007). 8-week-old female NSG mice (Jackson Laboratory) were injected into the dorsal fold with 1x10 6 K562 cells of the human chronic myeloid leukemia (CML) line in a volume of 100 mI (0.5% of mouse weight). On day 3 after cell administration, mice were randomly assigned to each group and on days 4-17 of the experiment, the selected solution was administered intraperitoneally once a day according to the schedule shown in Table 2. On day 21 , the experiment was terminated, the blood, tumors and remaining tissues were excised and fixed in buffered formalin for further analysis. Blood was analyzed for morphology. Subcutaneous tumors and body weight were measured daily during the experiment.
- CML chronic myeloid leukemia
- AML acute myeloid leukemia
- PBMC peripheral blood mononuclear cells
- CD34(+) peripheral blood mononuclear cells
- Blood samples from patients were collected by venipuncture and blood collection into BD Vacutainer EDTA-K2 tubes and transported to the laboratory within 1-2 hours.
- Interphase PBMC cells were washed twice with BSS, the resulting cells were counted and resuspended in the medium recommended by the manufacturer for CD34(+) cell isolation (EasySepTM Human CD34 Positive Selection Kit II, StemCell Technologies). Isolated CD34+ cells after counting and viability assessment using Muse Cell Analyzer (Millipore) were cultured in StemProTM -34 SFM medium with fresh addition of the recommended cytokines IL-3 (50ng/ml_), GM-CSF (25ng/ml_) and SCF (100 ng/mL). For experiments with BK124.1 , cells were always pre-incubated for 24 hours. The purity of the CD34+ fraction was assessed using an APC antibody against human CD34 antigen (Biolegend) and propidium iodide (Sigma) and the corresponding isotype control (Biolegend).
- cells were harvested after 24 hours and stained with anti-human CD34-APC antibody, anti-human CD38-PE (Biolegend, San Diego, CA, USA) and propidium iodide according to the manufacturers’ protocols.
- cells were treated with 2.5 mM or 5 mM BK124.1 , or DMSO, harvested after 24 hours, and stained with anti-human CD34-APC antibody, anti-human CD38-PE and Annexin V-FITC kit according to the manufacturer's' protocol. Appropriate isotype controls were included. In both cases stained cells were immediately analyzed on a BD LSRFortessa flow cytometer (BD, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (BD, Franklin Lakes, NJ, USA). Purity was analyzed with doublet exclusion on live cells only. Analysis of apoptosis was performed with doublet exclusion and for each cell population (CD34+/CD38+ or CD34+/CD38-) separately. Experiments were performed at the Laboratory of Cytometry, Nencki Institute of Experimental Biology.
- TGI Tumor Growth Inhibition
- TGI 100 - ⁇ (mean tumor volume in treatment group / mean tumor volume in control) x 100 TGI (%). Such calculated value represents the percentage that the tumors obtained in a given variant are smaller than the tumors observed for the control group.
- BK 124.1 a liquid pharmaceutical formulation of BK124.1 was optimized.
- Formulation of BK 124.1 was divided into two steps. The first step consisted in dissolving BK 124.1 in a mixture of 50% lipophilic polymer/50% polar compound and obtaining its concentrate, the second step consisted in preparing from the obtained concentrate a diluted solution of BK124.1 for intravenous or intraperitoneal administration once or twice a day and administering different doses of the active substance. The second step was also intended to facilitate filtration of the solution and increase the fluidity of the compound.
- the prepared concentrate was diluted with aqua pro injectione (water for injection) to obtain a concentration of 10% Solutol HS 15/10% ethanol.
- Such diluted solution was fluid, did not settle on the walls, and was convenient for filtration and for portioning the preparation.
- This solution was therefore sterilized by membrane filtration on a 0.2 pm filter (Minisart PES; Sartorius) and pipetted into sterile tubes of 2 ml. each, and these tubes were stored at room temperature for 48 h until used for injection in animals.
- the described protocol makes it possible to prepare a BK124.1 solution at a concentration of 2 mg/mL, which after filtration is stable, easy to store, sterile and apyrogenic, easy to dose, and therefore meets the necessary requirements for a drug administered in vivo. Moreover, solution of the compound thus prepared is also easy to sell and transport. The solution thus prepared was used for direct administration to the experimental animals.
- the two-step preparation of BK 124.1 described above permits oral and intraperitoneal administration in animals and humans.
- a third dilution step of a solution of 10% ethanol and 10% Solutol HS 15. This is based on the experiments made by the present inventors in a rat model, where administration (especially repeatedly) of an aqueous solution of 10% ethanol and 10% Solutol HS 15 alone, without BK 124.1 , to the tail vein resulted in necropsy of the tail.
- the described BK 124.1 solution should be further diluted in, for example, 5% glucose solution or 0.9% sodium chloride solution, e.g., in polyethylene, polypropylene or glass vessels, but not in PVC vessels. Only a clear and colorless solution should be administered.
- concentration of BK 124.1 in the solution for intravenous infusion may be in the range from 0.004 mg/mL to 0.1 mg/mL.
- Therapeutically effective doses of BK124.1 or its combinations with IM in the treatment of CML/AML may be determined by a skilled physician based on clinical findings, and adjusted for disease progression, patient age and weight, and risk of disease progression for a particular patient.
- FIG. 1 depicts a representative immunoblot showing the change in the level of selected proteins observed in K562 cells treated for 4, 8, 16, 24 and 48 hours with 0.1% DMSO solution or BK124.1 at a concentration of 5 mM in 0.1% DMSO.
- the first changes in protein levels under treatment with BK 124.1 were observed after 4 and 8 hours. Namely, the levels of such pro-life and cell division-stimulating proteins as STAT5 and AKT, as well as mTOR and P65 (other names: NF-KB, RelA) decreased, whilst the FOX03A and P21 protein levels increased (Fig. 1).
- P21 is a protein that, depending on the sub-cellular location, performs different functions. When localized in nucleus, it can induce apoptosis.
- FOX03A is a transcription factor that may play a role in initiating P21 transcription.
- Immunoblots were also performed to determine the FOX03A and P21 protein levels in the nuclear and cytoplasmic fractions of K562 cells treated for 8 or 24 hours with a solution of 0.1% DMSO or with BK124.1 at a concentration of 5 mM.
- the cytoplasmic and nuclear fractions of the cells were separated by a standard method of cell fractionation, in which, after homogenization, the cell lysate was subjected to differential centrifugation.
- the experiment was carried out in at least 3 independent biological repeats, Fig. 2 represents one representative immunoblot (Fig. 2).
- K562 cells were treated for 24 and 48 hours with BK124.1 at a concentration of 5 mM, which corresponded to a dose of 2 x IC50 for this compound.
- Control cells were treated with 0.1% DMSO solution at the same time points.
- the cells were stained with propidium iodide (PI) and analyzed by flow cytometry. The results are shown in Fig. 3.
- PI propidium iodide
- K562 cell line was first established, cells of which strongly expressed MDR1 (K562-MDR1), (methodology outlined above, in Materials and Methods section). The MTT assay of K562 and K562-MDR1 cell viability was then performed. This test showed no statistically significant differences in the cytotoxic activity of BK124.1 against K 562 (primary) and K562-MDR1 cells (Fig. 4).
- K562-MDR1 cells were incubated at a concentration equivalent to 2xlC 5 o with BK124.1 (5 mM) or with taxol (1 pM) or with vincristine (1 pM) for 24 hours, and then cells were stained with propidium iodide and the antibody against P-glycoprotein (MDR1) to assess PgP (MDR1) levels and cell survival. Results (Fig.
- K562-MDR1 cells were also sensitive to a lower concentration of BK124.1 (2.5pM), in which a high percentage of cells in early phase of apoptosis was also observed.
- BK124.1 induces an increase in MDR1 expression, however this fact does not change the high proapoptotic activity of this compound, resulting in cell population death.
- the cells were incubated for 24 hours with BK124.1 at doses of 2.5 pM and 5 pM in 0.1% DMSO or in 0.1% DMSO as a control. After incubation, the cells were labeled with Annexin V/propidium iodide and subsequently the level of apoptosis was assessed by flow cytometry.
- BK124.1 Pharmacokinetics of BK124.1 was studied in the rat (Wistar) body after intravenous (IV) administration of 5 mg/kg or 10 mg/kg dose of BK124.1 in a 10% Solutol HS 15/ 10% ethanol solution (Fig. 9). Five individuals for each datapoint were used in pharmacokinetics studies in a rat model. For PK measurements in rats, blood was collected from the same individual at different time points. Concentration of BK124.1 in blood of rats was analyzed 5 min, 15 min, 30 min, 1 h, 2h, 6h, and 24h after a single administration of the compound. The concentration was measured by mass spectroscopy. Based on the obtained values of drug concentrations in the blood of animals, pharmacokinetic parameters were determined for BK124.1.
- mice The study of pharmacokinetics of BK124.1 following IV versus IP route administration in mice
- BK124.1 should be administered 1 or 2 times a day at the tested doses. This dosage regimen is desirable for long-term therapy.
- Toxicological parameters such as physiological and morphological changes at the site of administration of the compound, effects of the compound on the morphology of internal organs such as liver, kidney, heart, spleen, effects of the compound on animal blood count and body weight (body weight was measured every 48 h), and effects of the compound on animal behavior were considered.
- mice with single intravenous administration of BK124.1 in 10% Solutol HS 15/ 10% ethanol solution at doses of 5 mg/kg body weight, 10 mg/kg, and 20 mg/kg.
- BK124.1 in a 10% Solutol HS 15/ 10% ethanol solution can be administered intraperitoneally in long-term, 1x or 2x aday, and which dose of the compound is non-toxic.
- BK124.1 was administered to animals on days 4-17 of the 21 -day experiment according to the dosing regimen described in Materials and Methods (Table 1 ).
- Toxicity at a dose of 40 mg/kg when administered once or twice a day was found consisting in decrease in the average weight of the animals, and based on observed change in the behavior of the animals.
- For a dose of 20 mg/kg as well as 30 mg/kg body weight administered IP once a day no changes in animal body weight were observed compared to control animals not treated with the compounds. There were also no changes in the behavior of these animals, in the appearance of their internal organs as well as in blood count parameters. It is summarized that BK 124.1 can be safely administered every 24 h for two weeks via the intraperitoneal route at a dose of 20 mg/kg or 30 mg/kg body weight.
- Fig. 11 The average results for tumor weight at the end of the experiment are also shown in Fig. 11.
- the change in theoretical mass of the tumor over the course of the experiment is shown in Fig. 12a and Fig. 12b.
- Each of the variants used was statistically significant as compared to the control group. In contrast, there were no statistically significant differences in tumor size when BK124.1 or BK124.1 and imatinib were administered to mice as compared to both control compounds.
- Fig. 14 selected representative images of isolated tumors (21 days after cell administration) are shown.
- BK124.1 can be administered to the patient as a single unit dose once a day or in multiple daily doses.
- the therapeutically effective daily dose of BK124.1 for monotherapy in adult may range from 10 to 30 mg/kg body weight.
- BK124.1 induced apoptosis in CD34+/CD38- leukemia stem cells from all the patients.
- the apoptotic death of CD34+/CD38- leukemia stem cells in response to BK124 was dose dependent and higher at 5 mM BK124.1 concentration.
- BK124.1 was particularly detrimental to CD34+/CD38- LSC from 2 CML and 1 AML patients.
Abstract
The subject of the invention is the BK124.1 compound of Formula I, for use in the treatment of cancer, wherein the cancer is leukemia or another cancer characterized by a clonal increase in the number of white blood cells, wherein the cancer shows resistance to previous treatments, in particular multidrug resistance.
Description
Dicarboximide derivative for use in cancer treatment
Field of the invention
The present invention relates to derivative of dicarboximide for use in cancer treatment, in particular in the treatment of leukemias (e.g. chronic myeloid leukemia (CML) or acute myeloid leukemia (AML)) and other diseases characterized by clonal hyperplasia and impaired proliferation of white blood cells, such as leukemias. Furthermore, pharmaceutically acceptable formulations have been developed enabling the use of the active ingredient in liquid compositions, their sterilization and stable storage. The compound to be used according to the invention can be used as monotherapy or in combination treatment, e.g. together with a tyrosine kinase inhibitor, such as imatinib.
Background art
Leukemias remain an unsolved problem of clinical medicine and pharmacology in the 21st century. Even though a number of clinically useful anti-cancer compounds are available on the market, many problems remain without an effective solution. The main challenges in leukemia therapies are the shortage of drugs that specifically act on cancer cells and do not damage normal cells, and that have no negative side effects. Furthermore, a big problem is development of multidrug resistance which desensitizes cancer cells to current chemotherapeutics, and shortage of drugs effective against cancer-initiating stem cells, which can be an origin of cancer recurrence. There is research on anticancer drugs with a better pharmacological profile in relation to those currently used.
One of the most common diagnoses is chronic myeloid leukemia (CML), which is a disorder involving clonal proliferation of a neoplastically transformed stem cell of bone marrow and accumulation of the neoplastically altered cells in blood. [1 Faderl 1999]. A characteristic feature of CML cells, present in more than 90% of patients, is the presence of mutual translocation between the long arms of chromosomes 9 and 22 [2 Deininger 2000, 3 Kurzrock 2003]. As a result of displacement, a short chromosome 22, the so-called Philadelphia chromosome (Ph) and an elongated chromosome 9 are formed. The effect of translocation is the transfer of the ABL oncogene (located on chromosome 9) to a strictly defined breaking site (breakpoint cluster region-BCR) on chromosome 22 and the formation of the fusion BCR-ABL gene. This gene encodes a protein that exhibits abnormal, increased activity of receptor tyrosine kinase (TK). BCR-ABL TK expression is the primary driving factor of neoplastic transformation in CML (3 Kurzrock 2003, 4 Colicelli 2010, 30 Thompson 2016, 31 Jabbour E 2018). BCR-ABL can transform myeloid progenitor cells into cancer cells and drives development in 95% of CML cases. BCR-ABL promotes leukemogenesis by activating
further signaling proteins that increase survival and proliferation of cancerously altered cells [4 Colicelli 2010] These pathways include, among others, RAS/mitogen-activated protein kinase ( RAF/M EK/ERK), phosphatidylinositol/AKT 3-kinase cascades (PI3K/AKT), and JAK/STAT signaling cascades [5 Steelman 2004]
The first-line drugs approved for use in humans in CML therapy are hydroxyurea, available as Hydroxyurea Medac, among others, and Imatinib (IM), available as Glivec®, among others. IM binds to the kinase domain of ABL kinase and inhibits the phosphorylation of its substrates. In IM therapy, there is a significant improvement in patient survivability, especially when used at an early stage. In recent years, CML treatment has been optimized by using therapy with compounds from the group of new generation tyrosine kinase inhibitors, having different activity against leukemic cells. The treatment of CML and other leukemias with imatinib was accompanied by therapies with other drugs from this group such as: dasatinib, nilotinib, bosutinib, ponatinib. In spite of introduction of new drugs in CML therapy, there is a need to personalize therapy in terms of dose selection as well as selection of the appropriate drug which, while maintaining efficacy, would avoid or at least reduce their side effects [27 Rea 2015, 28 Steegmann 2016]. In addition, the aim of the nowadays used therapy is only to prevent the progression of the disease, i.e. the transition from the chronic phase to the advanced stage and to bring the peripheral blood parameters to a state close to normal. IM and other tyrosine kinase inhibitors do not show satisfactory efficacy in the advanced stage of the disease, which is the so-called blast crisis, as well as resistance leading to relapse may develop during therapy with these compounds [6 An 2010, 7 Bubnoff 2003]. Cancer recurrence caused by clonal growth of cancer-initiating stem cells in patients after treatment also remains a problem.
The golden standard for the treatment of CML and other leukemias such as Ph+ acute lymphoblastic leukemia (ALL Ph+) is IM therapy. IM is a low-molecular inhibitor of protein tyrosine kinases that strongly inhibits the activity of BCR-ABL tyrosine kinase (TK) and many other receptor tyrosine kinases, e.g., Kit, a stem cell growth factor (SCF) receptor encoded by c-Kit proto-oncogene, colony-stimulating factor receptors (CSF-1 R), and 17 alpha and beta receptors for platelet-derived growth factor (PDGFR-alpha and PDGFR-beta). Despite its high efficacy, this drug does not eliminate cancer-initiating stem cells from the patient's body (11 Hamilton 2012, 12 Andersson 1979). Moreover, in about 3% of patients lack of response to IM treatment is observed, which is associated with primary drug resistance. In addition, during the IM treatment, an increase in acquired (secondary) resistance is observed, which can be observed in about 20% of patients in the chronic phase of CML.
The challenge of modern therapy is especially resistance to IM, which is observed much more often in patients in advanced stages of CML, especially in the phase of blast crisis
(70% of refractory patients) (13 O'Hare 2006, 14 Apperley 2007a, 15 Apperley 2007b, 16 Kantarjian 2006).
The most common cause of resistance (35^45%) is the occurrence of point mutations within the BCR-ABL gene. These mutations can lead to a permanent change in kinase conformation into its active form, not recognized by the drug, or affect amino acids directly involved in the formation of bonds with IM (17 Lahaye 2005, 18 Hochhaus 2002, 19 Brandford 2003).
Another cause of resistance (10-15%) is the T315I mutation associated with replacement of threonine by isoleucine. Threonine at position 315 forms a hydrogen bond with IM that stabilizes the enzyme-drug complex. Replacement of threonine by isoleucine prevents formation of the binding and consequently greatly reduces the potency of the drug (14 Apperley 2007 a, 15 Apperley 2007 b, 20 Nardi 2004, 29. Tamai 2016).
Important mechanisms of resistance to IM also include: gene amplification within BCR- ABL, clonal evolution consisting in arising of genetic changes leading to the activation of new signal conduction pathways, the presence of a population of stem cells insensitive to the drug, the emergence of mechanisms leading to the reduction of intracellular concentration of IM (binding of the drug by alpha-1 -acid glycoprotein, reduction of hOCT 1 - human organic cation transporter - expression, increase in expression of MDR1 which is responsible for the active ejection of the drug from cells) (17 Lahaye 2005, 18 Hochhaus 2002).
Therapy with the use of IM effectively treats many patients, but during the therapy development of resistance to IM is observed (Valenti, Biologies. 1 (4): 433 L 148, 2007). As mentioned above, resistance may be the result of one or more mechanisms, including molecular resistance caused by a mutation in the BCR-ABL gene, or independent BCR-ABL resistance, e.g. associated with stem cell-specific survival factors, e.g. increased signaling of the RAF/MEK/ERK pathway, which is not dependent on RAS but is initiated by PKC. CML patients are considered resistant to IM when the response is lost or not observed after a daily dose of >400 mg imatinib (Valenti, 2007; Kantarjian etal., Blood. 101 (2): 473, 2003; Baccarani et al., Blood 108 (6): 1809-20, 2006). CD34(+) CML stem cells are also a reservoir of cells with mutations that confer resistance to IM (24. Sorel 2004).
The present invention relates to the use of 4-[2-hydroxy-3-(propan-2-ylamino)propylj- 1 ,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02'6] dec-8-en-3,5,10-trione, also described as BK124.1 , in therapy of CML or AML and other leukemias. BK124.1 is a derivative of compounds from the group of dicarboximides. The synthesis of the compound was disclosed and described in the patent applications EP2687509, EP2687509, PL400000. In addition, the synthesis and cytotoxic activity against selected cell lines is described in the publication (23, Kuran 2016).
The BK124.1 compound is defined by the Formula I:
(Formula I)
An example of a pharmaceutically acceptable salt of the compound of formula I is:
Studies performed on normal cell lines (HUVEC) as well as leukemic cell lines (e.g. HL-
60, K562) using BK124.1 showed selective, high cytotoxicity of this compound against human leukemia cell lines: CML leukemia line (K562 cells) and AML leukemia line (HL-60 cells), and yet low cytotoxicity against other cancer lines and non-cancer cells (23 Kuran).
The inventors of the present solution have also developed a system of solvents neutral to an organism to enable administration of BK124.1 in vivo. Pharmacokinetic parameters and therapeutic window parameters for BK124.1 were determined, including the route of administration of the compound to the body and the dose of the compound that conditions activity in reducing growth of cancer cells in the absence of toxicity (on the example of a mouse model of human chronic myeloid leukemia CML - immunodefective mice after xenotransplantation of human CML leukemia cells). BK124.1 has been shown to be highly cytotoxic against hematopoietic blood stem cells (cells that initiate cancerogenesis) obtained directly from patients with leukaemia. High cytotoxic activity of BK124.1 against multidrug resistant leukemic cells was also demonstrated. Also the molecular mechanism of cytotoxicity of the compound in human CML cells has been described (described mechanism of action of BK124.1 is therapeutically justified and clinically interesting, as confirmed in the present application by demonstrating the efficacy of BK124.1 in monotherapy and in IM-combination therapy in a mouse model).
The BK124.1 substance is characterized by very untoward rheological properties from the technological point of view of producing oral solid as well as liquid forms of the compound. As disclosed in the patent applications EP2687509, EP2687509, PL400000, the BK124.1 substance is in the form of white non-hygroscopic powder having electrostatic properties. In addition, this compound is characterized by poor solubility in aqueous solutions. The solubility of the compound at the synthesis stage was found only for 100% DMSO (23 Kuran).
In the formulation process, a form that can be used in long-term therapy was prepared. In addition, the form of BK124.1 as a concentrate is suitable for administration, e.g. once or twice a day, and can be administered at different doses to help the patient comply with the recommendations for use as well as to facilitate the doctor to select an appropriate therapeutic dose.
Moreover, the preparation gives the possibility of controlled administration of the drug into the circulatory system and achieving the therapeutic concentration of the compound in blood during treatment. Another problem with a long-term therapy is the need to determine the optimal dose that the patient can tolerate. If such a dose is not established, this may lead to reduction in the effectiveness of the drug being administered.
An aim of the present invention is therefore also providing preparations suitable for long-term administration of an active compound of formula I. According to the results of animal studies, the compound of formula I can be administered, for example, by intraperitoneal or oral or intravenous routes.
In one aspect, a new type of therapy for a patient with chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) or other leukemia in BK124.1 monotherapy and a new type of therapy for a patient with IM-resistant CML or AML were disclosed.
In another aspect, the invention provides a new method of treatment chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) or other leukemia in a mammal subject, that involves administering a combination of a BCR-ABL inhibitor and BK124.1 . In this method of treatment, the patient does not resign from taking a BCR-ABL inhibitor, e.g. IM, and BK124.1 is added to the therapy.
In another aspect, the invention provides an additional type of therapy for a patient with chronic myeloid leukemia (CML) or acute myeloid leukemia (AML) or another disease characterized by a clonal increase in the number of white blood cells that is treated with a BCR-ABL inhibitor or other drug, e.g. a MEK inhibitor.
Thus, the described combination of IM with BK124.1 could potentially treat CML or AML or other leukemia as opposed to the current, e.g. IM monotherapy, which is only effective in the long-term treatment of CML.
This hypothesis is supported by the results of an in vitro study in a model human myeloid leukemia using K562 cells, where BK124.1 was shown to slow the growth of tumor cells regardless of the level of MDR1 (Multi Drug Resistance Protein 1 ) activity. The antitumor activity of IM, including both inhibition of proliferation and induction of apoptosis and activation of additional signaling pathways, may have a beneficial effect on BK124.1 activity.
The subject of the invention is therefore a compound of formula I:
or its pharmaceutically acceptable salt for use in the treatment of cancer, wherein the cancer is leukemia or another cancer characterized by a clonal increase in the number of white blood cells, wherein the cancer shows resistance to previous treatments, in particular multidrug resistance.
Resistance to previous treatment, within the meaning of this invention, includes both primary and secondary (acquired) resistance, developing after exposure of the tumor to a given therapy. With resistance, the cancer responds to therapy in an unsatisfactorily way or not at all, or the efficiency of therapy significantly weakens over time, which can lead to recurrence of the disease.
Multidrug resistance (MDR) is defined as the acquisition by cancer cells of simultaneous insensitivity to several groups of different therapeutic agents, which develops in response to the use of even a single anticancer agent.
Preferably, the cancer is myeloid leukemia, in particular, chronic myeloid leukemia or acute myeloid leukemia.
Preferably, the individual to be treated suffers from chronic myeloid leukemia or acute myeloid leukemia and is in the phase of blast crisis. Preferably, leukemia is chronic myeloid leukemia or acute myeloid leukemia with multidrug resistance or resistance to imatinib.
Preferably, a compound of formula I, prior to administration to the individual to be treated, is dissolved in a mixture containing at least one pharmaceutically acceptable water- soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer, and the concentrate thus obtained is then used to dilute and prepare the final form for administration, which is administered to the individual to be treated.
Preferably, the pharmaceutically acceptable water-soluble polar compound comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
Preferably, the pharmaceutically acceptable lipophilic polymer comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
Particularly preferably, the compound of formula I, prior to administration to the individual to be treated, is dissolved in a mixture containing ethanol and macrogol-15- hydroxy stearate (e.g., Solutol® HS 15), the resultant concentrate is then used to dilution and preparation of the final formulation which is administered to the individual to be treated.
Preferably, the prepared concentrate is diluted in an aqueous solution, and then sterilized, the resultant diluted form of the drug is administered to the individual to be treated.
Preferably, a compound of formula I is administered orally or intraperitoneally in a mixture containing ethanol 10% : macrogol-15-hydroxystearate 10%.
The given values of ethanol 10% : macrogol-15-hydroxystearate 10% refer to a mixture obtained by diluting the concentrate of dissolved compound of formula I, e.g. in water (such as water for injection) or in physiological salt solution.
Preferably, the resulting concentrate diluted in an aqueous solution is then re-diluted, e.g. in a 5% glucose solution or a 0.9% sodium chloride solution, and the resulting formulation is administered intravenously.
Preferably, the compound of formula I is administered to an individual at least once a day, at a daily dose in the range of 10-30 mg/kg body weight.
Preferably, the individual to be treated has previously undergone therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
Preferably, another BCR-ABL kinase inhibitor was imatinib.
Preferably, a compound of formula I is used as monotherapy.
In another preferred embodiment, the compound of formula I is used in combination with therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
Preferably, another BCR-ABL kinase inhibitor is imatinib.
The present inventors have shown that it is also effective to use the compound of formula I together with imatinib. This implies that for patients who have previously been treated with this compound and who develop or may develop resistance to treatment, it is possible to introduce treatment with the compound of formula I without withdrawing imatinib therapy.
The compound of formula I can be used in an individual in need thereof, preferably in mammals, while particularly preferably, an individual is a human.
The subject of this disclosure is also a new formulation of the compound of formula I, which allows not only its sufficiently effective dissolution, in a solvent system that is pharmaceutically acceptable, but to obtain a solution that can be sterilized by filtration at the same time. In addition, it is important that it is possible to dissolve a sufficient amount of the compound to obtain a concentrate that will remain sufficiently stable for the shelf life without precipitation of the compound. The compound of formula I is a substance that is hardly soluble. As the present inventors have shown, for the solutions in DMSO, solubility was observed only in 100% solvent. However, the solubility obtained was already unacceptable in the case of a small dilution of DMSO with water. Developing an appropriate pharmaceutically acceptable formulation that provides both sterility and storage stability, also in the form of a concentrate, was a major challenge.
Thus, the compound of formula I has also been disclosed:
or a pharmaceutically acceptable salt thereof, e.g.:
for use in the treatment of cancers, wherein a compound of formula I, before administration to the individual to be treated, is dissolved in a mixture containing at least one pharmaceutically acceptable water-soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer, preferably wherein the pharmaceutically acceptable water- soluble polar compound is ethanol and the pharmaceutically acceptable lipophilic polymer is macrogol-15-hydroxystearate, and the concentrate thus obtained is then used to dilute and prepare the final form for administration, which is administered to the individual to be treated.
Disclosed herein is a method of formulating BK 124.1 into a concentrate which can be then diluted and administered, e.g., intraperitoneally or orally, or after further dilution also, e.g., intravenously, as by infusion, or it can be used to make another pharmaceutical form.
The first step of formulating according to the preferred embodiment of the invention consists in dissolving BK124.1. After testing several possible schemes of proceeding, it was found that especially preferably in order to dissolve BK124.1 , a dose thereof, preferably in the range of up to 3 mg (optimally 1 mg) is measured into a transparent, glass or plastic vial, then a mixture of a water-soluble polar compound and a lipophilic polymer is added, with the water- soluble polar compound used in the amount of 40-60% and the lipophilic polymer used in the amount of 40-60%. For example, a water-soluble polar compound and a lipophilic polymer can be in a ratio of 50%/50%. In the embodiment, a mixture used to dissolve a compound of formula I is obtained by mixing Solutol HS 15 and anhydrous ethanol 99.8%, e.g. so as to obtain a solution of 50% Solutol HS 15/ 50% ethanol. The mixture is gently shaken for completely wetting the drug product. For example, the vial is rotated/shaken for a period of time (e.g. several minutes or e.g. 1-2 hours) to obtain a transparent solution. In addition, to increase solubility, the vial can be heated on a water bath at 30-40°C. The solution prepared is carefully inspected to ensure that the product has been dissolved and that the solution does not contain particulate matter. It is preferable to use clear and colorless solutions, without particulate matter. BK124.1 concentrate thus prepared is colorless.
The second step involves dilution of the BK124.1 concentrate obtained in the first step using a compatible solvent, such as, e.g. water for injection, aqueous solution, saline,
physiological multi-electrolyte solution, 5% glucose solution, saline mixture with 5% glucose solution in a ratio of 2:1 , lactated and non-lactated Ringer's solution, and their modifications, etc. Preferably, the concentrate is diluted so that in the resulting solution the content of the water-soluble polar compound and the lipophilic polymer is 10% or less, respectively.
The concentrate solution thus obtained can be sterilized, for example, by filtering on a 0.2 pm filter. As a part of formulating, stability studies of the prepared concentrate were carried out and it was found that the form prepared in this way allows the concentrate to be stored for at least 30 days without affecting its parameters. An additional advantage of the concentrate is the ease of selecting its doses, which is important for therapeutic applications. The solution thus obtained may be used for administration to the individual to be treated, e.g. intraperitoneally or orally.
For the purpose of intravenous administration, for example, an additional dilution step is preferred, e.g. in 5% glucose solution or 0.9% sodium chloride solution, preferably in polyethylene, polypropylene or glassware, but not in PVC vessels. Only a clear and colorless solution should be administered. The concentration of the compound of formula I in the solution for intravenous infusion may preferably be in the range from 0.004 mg/mL to 0.1 mg/mL.
Dissolution of the therapeutic compound should be performed under aseptic conditions. If the prepared concentrate does not contain preservatives, it is used for single use, the unused solution should be discarded. The addition of preservatives such as e.g. benzalkonium chloride, thiomersal, chlorocresol, benzyl alcohol, extends its shelf life. It is also recommended to sterilize said solution, e.g. by filtering on a 0.2 pm filter.
The present inventors have found that mixtures of compounds with low in vivo toxicity containing at least one pharmaceutically acceptable water-soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer can be used to solubilize the BK124.1 compound. A pharmaceutically acceptable lipophilic polymer should constitute from 40% to 60% by weight of the entire solution, optimally 50%. At least one pharmaceutically acceptable polar compound should be present in the mixture, in a proportion of 40% to 60% by weight respectively, based on the weight of the solution, optimally 50%.
Examples of a pharmaceutically acceptable lipophilic polymer are compounds including: N-vinyllactam homopolymer, N-vinyllactam copolymer, cellulose ester, cellulose ether, polyalkylene oxide, polyacrylate, polymethacrylate, polyacrylamide, polyvinyl alcohol, vinyl acetate polymer, oligosaccharide and polysaccharide Solutol HS 15 (macrogol-15- hydroxystearate; Polyethylene glycol (15)-hydroxystearate); CAS No: 70142-34-6).
The soluble form of BK124.1 thus obtained can be further dissolved in aqueous solutions such as saline. Once dissolving, these solutions can then be administered to the
body of the individual to be treated, e.g. by intravenous route, or can be used to prepare another pharmaceutical form of the compound (e.g. granules, tablets, capsules).
The invention encompasses development of a pharmaceutical formulation containing BK124.1 for administration once or twice a day, wherein the formulation contains the active compound BK124.1 in a liquid form. The developed formulation allows dosing BK124.1 , for example, at intervals of 12 or 24 hours, which is sufficient to obtain therapeutic levels of the compound in blood within 12-24 hours after administration.
It is noted here that BK124.1 was the only one of the group of dicarboximide derivatives (see EP2687509, EP2687509, PL400000) for which it was possible to develop a formulation that provides not only the possibility of easy administration of the compound, but also to maintain stability and effectiveness.
Throughout this description, the term "pharmaceutically acceptable" refers to substances, compounds, mixtures, solutions, preparations, etc. which are generally not toxic or cause no damage to the individual to whom they are administered.
The term "pharmaceutically acceptable salt", as used herein, means the salts of the compound according to the invention which are safe and effective for topical use in mammals and which continue to retain its desired biological activity. Pharmaceutically acceptable salts include salts of acidic or alkaline groups. Pharmaceutically acceptable salts are, for example, but not limited to, hydrochloride, chloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulfate, phosphate, hydrogen phosphate, isonicotinate, acetate, lactate, salicylate, citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate, p-toluenesulfonate and pamoate (that is, 1 ,1'-methylene- bis-(2-hydroxy-3-naphthoate).
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention belongs. Described herein are methods and materials for use in the present invention; other appropriate methods and materials known in the art may be used as well. The materials, methods and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries and other references mentioned herein are incorporated in their entirety by reference.
The characteristics of the invention will be described in detail with reference to figures and examples.
Brief Description of the Drawings
FIG. 1 depicts a representative immunoblot illustrating the change in the level of selected proteins in human leukemia K562 cells treated for 4, 8, 16, 24 and 48 hours with 0.1% DMSO solution or BK124.1 at a concentration of 5 mM in 0.1% DMSO.
FIG. 2 depicts a representative immunoblot documenting the level of FOX03A and P21 proteins in the nuclear and cytoplasmic fractions of K562 cells treated for 8 or 24 hours with BK124.1 at a concentration of 5 mM in 0.1% DMSO. During incubation of K562 cells with BK124.1 , an increase in the level of FOX03A and P21 proteins is observed, mainly in the nuclear fraction, where P21 performs proapoptotic functions. The purity of separated fractions documents the level of control proteins: ACTB (beta-actin) - marker of cytoplasmic fraction, LMNB1 (laminin B1 ) - marker of nuclear fraction.
FIG. 3 shows the effect of BK 124.1 on the cell cycle analyzed by flow cytometry after staining with propidium iodide (PI). After treatment with BK124.1 , K562 cells were found to be arrested in the G2/M phase of the cell cycle. Representative histograms show the change in the cell cycle of K562 cells treated for 24 hours (A) or 48 hours (B) with 0.1% DMSO or BK124.1 at a concentration of 5 mM in 0.1% DMSO.
FIG. 4 presents the results of the MTT viability test for cells with multidrug resistance MDR1 of the K562-MDR1 line and for primary K562 cells (without multidrug resistance) treated with BK124.1 in a concentration range of 0.1-10 mM in 0.1% DMSO. BK124.1 exhibits equally high cytotoxicity/lethality against both cell types, evidencing unreduced sensitivity of K562- MDR1 cells to BK124.1 (IC50 for K562-MDR1 = 2.2 mM; IC50 for K562 = 2.5 mM). The results show the mean ± SD absorbance in wells with BK124.1 relative to absorbance in control wells with 0.1% DMSO for 3 independent experiments.
FIG. 5 illustrates the effect of BK124.1 as compared to the reference compounds: taxol and vincristine on cells with K562-MDR1 multidrug resistance after 24 h incubation. Treatment of K562-MDR1 cells with BK124.1 results in an increase of the percentage of MDR1 -positive cells (left panel) while maintaining high cytotoxicity of this compound (right panel). During the experiment, K562-MDR1 cells were treated with 0.1% DMSO or compounds such as BK124.1 , taxol and vincristine dissolved in 0.1% DMSO at a concentration of 2xlC5o, respectively. The cells were stained and analyzed by flow cytometry. (A) The plot shows the average percentage of MDR1 -positive cells ± SEM. (B) The plot shows the average percentage of living cells ± SEM, cells were stained with propidium iodide. The experiments were performed in at least 3 independent biological repeats, each in a technical duplicate. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001 .
FIG. 6 shows that BK124.1 has high cytotoxic activity and causes cell death via apoptosis regardless of the presence of MDR1 multidrug resistance in cells. Example on K562- MDR1 cells. A) Representative flow cytometry dot plots showing K562-MDR1 cells stained
with Annexin V and propidium iodide (PI) at different stages of apoptosis following administration of BK124.1 at a concentration of 2.5 mM or 5 mM in 0.1% DMSO. (B) The plot shows the average percentage of living K562-MDR1 cells ± SEM. (C) The plot shows the average SEM ± for the percentage of K562-MDR1 cells in the early and late stages of apoptosis. The experiments were performed in at least 3 independent biological repeats, each in a technical duplicate. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001.
FIG. 7 shows the results of the MTT viability test for CD34(+) stem cells isolated from the blood mononuclear cell fraction (PBMC) of patients with chronic myeloid leukemia (CML). The analysis was performed for BK124.1 in a concentration range 0.1 mM - 5 mM. The results indicate a very high cytotoxicity of BK124.1 against CD34+ cells. The calculated IC50 value = 1.5 mM. The graph shows the average viability of the cells ± SD for five patients (n = 5). The test for 5 mM BK124.1 was performed in 3 patients. The values of cell viability for each patient individually are presented as a black triangle. Cell viability was calculated on the basis of absorbance in wells with the compound relative to absorbance in control wells with 0.1% DMSO. Normal distribution was verified using the Shapiro-Wilktest, statistical analysis was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001.
Fig. 8 shows that BK124.1 induces death of CML patient’s CD34+ stem cells via apoptosis after 24 h of incubation with 2.5 mM or 5 mM BK124.1. In the experiment, CD34+ cells isolated from a CML patient were treated for 24 hours either with a 0.1% DMSO solution or BK124.1 dissolved in 0.1% DMSO at a concentration of 2.5 mM or 5 mM, and then the number of cells in early and late apoptosis (dead cells) was determined using FITC Annexin V labeling and flow cytometry. The graph shows the average percentage of CD34(+) cells ± SEM (A) alive and (B) in early apoptosis and (C) in late apoptosis (dead cells). The experiment was performed in at least 3 independent repeats, each in a technical duplicate. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001 .
FIG. 9 presents the results of a pharmacokinetic study of BK124.1 in rats (Wistar breed) after 5 min, 15 min, 30 min, 1 h, 2h, 6h and 24h from a single intravenous administration of the compound at a concentration of 5 or 10 mg/kg body weight. The upper panel shows the average concentrations of the compound in blood ± SD, the lower panel shows individual results for each rat.
FIG. 10 illustrates no effect of the carrier used in the administration of BK124.1 (10% Solutol FIS 15 / 10% ethanol) on tumor growth in a mouse xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells). Experimental conditions were given in Table 2.
Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001.
FIG. 11 shows antileukemic activity of BK124.1 in a mouse xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells) as determined by measuring tumor size at the end of the experiment. Administration of the compounds took place on days 3 - 17 after implantation of K562 cells. The experiment was completed on day 21. The diagram of the experiment was given in Table 2, and the results are summarized in Table 7. Each of the variants used was statistically significant compared to the control group, which was not treated with any compound. However, there were no statistically significant differences in tumor size for each of the compounds tested. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001.
FIG. 12 shows mean theoretical mass of the tumor observed during the experiment with BK124.1 administration in a xenogeneic model of CML leukemia (NSG mice with implanted human K562 cells). The formula for the theoretical mass of the tumor and the exact diagram of the experiment are presented in Table 2. Administration of the compounds was carried on days 3 - 17 after implantation of K562 cells. The experiment was completed on day 21. FIG. 13a shows the results compared to the control not treated with any compound, Fig. 13b provides a more detailed comparison of the effects of the individual compounds tested.
FIG. 13 provides an exemplary plot of the change in average weight of animals during the experiments presented in Fig. 13 testing the effect of compounds on tumor growth. The weight of animals receiving the test compound - BK124.1 at a dose of 20 mg/kg (IP) was found to be similar to that of animals receiving imatinib. The weight of animals not treated with compounds was increased due to the unlimited growth of tumor mass. The graph shows the results for an experiment in which the number of animals in each group N = 4. To maintain the readability of the graph, there are no standard deviations included.
FIG. 14 shows selected representative images of isolated tumors in the experiment depicted on Fig. 12 - 14. The tumors were isolated after 21 days from the K562 cell implantation.
Fig. 15 shows representative flow cytometry images (A) and percentages of cells (B) in subpopulations identified based on CD34/CD38 antigens in cells from PBMCs of CML and AML patients before any treatment (purity) and after treatment for 24 h with BK124.1 at a concentration of 2.5 microM and 5 microM. Data from individual patients is presented. Fig. 15C illustrates results of apoptosis assessment with Annexin V-FITC/Propidium iodide flow cytometry assay in CD34+/CD38+ and CD34+/CD38- cell subpopulations from CML or AML patients after treatment for 24 h with 0.1% DMSO or BK124.1 at the concentration of 2.5 mM or 5 mM.
Detailed description of the invention
Methods of CML treatment
Described herein are the methods of CML treatment based on BK124.1 monotherapy and combination therapy with imatinib (IM), which is an inhibitor of BCR-ABL kinase. This type of therapy can be used in treating or reducing the risk of IM resistance.
Described is a method for formulating and preparing a BK124.1 concentrate allowing administration of different doses. The preparation of BK124.1 thus prepared can be used in the treatment of cancer, in a preferred variant, e.g. leukemias, including CML.
The methods of using BK124.1 in monotherapy and in combination therapy for the CML treatment have been described. Any method known in the art can be used to diagnose CML, including detecting the presence of Philadelphia chromosome (Ph) translocation in the patient's leukemia cells. Other diagnostic methods can also be used. The CML stem cells described herein are characterized as bone marrow cells of the CD34(+) phenotype with the Ph chromosome present. Routine methods can be used to detect expression levels as well as to determine the phenotype of the patient's cells.
While the methods described herein refer, for example, to the treatment of CML, these methods can also be used to treat other BCR-ABL+ IM-resistant leukemia, e.g. Ph+ acute lymphocytic leukemia (-20% in adults, 5% in children), Ph+ acute myeloid leukemia (-2%) and potentially KIT+ gastrointestinal stromal neoplasms (GIST, as IM can also inhibit KIT kinase).
BK124.1 can be used in a combination therapy with other BCR-ABL inhibitors.
Many compounds that inhibit the activity of BCR-ABL tyrosine kinase are known, one of the most commonly used in medicine in CML therapy is imatinib (GLEEVEC). Further compounds used in medicine are Nilotinib (AMN107, Tasigna); Dasatinib (BMS-345825, Sprycel); Bosutinib (SKI-606, Bosulif); Ponatinib (AP24534 lclusig) (31 Jabbour 2018, 32 Soverini S 2019).
The diversity of mechanisms of resistance to IM creates the need to develop new therapeutic strategies that would prevent the development or overcome existing resistance in CML patients. Prominent in therapy are molecules belonging to the new generation of BCR- ABL inhibitors, which have not yet entered common clinical practice for CML and ALL therapy. They can also be used in a combination therapy together with BK124.1. These compounds are Bafetinib (INNO-406), Rebastinib (DCC-2036), Tozasertib (VX-680, MK-0457), Danusertib (PHA-739358), HG-7-85-01 , GNF-2 and -5, and 1 ,3,4 thiadiazole derivatives. These inhibitors are promising candidates for therapy because they are characterized by greater potency and high activity to many mutant domains of BCR-ABL kinase as compared to IM (33. Rossari F 2018 ).
BK124.1 affects the constitutive signaling of BCR-ABL1 kinase, known as the main cause of CML. This signaling includes pathways such as PI3K/AKT and JAK/STAT.
The activity of BK124.1 is associated with induction of the apoptosis process in target cells. In the first stages of activity this compound causes a decrease in STAT5, AKT, mTOR and P65 protein level. At the same time, an increase in F0X03A and P21 protein level is observed. On this basis, it can be assumed that as a result of the activity of BK124.1 , there is an increase in F0X03A protein level, which initiates an increase in P21 protein synthesis. In the next stage, these proteins are transported from the cytoplasm to the cell nucleus. Increasing the level of nuclear P21 triggers apoptosis, a natural process of programmed cell death, decreasing the cell population without the occurrence of an inflammatory process. Also importantly, the mechanism of action of this compound is therefore significantly different from that of kinase inhibitors such as imatinib, allowing for use thereof when resistance to treatment with these inhibitors develops.
The methods described herein include the manufacture and use of pharmaceutical compositions that include a dicarboxyimide derivative, e.g. BK124.1 , and optionally also a BCR-ABL inhibitor, e.g. IM, as active ingredients. The pharmaceutical compositions themselves are also included.
The pharmaceutical compositions usually comprise a pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier" as used herein includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like, compatible with pharmaceutical administration.
Pharmaceutical compositions are typically formulated to be compatible with the intended route of administration. Examples of routes of administration include parenteral administration, e.g. intravenous, intradermal, subcutaneous, oral (e.g. inhalation), percutaneous (topical), mucosal, intraperitoneal and rectal.
The methods for formulating suitable pharmaceutical compositions are known in the art and are described in the Polish Pharmacopoeia or in the European Pharmacopoeia, for example, solutions or suspensions used for parenteral, intradermal or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulphite; chelating compounds such as versenic acid; buffers such as acetates, citrates or phosphates and tonicity-adjusting agents such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral preparation may be closed in ampoules, disposable syringes or multidose vials made of glass or plastic.
Pharmaceutical compositions suitable for use for injection may include sterile aqueous solutions when the active ingredients are water-soluble, or dispersions and sterile powders for the preparation ex-tempore of sterile solutions or dispersions for injection. For intravenous administration, suitable carriers include saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be liquid to such an extent that there is an ease of injection. It should be stable under manufacturing and storage conditions and must be protected from the polluting effects of microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol and the like) and suitable mixtures thereof. Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the event of dispersion and by using surfactants. Preventing the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, timerosal and the like. In many cases, it will be advantageous to include isotonic agents in the composition, for example, sugars, polyalcohols, such as mannitol, sorbitol, sodium chloride. Prolonged absorption of the injectable composition can be achieved by including an agent that delays absorption in the composition, for example, aluminum monostearate and gelatin and corresponding mixtures thereof. Appropriate fluidity can be maintained, for example, by applying a coating such as lecithin, by maintaining the required particle size in the event of dispersion and by using surfactants.
Sterile injectable solutions can be made by introducing the active compound in the required amount into a suitable solvent together with one or a combination of the ingredients listed above, as required, and then sterilization by filtration.
Dispersions are generally produced by incorporating the active compound into a sterile carrier that contains the primary dispersion medium and optionally other components from those listed above. In the case of sterile powders for the manufacture of sterile injectable solutions, the advantageous ways of manufacturing are vacuum drying and freeze drying, which give the active ingredient powder plus optionally any additional desirable component from its previously sterile filtered solution.
Oral compositions in general contain an inert diluent or edible carrier. For oral therapeutic administration, the active compound can be combined with excipients, and used in the form of tablets, lozenges or capsules, e.g. gelatin capsules. Oral compositions can also be made using a liquid carrier for use as a mouthwash. Pharmaceutically compatible binders and/or adjuvant materials can be included as part of the composition. Tablets, pills, capsules,
lozenges and the like may contain any of the following ingredients or compounds having similar nature: a binder such as microcrystalline cellulose, tragacanth gum or gelatin; an excipient such as starch or lactose, a disintegrant such as alginic acid, Primogel or maize starch; a lubricant such as magnesium stearate or Sterotes; a lubricant, such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin or a flavoring agent such as peppermint, methyl salicylate or orange flavor.
For inhalation, the compounds can be provided as an aerosol from a pressurized container or dispenser that contains a suitable propellant, e.g. a gas such as carbon dioxide, or from a nebulizer.
Systemic administration of the therapeutic compound, as described herein, can also be carried out through the mucous membranes or transdermally. For administration through mucous membranes or transdermally, penetrating agents suitable for the barrier to be penetrated are used in the preparation. Such penetrants are generally known in the art and include, for example, for administration through mucous membranes, detergents, bile salts and fusidic acid derivatives. Administration through the mucous membranes can be achieved by using nasal sprays or suppositories. For transdermal administration, active compounds are formulated in ointments, lotions, gels or creams generally known in the art.
Pharmaceutical compositions can also be produced in the form of suppositories (e.g. with conventional vehicles for suppositories such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
In one form, the active compound(s) is (are) produced with carriers that will protect therapeutic compounds from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as [poly(ethylene-co-vinyl acetate)], polyanhydrides, Polyglycolide, collagen, poly(ortho-esters) and Polylactide [poly(lactic acid)] can be used. Such preparations can be manufactured using standard techniques or can be obtained commercially, e.g. from Alza Corporation and Nova Pharmaceuticals, Inc. They can be produced according to the methods known to persons skilled in the art, according to the requirements and specifications described in the European Pharmacopoeia.
Pharmaceutical compositions can be contained in ampoules, container, package or dispenser along with instructions for administration.
The therapeutic dose is the amount of compound sufficient to obtain beneficial or desirable therapeutic results. This dose may be the same or different from the prophylactic dose, which is the amount necessary to prevent the onset of symptoms of the disease or onset
of the disease. The therapeutic dose may be administered in one or more administrations, applications or doses. The therapeutic dose is characteristic of each compound, it can be administered once or more times a day, in addition, therapy can be continuous or can be used once or more times a week. The choice of therapeutic dose depends on the severity of the disease or disorder, previous treatment, general health and factors such as body weight, age of the patient and other accompanying diseases. Therefore, the therapeutic dose is selected by a person skilled in the art, who determines the dosage and time required to effectively achieve therapeutic effects.
Dosages, toxicity and therapeutic efficacy of compounds can be determined by standard procedures, which are in vitro experiments in cell cultures and in vivo experiments in animals. For this purpose, LD50 (lethal dose for 50% of the population) and ED50 (therapeutically effective dose in 50% of the population) can be determined. Based on the ratio between the toxic and therapeutic doses, therapeutic index is determined, which is expressed as the LD50/ED50 ratio. Compounds are considered therapeutically beneficial when their therapeutic indicators are high. Another parameter used may be the IC5o (the concentration at which proliferation/viability of tumor cells is inhibited by 50%, relative to the control). Toxic compounds may be used in therapy, but at the design stage, appropriate drug transport systems are created, which direct the molecules to the affected area. Such systems are created to minimize potential damage to normal cells and thus reduce side effects.
Data from cell culture and animal studies can be used to create a dosage range for humans. Such dosage is in the range of concentrations of compounds in the circulation, for which low toxicity or no toxicity is observed. The dosage may vary within that range depending on the dosage form and the route of administration used. The therapeutically effective dose has been initially estimated based on tests on cell cultures. Then the dose was tested in animal models, in which the IC5o value (i.e., the concentration of the test compound that inhibited symptoms at 50%) was determined. In addition, the activity of the compound tested was compared with the activity of the preparations used in therapy. Such information was used to determine therapeutic doses for humans.
In combination therapy, FDA-approved doses can be used, e.g., up to a maximum of 800 mg a day of IM and for BK124.1 based on in vitro, in vivo (mouse model), and ex vivo (patient blood model) experiments performed and described below, in which the optimal dosage without adverse effects is administration of BK124.1 twice a day at a daily dose of 20 to 30 mg/kg body weight.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
MATERIALS AND METHODS
The following examples use the following materials and methods.
Study Design
The overall object of the study was to understand the mechanisms underlying the activity of BK124.1 and to investigate its potential independent anti-cancer effects and synergistic effects on the increase in cytotoxicity of imatinib used in the treatment of CML and other leukemias, e.g. AML. The study was divided into an in vitro stage conducted on cell lines and an in vivo stage in which the compound was tested in a mouse model. These studies used, among others, the human CML cell line K562 (BCR-ABL+), a xenogeneic mouse model of human CML leukemia (NSG mouse with K562 cells administered), human cells isolated from the blood of individuals diagnosed with CML and AML. The study consisted of a series of controlled laboratory experiments in which a number of parameters such as cell viability, apoptosis, cell signaling pathway activity and leukemia progression were analyzed. For in vitro experiments on BCR-ABL+ cells (K562), all quantitative data were collected from experiments performed in at least three biological replicates.
For in vivo experiments, animals were randomly assigned to each group, and the results were analyzed in an unblinded manner. Animal sample sizes were chosen based on the procedure established in previous publications. To achieve statistical significance, the animal groups studied consisted of at least 4 individuals. K562 (BCR-ABL+) cells were used for mouse experiments.
Human CML cell line K562 cultures
CML cell line K562 (BCR-ABL+) was purchased from an ATCC supplier. Cells were cultured in RPMI 1640 medium containing HEPES (Biiest) with 2 mM GlutaMAX (Gibco), supplemented with 10% fetal bovine serum (Sigma) and 100 pg/mL penicillin and 100 U/mL streptomycin (Gibco). Cell viability was monitored using the Muse Cell Analyzer (Millipore).
Obtaining human leukemia K562 cells with multidrug resistance
K562 cells with multidrug resistance type 1 (MDR1) were selected in the Laboratory for Preclinical Research of Elevated Standard (Marceli Nencki Institute of Experimental Biology, Polish Academy of Sciences) according to the protocol published by Tsuruo (26 Tsuruo 1983). K562 cells were cultured in increasing concentrations of vincristine starting at the IC50 determined by MTT assay (Sigma). The final concentration of DMSO in the culture was 0.1%.
Vincristine dose was doubled every 2-3 weeks after the cells reached at least two confluences. Cell viability was above 90%, which was confirmed using the Muse Cell Analyzer (Millipore).
MTT assay for cell viability
K562 or K562-MDR1 cells were seeded into 50 pi 96-well dishes in the amount of 7x103 cells per well. On the following day, the test compounds dissolved in 0.1% DMSO were added to the cells. After 24 hours, 50 pi of medium with appropriate drug concentration was added to each well. The final DMSO concentration per well was 1% for K562 and K562-MDR1 , and 0.1% for CD34(+). After 48 hours, 20 mI of Tetrazolium Bromide (Thiazolyl Blue) at a concentration of 5 mg/mL was added to each well. Cells were incubated for 2-3 hours in a cell culture incubator and then 10% SDS, 1% HCI were added to each well to lyse the cells and dissolve the formazan crystals. Plates were left overnight at 37°C and further analyzed on iMark Microplate absorbance reader (BioRad). Results were calculated relative to control cells treated with DMSO alone.
Analyses by flow cytometry methods
Annexin V / propidium iodide - apoptosis measurement
K562 or K562-MDR1 cells were seeded in a 6-well plate at a concentration of 0.1 - 0.2 x106 cells/mL. CD34(+) cells were seeded at a concentration of 0.25 x106 cells/mL in a 24-well plate. After 24 hours of pre-incubation, the test compound dissolved in DMSO was added to achive a final concentration of DMSO of 0.1%. After 24 hours of incubation, cells were harvested and stained according to the manufacturer's protocol using FITC Annexin V apoptosis detection kit (BD Pharmingen). Stained cells were immediately analyzed on a BD FACSCalibur flow cytometer, (USA) using CellQuest software (BD Biosciences, USA).
Propidium iodide labeling and cytometric analysis of cell cycle profile
Cells were seeded at a concentration of 0.1 - 0.2 x106 cells/mL in a 6-well plate. After 24 hours of pre-incubation, the test compound - BK124.1 in 0.1% DMSO was added to the cells, and then the cells were further incubated for 24 or 48 hours. After incubation, cells were washed twice with ice-cold PBS solution (without Mg and Ca), resuspended in ice-cold 70% ethanol solution, and placed at -20°C for 24 hours. After this time, ethanol was washed off, the cells were resuspended in 50 pg/mL propidium iodide (Sigma) with 50 pg/L DNAase free RNase in PBS and incubated at 37°C for 30 minutes. The cell cycle was analyzed by flow cytometry on a FACSCalibur cytometer (Becton Dicknson, USA) using ModFit LT 3.2 software (Verity Software Flouse, USA).
Cytometric analysis of the presence of P-glvcoprotein responsible for MDR1 resistance
Cells were seeded at a concentration of 0.1 - 0.2 x106 cells/mL in a 6- or 24-well plate. After 24 hours of pre-incubation, a solution of 0.1% DMSO or the test compounds in 0.1% DMSO were added to the cells. The cells were then incubated for 24 hours. After this time, cells were harvested, washed with 0.5% BSA in PBS (Mg2+ and Ca2+ free), and labeled for 30 min on ice with FITC Mouse Anti-Human P-glycoprotein (CD243) clone 17F9 (BD Pharmingen) or an appropriate isotype control. After incubation with the antibody, cells were washed with PBS and analyzed with a BD FACSCalibur flow cytometer using CellQuest software (BD Biosciences).
Preparation of cell lysates and analysis of protein levels by immunoblotting
K562 cells were seeded at a concentration of 0.1 - 0.2 x106 cells/mL in a 6-well plate. After 24 hours of pre-incubation, the test compound - BK124.1 dissolved in 0.1% DMSO at the analyzed concentration was added and incubated for a specified time. In all experiments using this technique, the final DMSO concentration was 0.1%. After incubation, the cells were collected and lysed using a RIPA (Sigma) lysis buffer supplemented with complete Protease Inhibitor (Roche) and PhosSTOP (Roche). Protein concentration in cell lysates was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Lysates prepared for immunoblotting (Western blotting) analysis contained the same amount of protein (20 pg) in equal final volume. Protein lysates were then incubated for 5 min in Laemmli buffer (Biorad) at 95°C. SDS PAGE electrophoresis was performed using TGX StainFree FastCast (Biorad) in TGS Running Buffer (Biorad). After electrophoretic separation, transfer of proteins to membranes (AppliChem) in Towbin buffer was performed at 4°C for 1 h at 100 V. Both electrophoresis and transfer used BioRad apparatus. Membranes (AppliChem) were blocked with 5% non-fat milk powder and then incubated with primary antibodies (Cell Signaling Technology) overnight at 4°C with gentle shaking. HRP-conjugated anti-mouse and anti-rabbit secondary antibodies (Cell Signaling Technologies) were used to visualize the labeling results. Blots were developed and analyzed densitometrically using Clarity Western ECL Substrate from ChemiDoc XRS+ (Biorad). Protein levels normalized to controls were determined using ImageJ software.
Cytoplasmic and nuclear fraction isolation
K562 cells were plated at a concentration of 1.5 c 105 cells/mL in a 6-well plate. After 24 h of pre-incubation, test compound - BK124.1 , dissolved in DMSO, was added at the analyzed concentration and incubated for the specified time. In all experiments using this technique, the final DMSO concentration was 0.1%. Cells were harvested and washed with ice cold PBS. In order to isolate the cytoplasmic fraction, cells were resuspended in hypotonic buffer (10 mM HEPES pH 7.9; 10 mM KCI, 0.1 mM EDTA pH 8; 0.1 EGTA pH 8) supplemented
with complete Protease Inhibitor and PhosSTOP (Sigma-Aldrich, St. Louis, MO, USA). Cells were incubated on ice for 15 minutes, followed by addition of 25 pL of 5% NP-40 (Sigma- Aldrich, St. Louis, MO, USA) to each lysate and the tube was intensively vortexed for 10 seconds. Tubes were then centrifuged at 4 °C, 1,000 c g for 15 minutes. Cytosol fraction was collected as supernatant. The pellet was washed twice with hypotonic buffer and resuspended in hypertonic buffer (20 mM HEPES pH 7.9; 0.4 M NaCI, 1 mM EDTA pH 8; 1 mM EGTA pH 8) supplemented with complete Protease Inhibitor and PhosSTOP. Lysate was incubated for 30 minutes on ice, followed by centrifugation at 4 °C, 10,000 c g for 15 minutes. Nuclear fraction was collected as the supernatant. Protein concentration in cell lysates was measured using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Lysates contained the same amount of protein (at least 15 pg) in the same final volume. The purity of the separated fractions is documented by the level of control proteins: ACTB (beta- actin) - cytoplasmic fraction marker (Cell Signaling Technology, Danvers, MA, USA), LMNB1 (laminin B1 ) - nuclear fraction marker (Santa-Cruz Biotechnology, Dallas, TX, USA). SDS- PAGE Western Blot procedure was performed as described for whole cell lysates.
Experimental Animals
The experimental protocols used on animals have been approved by the Ethical Committee for Animal Experiments and are in accordance with national and European Union regulations on the planning and performing animal experiments. The groups and numbers of animals used for the experiments are shown in the tables in the description of experiment.
Assay of BK124.1 toxicity in vivo
Assay of in vivo toxicity of the tested compounds was performed in a mouse model. 8- week-old female Balb/c nude mice were randomly assigned to 5 groups of 4 mice each. On days 3-17, the selected solution was administered intraperitoneally as per protocol according to the schedule shown in Table 1.
On day 21 , the experiment was terminated, and blood and organs were collected for further analysis. Blood morphology was assessed by analyzing the following parameters: red blood cells [erythrocytes, RBC] monocytes [MONO], white blood cells [leukocytes WBC], lymphocytes [LYMPH], hematocrit [HCT], platelets [thrombocytes PLT], red blood cell volume distribution [RDW], mean corpuscular hemoglobin [MCH], mean corpuscular hemoglobin concentration [MCHC]
The obtained parameter values were compared with the values observed in the control group of animals. Body weight was measured daily during the experiment.
Investigation of tumor growth profile in a xenogeneic NSG mouse model
A xenogeneic NSG mouse model was generated as previously described (21 Huang R 2018, 22 Zhao WH 2017, 25 Vlahovic G 2007). 8-week-old female NSG mice (Jackson Laboratory) were injected into the dorsal fold with 1x106 K562 cells of the human chronic myeloid leukemia (CML) line in a volume of 100 mI (0.5% of mouse weight). On day 3 after cell administration, mice were randomly assigned to each group and on days 4-17 of the experiment, the selected solution was administered intraperitoneally once a day according to the schedule shown in Table 2. On day 21 , the experiment was terminated, the blood, tumors and remaining tissues were excised and fixed in buffered formalin for further analysis. Blood was analyzed for morphology. Subcutaneous tumors and body weight were measured daily during the experiment.
Measurement of tumor growth
During the experiment, the theoretical tumor volume [mm] was calculated according to the formula (a2 x b) * 0.5 , with a = shorter tumor size [mm], b = longer tumor size [mm]. Tumor growth was measured with an electronic caliper.
Study of human cells from patients with CML and AML
Blood samples were collected from patients diagnosed with chronic myeloid leukemia (CML) or with acute myeloid leukemia (AML) treated at the Hematology Department of the University Clinical Center of Warsaw Medical University, Banacha Street, Warsaw. The experimental protocols used for collection and analyzing blood plasma samples were approved by the Ethics Committee for Human Research at the Central Clinical Hospital of the Ministry of Internal Affairs and Administration in Warsaw and are in accordance with national and European Union regulations and the Code of Ethical Principles for Medical Research developed with the participation of members of the World Medical Association. Peripheral blood samples were collected from all subjects after obtaining written informed consent from patients or their legal representatives. Patients were diagnosed by qualified clinic staff. The
diagnosis was based on history, blood parameters, and molecular testing. CML patient characteristics are shown in Table 3.
Extraction and storage of peripheral blood mononuclear cells (PBMC) and blood stem cells CD34(+)
Blood samples from patients were collected by venipuncture and blood collection into BD Vacutainer EDTA-K2 tubes and transported to the laboratory within 1-2 hours. The resulting blood was diluted 1 :1 with a balanced salt solution (0.01% anhydrous D-glucose, 5 mM CaCI x 2H20, 98 mM MgCI2 x 6 H20, 0.54 mM KCI, 14.5 mM Tris, pH = 7.6 in 0.9% saline) and centrifuged on a Ficoll-Paque Plus (GE Healthcare). Interphase PBMC cells were washed twice with BSS, the resulting cells were counted and resuspended in the medium recommended by the manufacturer for CD34(+) cell isolation (EasySep™ Human CD34 Positive Selection Kit II, StemCell Technologies). Isolated CD34+ cells after counting and viability assessment using Muse Cell Analyzer (Millipore) were cultured in StemPro™ -34 SFM medium with fresh addition of the recommended cytokines IL-3 (50ng/ml_), GM-CSF (25ng/ml_) and SCF (100 ng/mL). For experiments with BK124.1 , cells were always pre-incubated for 24 hours. The purity of the CD34+ fraction was assessed using an APC antibody against human CD34 antigen (Biolegend) and propidium iodide (Sigma) and the corresponding isotype control (Biolegend).
Analysis of human CD34+ cells from CML patients
To analyze the effects of BK124.1 and IM on blood cells in CML samples, frozen cells from a patient were first heated for 10 minutes at37°C. Then 1 ml of pre-heated IMDM (Iscove's Modified Dulbecco's Media) defrosting medium with 5% FBS was added, the cells were washed with 10 ml of medium (IMDM with 5% FBS). The cells so thawed were centrifuged at 300 g for 10 min. The cell pellet was then resuspended in an appropriate volume of IMDM culture medium from [STEMCELL Technologies] and incubated at 37°C. After 36-48 hours of culture, the cells were used for the experiment. During the experiment, cells were treated for 24 or 48 hours with the solutions: DMSO, 5 mM imatinib IM, or 5 mM BK124.1. Cell viability in the target population was then calculated by multiplying the percentage of the target population that was obtained by FACS analysis and the total number of viable cells, as determined by trypan blue staining.
Analysis of purity and measurement of apoptosis in CD34+/CD38- leukemia stem cells isolated from blood of CML or AML patients
CD34+ cells isolated from peripheral blood of additional CML or AML patients, as described above, were seeded at a concentration of 2.5 c 105 cells/m L in a 24-well plate and cultured for 24 hours in StemPro™-34 SFM medium with fresh addition of cytokines as described above. For the purpose of purity analysis cells were harvested after 24 hours and stained with anti-human CD34-APC antibody, anti-human CD38-PE (Biolegend, San Diego, CA, USA) and propidium iodide according to the manufacturers’ protocols. For the purpose of apoptosis analysis, cells were treated with 2.5 mM or 5 mM BK124.1 , or DMSO, harvested after 24 hours, and stained with anti-human CD34-APC antibody, anti-human CD38-PE and Annexin V-FITC kit according to the manufacturer's' protocol. Appropriate isotype controls were included. In both cases stained cells were immediately analyzed on a BD LSRFortessa flow cytometer (BD, Franklin Lakes, NJ, USA) and analyzed with FlowJo software (BD, Franklin Lakes, NJ, USA). Purity was analyzed with doublet exclusion on live cells only. Analysis of apoptosis was performed with doublet exclusion and for each cell population (CD34+/CD38+ or CD34+/CD38-) separately. Experiments were performed at the Laboratory of Cytometry, Nencki Institute of Experimental Biology.
Statistics
All statistical data collected from in vitro experiments are from experiments performed in at least three biological replicates. Statistical data are expressed as mean +/- standard deviation (SD). Animal experiments are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed using Graph Pad Prism. For patient experiments, the normality of the distribution for the material was verified using the Shapiro-Wilk test. Statistical analysis for this experiment was performed using one-way ANOVA with post-hoc Dunnett’s
test * P <0.05, ** 0.001 <P <0.05, *** 0.0001 <P <0.001 , **** P <0.0001. Tumor Growth Inhibition (TGI) analysis was performed by analyzing the percentage of tumor growth inhibition for each experimental variant. TGI [%] was calculated according to the formula: TGI = 100 - {(mean tumor volume in treatment group / mean tumor volume in control) x 100 TGI (%). Such calculated value represents the percentage that the tumors obtained in a given variant are smaller than the tumors observed for the control group.
Example 1
Formulating BK124.1 for in vivo administration At the compound synthesis stage, solubility of BK124.1 was found only for 100%
DMSO. A number of solvents were analyzed to obtain a soluble pharmaceutical form of BK124.1 for in vivo administration. For further experiments, a solvent that is non-toxic and guarantees stability of the pharmaceutical formulation for a minimum of 7 days was selected. The results are presented in Table 4.
Based on a series of experiments, obtaining a liquid pharmaceutical formulation of BK124.1 was optimized. Formulation of BK 124.1 was divided into two steps. The first step consisted in dissolving BK 124.1 in a mixture of 50% lipophilic polymer/50% polar compound and obtaining its concentrate, the second step consisted in preparing from the obtained
concentrate a diluted solution of BK124.1 for intravenous or intraperitoneal administration once or twice a day and administering different doses of the active substance. The second step was also intended to facilitate filtration of the solution and increase the fluidity of the compound.
In the first step, 50 mI_ of Solutol HS 15 and 50 mI_ of anhydrous 99.8% ethanol were added to 1 mg of BK 124.1 to obtain a 50% Solutol HS 15 / 50% ethanol solution; the entirety was shaken (1-2 h) at 30-40°C until BK 124.1 was completely dissolved. The solution thus prepared was found to be stable for 48 hours at 30-36 °C. At room temperature, however, precipitation of the compound (increase in opalescence) is observed after only 6-12 h. This concentrate is viscous, settles on the walls, and has poor properties in terms of filtration and portioning the formulation. Therefore, such a concentrate must not be administered intravenously or intraperitoneally without dilution. Therefore, in the second step, the prepared concentrate was diluted with aqua pro injectione (water for injection) to obtain a concentration of 10% Solutol HS 15/10% ethanol. Such diluted solution was fluid, did not settle on the walls, and was convenient for filtration and for portioning the preparation. This solution was therefore sterilized by membrane filtration on a 0.2 pm filter (Minisart PES; Sartorius) and pipetted into sterile tubes of 2 ml. each, and these tubes were stored at room temperature for 48 h until used for injection in animals.
The described protocol makes it possible to prepare a BK124.1 solution at a concentration of 2 mg/mL, which after filtration is stable, easy to store, sterile and apyrogenic, easy to dose, and therefore meets the necessary requirements for a drug administered in vivo. Moreover, solution of the compound thus prepared is also easy to sell and transport. The solution thus prepared was used for direct administration to the experimental animals.
Further experiments in experimental animals showed that the concentrations of 10% ethanol and 10% Solutol HS 15 used in the solution of BK124.1 were the maximum concentrations of these solvents that were well tolerated by animals in intraperitoneal administrations. In addition, the volume of the preparation in such solvent for intraperitoneal administration to the animals ranged from 200 mI to a maximum of 400 mI in the experiments, consistent with the generally recommended administration volumes for these animals.
The two-step preparation of BK 124.1 described above permits oral and intraperitoneal administration in animals and humans. However, for administration by the intravenous route, including intravenous infusion in humans, it is optimal to further reduce ethanol and solute concentrations in the final formulation by a third dilution step of a solution of 10% ethanol and 10% Solutol HS 15. This is based on the experiments made by the present inventors in a rat model, where administration (especially repeatedly) of an aqueous solution of 10% ethanol and 10% Solutol HS 15 alone, without BK 124.1 , to the tail vein resulted in necropsy of the tail. For intravenous administrations in animals and humans, the described BK 124.1 solution should be further diluted in, for example, 5% glucose solution or 0.9% sodium chloride solution,
e.g., in polyethylene, polypropylene or glass vessels, but not in PVC vessels. Only a clear and colorless solution should be administered. The concentration of BK 124.1 in the solution for intravenous infusion may be in the range from 0.004 mg/mL to 0.1 mg/mL. Therapeutically effective doses of BK124.1 or its combinations with IM in the treatment of CML/AML may be determined by a skilled physician based on clinical findings, and adjusted for disease progression, patient age and weight, and risk of disease progression for a particular patient.
Example 2
Elucidation of the effect of BK124.1 on intracellular signaling associated with proliferation and apoptosis
The inventors chose to trace signaling pathways such as JAK/STAT5 and PI3K/AKT, which are the most well-known pathways associated with activation of BCR-ABL1 signaling in CML. For this purpose, the effects of BK124.1 at a concentration of 2xlC5o on K562 cells were studied during 4, 8, 16, 24, and 48 hour incubations. The experiments were performed by immunoblotting technique in at least 3 independent biological repeats. FIG. 1 depicts a representative immunoblot showing the change in the level of selected proteins observed in K562 cells treated for 4, 8, 16, 24 and 48 hours with 0.1% DMSO solution or BK124.1 at a concentration of 5 mM in 0.1% DMSO.
The first changes in protein levels under treatment with BK 124.1 were observed after 4 and 8 hours. Namely, the levels of such pro-life and cell division-stimulating proteins as STAT5 and AKT, as well as mTOR and P65 (other names: NF-KB, RelA) decreased, whilst the FOX03A and P21 protein levels increased (Fig. 1). P21 is a protein that, depending on the sub-cellular location, performs different functions. When localized in nucleus, it can induce apoptosis. FOX03A is a transcription factor that may play a role in initiating P21 transcription. An increase in the level of FOX03A was observed after 4 hours of incubation, the maximum increase was after 8 hours, and in the following hours a decrease in the level of this protein was observed. The increase in FOX03A level was accompanied by an increase in the P21 protein level, the elevated level of which persisted as long as 24 hours and decreased only after 48 hours. These results indicate that the increase in FOX03A levels initiated under the influence of BK 124.1 actually induces an increase in the P21 protein level in K562 cells.
Immunoblots were also performed to determine the FOX03A and P21 protein levels in the nuclear and cytoplasmic fractions of K562 cells treated for 8 or 24 hours with a solution of 0.1% DMSO or with BK124.1 at a concentration of 5 mM. For this purpose, the cytoplasmic and nuclear fractions of the cells were separated by a standard method of cell fractionation, in which, after homogenization, the cell lysate was subjected to differential centrifugation. The
experiment was carried out in at least 3 independent biological repeats, Fig. 2 represents one representative immunoblot (Fig. 2).
This analysis showed that the increase in the FOX03A and P21 protein levels occurs mainly in the nuclear fraction (Fig. 2). On this basis, it was suggested that the mechanism of activity of BK124.1 is related to P21 protein. In cells, during incubation with the test compound, cytoplasmic synthesis of P21 protein increases, which protein, when passing from the cytoplasm to the cell nucleus, stimulates the process of apoptosis. At the same time, BK 124.1 downregulates BCR-ABL kinase-dependent proteins that stimulate cell proliferation and viability.
Example 3
Demonstration of the effect of BK124.1 on the cell cycle
To further elucidate the mechanism of activity of BK124.1 , its effect on cell cycle profile was investigated. For this purpose, K562 cells were treated for 24 and 48 hours with BK124.1 at a concentration of 5 mM, which corresponded to a dose of 2 x IC50 for this compound. Control cells were treated with 0.1% DMSO solution at the same time points. After incubation, the cells were stained with propidium iodide (PI) and analyzed by flow cytometry. The results are shown in Fig. 3. During the study, the cells were found to be arrested in the G2/M phase of the cell cycle. Compared to control cells, there is an increase in the number of cells in the G2/M phase accompanied by a decrease in the number of cells in the G0/G1 and S phases after treatment with BK124.1.
In addition, after 48 hours of incubation (lower panel), the number of cells per cycle phase decreased significantly compared to the levels observed after 24 hours. These changes are likely due to activation of the apoptosis process after 24 hours, with a decreased number of cells in the G0/G1 phase compared to controls, while the number of cells in the G2/M phase was higher.
Example 4
Demonstration of BK124.1 cytotoxicity against K562 cells with acquired MDR1- type multidrug resistance.
We analyzed cytotoxicity of BK124.1 against cells with active and strong MDR1 expression, which may alter sensitivity of cells to the applied therapy. In order to achieve this, the K562 cell line was first established, cells of which strongly expressed MDR1 (K562-MDR1), (methodology outlined above, in Materials and Methods section).
The MTT assay of K562 and K562-MDR1 cell viability was then performed. This test showed no statistically significant differences in the cytotoxic activity of BK124.1 against K 562 (primary) and K562-MDR1 cells (Fig. 4).
Thus, the experiments lead to the conclusion that the activity of BK124.1 against the cells exhibiting MDR1 resistance mechanism does not decrease.
In the subsequent step, the cytotoxicity of the reference compounds, vincristine and taxol, was compared with that of BK124.1 against K562 and K562-MDR1 cells. For this purpose, K562-MDR1 cells were incubated at a concentration equivalent to 2xlC5o with BK124.1 (5 mM) or with taxol (1 pM) or with vincristine (1 pM) for 24 hours, and then cells were stained with propidium iodide and the antibody against P-glycoprotein (MDR1) to assess PgP (MDR1) levels and cell survival. Results (Fig. 5) showed that for taxol or vincristine concentrations of 2xlC5o, no increase in the percentage of MDR1 -positive cells is observed compared to control cells (Fig. 5A). In contrast, for BK124.1 a surprisingly high increase in the percentage of MDR1+ cells was observed. However, the increase in MDR1 activity after treatment with BK124.1 does not result in a decrease in the cytotoxicity of this compound. On the contrary, there is a statistically significant decrease in the percentage of viable K562-MDR1 cells treated with BK124.1 compared not only to control, but also to K562-MDR1 cells treated with taxol and vincristine. This result implies that even low expression of the PgP pump (MDR1 ) is sufficient to pump out a significant number of vincristine or taxol molecules, resulting in lesser K562-MDR1 cell mortality, whereas even increased PgP expression, under the influence of BK124.1 , is not sufficient to protect K562-MDR1 cells from death after treatment with BK124.1.
After the above results were obtained, an experiment was performed to check apoptosis level in cells treated and untreated with BK124.1 using Annexin V and propidium iodide staining. For this purpose, K562-MDR1 line cells were treated with 0.1% DMSO or BK124.1 at a concentration of 2.5 mM or 5 mM in 0.1% DMSO for 24 hours. The results of the cytometric analysis are shown in Fig. 6.
It was observed that after 24h incubation of K562-MDR1 cells with BK124.1 at a concentration of 2cIOdo (5pM), there is a significant increase in the number of cells in early and late apoptosis, well above 80%, while the level of viable cells decreased well below 20%. K562-MDR1 cells were also sensitive to a lower concentration of BK124.1 (2.5pM), in which a high percentage of cells in early phase of apoptosis was also observed.
In summary, it can be concluded that BK124.1 induces an increase in MDR1 expression, however this fact does not change the high proapoptotic activity of this compound, resulting in cell population death.
Example 5
Effects of BK124.1 on stem cells derived from CML patients
After confirming high activity of BK124.1 against K562 and K562-MDR1 CML cells,
the effects of the compound on leukemia stem cells isolated from patients were investigated. Cells were isolated from patients diagnosed with chronic phase CML. Diagnosed patients aged 39-79 years, mostly men, had been previously treated with imatinib (Table 3). In all cases, the diagnosis was confirmed by finding the presence of BCR-ABL1 transcript in the patient's cells. A cytotoxicity assay was performed on freshly isolated CD34(+) cells to determine their sensitivity to BK124.1. The assay was performed using the MTT method in a concentration range of 0.1 mM - 5 mM of BK124.1. In Fig. 7 mean CD34+ cell viability ± SD for five patients is shown. Cell viability was calculated on the basis of absorbance in wells with BK124.1 in 0.1% DMSO relative to absorbance in control wells with 0.1% DMSO alone (negative control). This experiment demonstrated high cytotoxicity of BK124.1 against leukemic CD34(+) stem cells of patients, as evidenced by an IC5o value of approximately 1 .5 pM and high reproducibility of results in all patients (Fig. 7).
Additionally, as in previous experiments in K562 cells treated with BK124.1 , the number of CD34+ stem cells from CML patients, the cells being in early and late apoptosis in response to selected concentrations of BK24.1 , was investigated (Fig. 8).
For this purpose, the cells were incubated for 24 hours with BK124.1 at doses of 2.5 pM and 5 pM in 0.1% DMSO or in 0.1% DMSO as a control. After incubation, the cells were labeled with Annexin V/propidium iodide and subsequently the level of apoptosis was assessed by flow cytometry.
The experiment showed (Fig. 8) that CD34(+) cells of patients treated with 0.1% DMSO control solution were in good condition, their viability reaching above 90%. Meanwhile, the viability of CD34(+) cells treated with BK124.1 solution was significantly lower for each of the BK124.1 doses analyzed, only a few percent of the cells could be considered as viable. In conclusion, CD34(+) cells isolated from patients treated with BK124.1 trigger the process of apoptosis, leading to destruction of their population.
Example 6
Determination of PK in a rat model.
Pharmacokinetics of BK124.1 was studied in the rat (Wistar) body after intravenous (IV) administration of 5 mg/kg or 10 mg/kg dose of BK124.1 in a 10% Solutol HS 15/ 10% ethanol solution (Fig. 9). Five individuals for each datapoint were used in pharmacokinetics studies in a rat model. For PK measurements in rats, blood was collected from the same individual at different time points. Concentration of BK124.1 in blood of rats was analyzed 5 min, 15 min, 30 min, 1 h, 2h, 6h, and 24h after a single administration of the compound. The concentration was measured by mass spectroscopy. Based on the obtained values of drug concentrations in the blood of animals, pharmacokinetic parameters were determined for
BK124.1.
The results indicate that after intravenous (IV) administration, BK124.1 persists in the bloodstream and undergoes gradual elimination in up to 24 hours (Fig. 9). No adverse reactions were observed immediately after administration of BK124.1 in 10% Solutol HS 15/ 10% ethanol solution.
Example 7
The study of pharmacokinetics of BK124.1 following IV versus IP route administration in mice
A series of experiments was then conducted to analyze pharmacokinetics and half-life ti/2 of BK124.1 in 10% Solutol HS 15/ 10% ethanol solution. The study was performed in a mouse model after intravenous (IV) administration at 20 mg/kg dose and after intraperitoneal (IP) administration at 20 mg/kg or 40 mg/kg dose. For each datapoint, blood from 4 animals was used, then the pharmacokinetic parameters of the test compound - BK124.1 were determined from the results obtained. Concentration of BK124.1 in blood was analyzed at 15 min, 6h, 12h, and 24h after a single administration of the compound. The concentration was measured by mass spectroscopy.
Based on the determined values of drug concentrations in peripheral blood, the half- life ti/2 of the test compound was determined (Table 5). Table 5. Pharmacokinetics of BK124.1 following IV versus IP route administration.
Based on the pharmacokinetic parameter ti/2 (half-life), it was concluded that BK124.1 should be administered 1 or 2 times a day at the tested doses. This dosage regimen is desirable for long-term therapy.
Example 8
Determination of non-toxic dose and route of non-toxic administration of BK124.1 repeatedly and in long-term in vivo in a mouse model
Subsequently, different doses of BK124.1 in 10% Solutol HS 15/ 10% ethanol solution were evaluated toxicologically after intravenous and intraperitoneal administration in 8-week- old female Balb/cOlaHsd-Foxn1nu (Balb/c nude) mice (Envigo sc). Toxicological parameters such as physiological and morphological changes at the site of administration of the compound, effects of the compound on the morphology of internal organs such as liver, kidney, heart, spleen, effects of the compound on animal blood count and body weight (body weight was measured every 48 h), and effects of the compound on animal behavior were considered.
No adverse effects were observed in mice with single intravenous administration
of BK124.1 in 10% Solutol HS 15/ 10% ethanol solution at doses of 5 mg/kg body weight, 10 mg/kg, and 20 mg/kg.
Some adverse effects were subsequently observed in mice with long-term intravenous (IV) administrations, once daily, every 24 h, for 14 days of BK124.1 substance in 10% Solutol HS 15/ 10% ethanol solution at doses of 5 mg/kg body weight, 10 mg/kg, and 20 mg/kg. Adverse effects included inflammation at the site of administration of the compounds after 3-4 days resulting in tail necropsy. The inflammation did not resolve when the compound was discontinued.
The reason for this adverse effect was probably small diameter of the capillaries in mice, which influenced the susceptibility to vascular blockage after contact of the composition used with blood proteins, rather than a direct toxic effect of the compound. It should be noted here that intravenous administration in rats (see Example 6) did not cause adverse reactions.
Subsequently, it has been investigated whether BK124.1 in a 10% Solutol HS 15/ 10% ethanol solution can be administered intraperitoneally in long-term, 1x or 2x aday, and which dose of the compound is non-toxic.
BK124.1 was administered to animals on days 4-17 of the 21 -day experiment according to the dosing regimen described in Materials and Methods (Table 1 ).
Toxicity at a dose of 40 mg/kg when administered once or twice a day was found consisting in decrease in the average weight of the animals, and based on observed change in the behavior of the animals. For a dose of 20 mg/kg as well as 30 mg/kg body weight administered IP once a day, no changes in animal body weight were observed compared to control animals not treated with the compounds. There were also no changes in the behavior of these animals, in the appearance of their internal organs as well as in blood count parameters. It is summarized that BK 124.1 can be safely administered every 24 h for two weeks via the intraperitoneal route at a dose of 20 mg/kg or 30 mg/kg body weight.
Example 9
Investigation the effect of carrier: 10% Solutol HS 15/ 10% ethanol on tumor growth in a xenogeneic model.
Effect of BK124.1 on tumor growth in a xenogeneic NSG mouse model (NOD.Cg- Prkdcscid N2rgtm1 Wjl/SzJ) was analyzed. For this purpose, 8-week-old female mice weighing 19-20 g were injected with 1 x106 K562 cells into the dorsal fold according to the previously described protocol (Materials and Methods). Then, on day 3 after cell administration, the animals were randomly divided into 2 groups of 4 individuals each and on days 4-17 of the experiment, a 10% Solutol HS 15/ ethanol 10% vehicle solution was administered once a day (IP) to one of the groups at a dose of 400 mI per mouse. After 21 days, the experiment was terminated, and blood and tumors were collected for further analysis. The results of the tumor size analysis are shown in Table 6 and collectively in Fig. 10.
Table 6. Effect of vehicle on tumor growth compared to control animals.
There were no significant statistical differences between tumor growth in control animals when compared to animals receiving intraperitoneal administration of 10% Solutol HS 15/ethanol 10% vehicle at a dose of 400 pl/mouse.
Example 10
Investigation the effect of BK124.1 on human CML cell proliferation in vivo (in a xenogeneic model)
Effect of BK124.1 on tumor growth in a xenogeneic NSG mouse model (NOD.Cg- Prkd( ad ll2rgtm1Wjl/ SzJ) was analysed. All experiments in the xenogeneic model were performed according to the same scheme, in which 8-week-old female mice weighing 19-20 g were injected with 1x106 human CML K562 line cells into the dorsal fold according to the protocol previously described in Materials and Methods. Then, on day 3 after cell administration, the animals were randomly divided into groups, and on days 4-17 of the experiment, a 10% Solutol HS 15/ ethanol 10% vehicle solution was administered once a day to one of the groups at a dose of 400 pi per mouse, and the other groups were administered with BK124.1 , positive control compounds: hydroxyurea and imatinib, or BK124.1 with
imatinib according to the data summarized in Table 7. After 21 days, the experiment was terminated, blood and tumors were collected for further analysis, and the results of tumor growth measurement are summarized in Table 7.
Table 7. Inhibition of tumor growth after treatment with BK124.1 in a xenogeneic model. Values are given for tumor measurements at day 21 after administration of K562 cells to animals.
The average results for tumor weight at the end of the experiment are also shown in Fig. 11. The change in theoretical mass of the tumor over the course of the experiment is shown in Fig. 12a and Fig. 12b. Each of the variants used was statistically significant as compared to the control group. In contrast, there were no statistically significant differences in tumor size when BK124.1 or BK124.1 and imatinib were administered to mice as compared to both control compounds.
A summary of the toxicity results after the end of the experiment is provided in Table 8 below. Changes in body weight of animals are also illustrated in Fig. 13.
Table 8. Results of the toxicity study of the compounds obtained at the end of the experiment. Parameters such as body weight, liver weight, white blood cell count (WBC) were analyzed. The reference value of WBC for mice is 3.0-15 x 109/L.
In Fig. 14 selected representative images of isolated tumors (21 days after cell administration) are shown.
The results indicate that BK124.1 at a dose of 20 mg/kg or 30 mg/kg has antitumor activity similar to the control compounds used in therapy.
In an in vivo xenogeneic model, human K562 cells were administered to immunodeficient animals. After treatment with BK124.1 at 20 mg/kg (IP), 30 mg/kg (IP) dose in a solution of 10% Solutol HS 15 with 10% ethanol administered once a day, there was a significant statistical inhibition of tumor growth, which was about 75% compared to the control. This inhibition was comparable with the activity of the reference compound - imatinib at 100 mg/kg administered once a day (p.o.). In addition, the combined effect of administering BK124.1 together with imatinib, at the same doses as the compounds administered individually, was examined. Combination therapy of the tested compounds with imatinib did not result in additional therapeutic effect, which, however, does not exclude creating such therapy for a selected group of patients in the future. For the tested BK124.1 compound (20mg/kg) (IP), and its combination with imatinib at 100 mg/kg (PO), there were no behavioral changes in the animals tested, no changes in blood count parameters and no changes in internal organs such as liver, spleen, kidney, lung, heart.
The results indicate that BK124.1 can be administered to the patient as a single unit dose once a day or in multiple daily doses. The therapeutically effective daily dose of BK124.1 for monotherapy in adult may range from 10 to 30 mg/kg body weight.
Example 11
The analysis of the apoptotic death triggered by BK124.1 in CD34+ cells from CML patients was deepened into the subpopulations of CML CD34+ cells distinguished by double labeling with anti-CD34 and anti-CD38 antibodies, particularly focusing on the CML CD34+/38- cells corresponding to tumor initiating leukemia stem cells (Fig. 15). Moreover, we extended the analysis to the CD34+/CD38- leukemia stem cells from patients with acute myeloid
leukemia (AML). The analysis involved CD34+/CD38- cells from additional newly diagnosed 3 CML and 2 AML patients. The percentages of cells in each CD34/CD38 subpopulation before and after treatment with BK124.1 are shown in Fig. 15 A, B. As shown in Fig. 15C, already within 24 h BK124.1 induced apoptosis in CD34+/CD38- leukemia stem cells from all the patients. The apoptotic death of CD34+/CD38- leukemia stem cells in response to BK124 was dose dependent and higher at 5 mM BK124.1 concentration. BK124.1 was particularly detrimental to CD34+/CD38- LSC from 2 CML and 1 AML patients. The individual differences in response among patients, both CML and AML, probably reflect variability of molecular mechanisms of LSC chemoresistance and of disease drivers in various patients.
The results obtained indicate that BK124.1 induces apoptosis in the rare CD34 + / CD38- CML-initiating stem cells as well as in CD34 + / CD38- AML-initiating stem cells. The results are shown in Fig. 15c.
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Claims
1. The compound of formula I:
or its pharmaceutically acceptable salt for use in the treatment of cancer, wherein the cancer is leukemia or another cancer characterized by a clonal increase in the number of white blood cells, wherein the cancer shows resistance to previous treatments, in particular multidrug resistance.
2. The compound of formula I for use according to claim 1 , wherein the cancer is myeloid leukemia, in particular chronic myeloid leukemia or acute myeloid leukemia.
3. The compound of formula I for use according to claim 2, wherein the individual to be treated, suffers from chronic myeloid leukemia and is in the phase of blast crisis or the individual to be treated, suffers from acute myeloid leukemia.
4. The compound of formula I for use according to any one of claims 1 -3, wherein the leukemia is chronic myeloid leukemia or acute myeloid leukemia with multidrug resistance or resistance to imatinib.
5. The compound of formula I for use according to any one of claims 1-4, wherein the compound of formula I, prior to administration to the individual to be treated, is dissolved in a mixture containing at least one pharmaceutically acceptable water-soluble polar compound and at least one pharmaceutically acceptable lipophilic polymer, and the concentrate thus obtained is then used to dilute and prepare the final form for administration, which is administered to the individual to be treated.
6. The compound of formula I for use according to claim 5, wherein the pharmaceutically acceptable water-soluble polar compound comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
7. The compound of formula I for use according to claim 5, wherein the pharmaceutically acceptable lipophilic polymer comprises from 40% to 60% by weight of the mixture used to dissolve the compound of formula I.
8. The compound of formula I for use according to any of claims 1 -7, wherein the compound of formula I, prior to administration to the individual to be treated, is dissolved in a mixture containing ethanol and macrogol-15-hydroxystearate, and the resultant concentrate is then used to dilution and preparation of the final formulation which is administered to the individual to be treated.
9. The compound of formula I for use according to claim 8, wherein the prepared concentrate is then diluted in an aqueous solution, and then sterilized, and the resultant formulation is administered to the individual to be treated.
10. The compound of formula I for use according to claim 9, wherein the compound of formula I is administered orally or intraperitoneally in a mixture containing ethanol 10% : macrogol-15- hydroxystearate 10%.
11. The compound of formula I for use according to claim 10, wherein the resulting concentrate diluted in an aqueous solution is then re-diluted, e.g. in a 5% glucose solution or a 0.9% sodium chloride solution, and the resulting formulation is administered intravenously.
12. The compound of formula I for use according to any one of claims 1-11 , wherein the compound of formula I is administered to an individual at least once a day, at a daily dose in the range of 10-30 mg/kg body weight.
13. The compound of formula I for use according to any one of claims 1-12, wherein the individual to be treated has previously undergone therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
14. The compound of formula I for use according to claim 13, wherein another BCR-ABL kinase inhibitor was imatinib.
15. The compound BK124.1 for use according to any of claims 1-14, wherein the compound of formula I is used as monotherapy.
16. The compound of formula I for use according to any of claims 1 -14, wherein the compound of formula I is used in combination with therapy with another BCR-ABL kinase inhibitor and/or MEK inhibitor.
17. The compound of formula I for use according to claim 16, wherein another BCR-ABL kinase inhibitor is imatinib.
18. The compound BK124.1 for use according to any one of claims 1-17, wherein the individual is a human.
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EP2687509A2 (en) * | 2012-07-16 | 2014-01-22 | Warszawski Uniwersytet Medyczny | Dicarboxyimides derivatives for use in the treatment of cancer |
WO2015175846A2 (en) * | 2014-05-16 | 2015-11-19 | University Of Massachusetts | Treating chronic myelogenous leukemia (cml) |
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EP2687509A2 (en) * | 2012-07-16 | 2014-01-22 | Warszawski Uniwersytet Medyczny | Dicarboxyimides derivatives for use in the treatment of cancer |
WO2015175846A2 (en) * | 2014-05-16 | 2015-11-19 | University Of Massachusetts | Treating chronic myelogenous leukemia (cml) |
Non-Patent Citations (3)
Title |
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CIESLAK MARCIN ET AL: "New Thalidomide-Resembling Dicarboximides Target ABC50 Protein and Show Antileukemic and Immunomodulatory Activities", BIOMOLECULES, vol. 9, no. 9, 4 September 2019 (2019-09-04), pages 446, XP055919153, Retrieved from the Internet <URL:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6770581/pdf/biomolecules-09-00446.pdf> DOI: 10.3390/biom9090446 * |
GRODNER BLAZEJ ET AL: "Capillary electrophoresis for the investigation of two novel aminoalkanol derivatives of 1,7-diethyl-8,9-diphenyl-4-azatricyclo[5.2.1.02,6] dec-8-ene-3,5,10-trione as potential anticancer drugs in water solution and serum", JOURNAL OF SEPARATION SCIENCE, vol. 43, no. 3, 28 November 2019 (2019-11-28), DE, pages 648 - 656, XP055919115, ISSN: 1615-9306, Retrieved from the Internet <URL:https://onlinelibrary.wiley.com/doi/full-xml/10.1002/jssc.201900648> DOI: 10.1002/jssc.201900648 * |
KURAN BOZENA ET AL: "New, Substituted Derivatives of Dicarboximides and their Cytotoxic Properties", ANTI-CANCER AGENTS IN MEDICINAL CHEMISTRY, 2016, 16, 852-864, 1 January 2016 (2016-01-01), XP055919088, Retrieved from the Internet <URL:http://www.eurekaselect.com/article/73887> [retrieved on 20220509] * |
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