Patent Application
20200281897
The present invention is comprised in the field of medicine and pharmacy, and relates to the use of new compounds and the pharmaceutically acceptable derivatives thereof in the production of a medicinal product for the prevention, relief, improvement, and/or treatment of cancer, more specifically for the treatment of hematological cancer, and even more preferably for the treatment of acute myeloid leukemia (AML) and monoclonal gammopathies generally, and multiple myeloma (MM) particularly.
Hematological cancer, also known as hematological neoplasms or hematological malignancies, are a heterogeneous group of malignant diseases affecting the blood, bone marrow, and lymph nodes.
The WHO classifies hematological neoplasms according to their myeloid or lymphoid origin (Vardiman and James W, 2009. Blood Journal 114:937-951).
Chronic myeloproliferative Neoplasms (CMN)
Myeloid and Lymphoid Neoplasms with Eosinophilia and PDGFRA, PDGFRB, or FGFR1 Abnormalities
Precursor Cell Lymphoid Neoplasms
Mature B-Cell Neoplasms
Mature NK-Cell and T-Cell Neoplasms
Hodgkin's Lymphoma
Acute myeloid leukemia (AML, or LMA in Spanish) is known by many other names, including acute myelocytic leukemia, acute myelogenous leukemia, acute granulocytic leukemia, and acute non-lymphocytic leukemia. It is the most common type of acute leukemia in adults. In normal conditions, the bone marrow produces cells called myeloblasts which, upon maturation, turn into granulocytes, i.e., cells responsible for defending the body against infections.
In AML, cells from the myeloid line (myeloblasts) proliferate abnormally, progressively invading the bone marrow and interfering with the production of normal blood cells, leading to medullary insufficiency and extramedullary tissue infiltration.
Often, AML is the final stage of other diseases such as chronic myeloproliferative syndromes or myelodysplastic syndromes. The incidence of AML is very high among patients with certain chromosomal abnormalities such as Down syndrome or Fanconi anemia.
AML is a disease of adults, although it can sometimes be observed in children. This type of leukemia represents 40% of all leukemias in the western world. The incidence of AML in Spain is estimated at 15 new cases per million of inhabitants per year.
AML patients have a median age of 64 years old and most patients are between 60 and 75 years old.
AML therefore represents a heterogeneous group of diseases caused by a clonal disorder resulting from genetic abnormalities in hematopoietic stem cells. Most patients have a poor prognosis. In this sense, only between 40% and 55% of adults over 60 years of age achieve complete remission, with long-term survival rates. For younger patients, about 60% to 80% achieve complete remission with the standard treatment. However, only 20% and 30% enjoy a long-term disease-free survival. Despite the new medicinal products that have been designed in recent years, relevant advances in terms of response or survival have not been obtained, and accordingly the standard treatment is still based on the conventional combination of cytarabine and anthracycline. Therefore, the identification of new active compounds in the treatment of this disease is a medical need that is yet to be satisfied.
Multiple myeloma (MM) is the second most common hematological neoplasm. In recent years, therapeutic improvements brought about by immunomodulatory drugs and proteasome inhibitors, among other agents, have allowed a significant progress in the control of this disease. However, MM is still considered incurable. Therefore, considerable efforts are being dedicated to uncover new drugs for improving the therapeutic arsenal available today.
Cannabinoids are the active components of Cannabis sativa (marijuana). The therapeutic interests of cannabinoids came following discovery of an endocannabinoid physiological control system in humans, based on cannabinoid receptors (CBs), referred to as CB1 and CB2. While CB1 is extremely abundant in the central nervous system (CNS), CB2 is almost only present in hematopoietic and immune cells, which also express CB1 although to a much lesser extent. Some subsets of hematopoietic cells show high levels of CB2, particularly B-cells, plasma cell precursors. Despite this information, research on the effect of cannabinoids in the hematopoietic system has been less extensive than in the CNS, and many characteristics of the CB2 function and regulation are still rather poorly characterized.
There is increasingly more evidence supporting that cannabinoids may be useful in the treatment of diseases such as glaucoma, osteoporosis, multiple sclerosis, pain, cardiovascular disorders, and neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases. Furthermore, they are used to mitigate vomiting associated with cancer treatment. One of the most exciting therapeutic interests of cannabinoid research lies in its potential antitumor activity. In this sense, some cannabinoids inhibit the proliferation of various tumor cells, such as glioma cell lines, both in vitro and in vivo. This effect seems to be mediated by the modulation of several signaling pathways involved in cell proliferation, survival, and apoptosis.
MM has an incidence rate of 4-5 per 100,000 inhabitants per year. The age of onset is about 65 years old, and although the therapeutic arsenal has been expanded in recent years with the development of new molecules such as proteosome inhibitors or immunomodulatory drugs (IMIDs), which have added to the conventional treatments such as melphalan and prednisone, as well as hematopoietic progenitor transplant, multiple myeloma is still considered an incurable disease.
In that sense, with the treatments available today, the five-year survival for multiple myeloma is still low, particularly when compared to other types of cancer. Furthermore, there is a need to take into account the side effects of the treatments. For this reason, there is a need to provide alternative treatments with respect to the current ones which do not affect normal cell viability, including the hematopoietic cells from healthy donors post-transplant.
A first aspect of the invention relates to the use of a compound, hereinafter compound of the invention, of general formula (I):
where R1 and R4 are members of the group consisting of hydrogen, halogen, nitro, or amino.
R2 is a member of the group consisting of propyl, butyl, pentyl, cyclohexylmethyl, phenethyl, naphthylmethyl, heterocycloalkyl, primary, secondary or tertiary amine, or substituted benzyl, wherein the phenyl group may contain 1 or 2 substituents of the group consisting of alkyl, hydroxy, methoxy, nitro, amino, or halogen;
R3 is a member of the group consisting of methyl, ethyl, propyl, pentyl, cycloalkylmethyl, cycloalkylethyl, dialkylaminoethyl, heterocycloalkylethyl, cycloalkylcarbonyl (carbonyl group attached to cycloalkyl), heteroarylcarbonyl (carbonyl group attached to heteroaryl), optionally substituted arylcarbonyl (carbonyl group attached to aryl), or optionally substituted aralkylcarbonyl (carbonyl group attached to aralkyl);
or any of the pharmaceutically acceptable salts, esters, tautomers, solvates, and hydrates thereof, or any of the combinations thereof, in the production of a medicinal product for the prevention, relief, improvement, and/or treatment of cancer. More preferably, it relates to the use of a compound of the invention for the production of a medicinal product for the treatment of cancer.
In a preferred embodiment of this aspect, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy.
In another preferred embodiment of this aspect of the invention:
In another preferred embodiment:
In an even more preferred embodiment, the compound of the invention is selected from the list consisting of:
Alternatively, the present invention also relates to a compound of general formula (II):
the pharmaceutically acceptable salts, tautomers, prodrugs, solvates, and hydrates thereof, where
In a preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R3 is selected from optionally substituted aryl, optionally substituted aralkyl, 2-thienyl, and 4-chloro-3-pyridyl.
In a more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R3 is selected from 1-naphthyl, 2-naphthyl, 4-tolyl, 3,4,5-trimethylphenyl, 2-benzyloxyphenyl, 3,4,5-trimethoxyphenyl, 2,3-dichlorophenyl, 2,3-difluorophenyl, 2,6-dichlorophenyl, 2,3,6-trifluorophenyl, 2-chlorophenyl, 3-fluorophenyl, 3-chloro-2-fluorophenyl, 4-biphenylol, 4-chlorobenzyl, 4-methoxybenzyl, and 1-adamantyl.
In an even more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R3 is selected from 1-naphthyl, 2-naphthyl, 2-benzyloxyphenyl, 2,3-dichlorophenyl, and 4-methoxybenzyl.
In a preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is selected from heterocycloalkyl, diisopropylamino, dimethylamino, and diethylamino.
In a more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is a heterocycloalkyl.
In an even more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is selected from piperidinyl, morpholinyl, and pyrrolidinyl.
In an even more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is piperidinyl.
In a preferred embodiment, the present invention relates to the use of a compound of general formula (II), where n is selected from 2 and 3.
In a preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is a heterocycloalkyl and R3 is selected from 1-naphthyl, 2-naphthyl, and substituted phenyl.
In a more preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is a heterocycloalkyl and R3 is selected from 1-naphthyl, 2-naphthyl, 2-benzyloxyphenyl, 2,3-dichlorophenyl, and 4-methoxybenzyl.
In another preferred embodiment, the present invention relates to the use of a compound of general formula (II), where R4 is a heterocycloalkyl, R3 is selected from 1-naphthyl, 2-naphthyl, or substituted phenyl, and R1 and R2 are independently selected from hydrogen and halogen.
In a preferred embodiment, the present invention relates to the use of a compound of general formula (I), where R4 is —NR5R6 and R3 is selected from heteroaryl, optionally substituted aryl, and optionally substituted aralkyl.
In a more preferred embodiment, the present invention relates to the use of a compound of general formula (I), where R4 is —NR5R6 and R3 is selected from 1-naphthyl, 2-naphthyl, and substituted phenyl.
In a more preferred embodiment, the present invention relates to the use of a compound of general formula (I), where R4 is —NR5R6, R3 is selected from 1-naphthyl, 2-naphthyl, or substituted phenyl and R1 and R2 are independently selected from hydrogen and halogen.
In a preferred embodiment, the compound of the invention is selected from the list comprising:
More preferably, the compounds of the invention used in the production of a medicinal product for the prevention, relief, improvement, and/or treatment of a hematological cancer are:
Even more preferably, the compound is PGN128 (2,4,6-trimethylphenyl)(3-(3-piperidinopropoxy)-1-indazolyl)ketone.
According to the present specification, any of the compounds defined above, i.e., those compounds corresponding to general formula (II), can also be referred to in this specification as “compound or compounds of the invention”.
In another preferred embodiment, the compounds of the invention described by formulae (I) and (II) do not include:
In another preferred embodiment of the first aspect of the invention, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In an even more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
A second aspect of the present invention relates to the use of a composition, hereinafter composition of the invention, comprising or consisting of a compound of the invention, or any of the pharmaceutically acceptable salts, esters, tautomers, solvates, and hydrates thereof, or any of the combinations thereof, for the production of a medicinal product for the prevention, relief, improvement, and/or treatment of cancer.
In a preferred embodiment of this aspect, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy. In a preferred embodiment of this aspect of the invention, the composition further comprises one or more pharmaceutically acceptable excipients, or consists of a compound of the invention and one or more pharmaceutically acceptable excipients. In another preferred embodiment, the composition further comprises another active ingredient. In a more preferred embodiment, the other active ingredient is selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony-stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any of the combinations thereof. In an even more preferred embodiment, the other active ingredient is dexamethasone. In another even more preferred embodiment, the other active ingredient is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalidomide or thalidomide.
In another preferred embodiment of this second aspect of the invention, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In a more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
A third aspect of the invention relates to a combined preparation, hereinafter combined preparation of the invention, comprising or consisting of:
a) component A which is a compound (compound of the invention) or a composition (composition of the invention) as defined in the present invention; and
b) component B which is an active ingredient selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony-stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any of the combinations thereof.
In an even more preferred embodiment, the active ingredient of (b) is dexamethasone. In another even more preferred embodiment, the active ingredient of (b) is melphalan. In another preferred embodiment, the combined preparation of the invention further comprises pharmaceutically acceptable excipients. In another preferred embodiment, the combined preparation of the invention comprises only those mentioned above as active ingredients, although it may comprise other pharmaceutically acceptable excipients and vehicles.
A fourth aspect relates to the use of the combined preparation of the invention in the production of a medicinal product for simultaneous, separate, or sequential use in therapy. A preferred embodiment of this aspect relates to the use of the combined preparation of the invention, where components A (a) and B (b) are administered simultaneously, separately, or sequentially for the prevention, relief, improvement, and/or treatment of cancer.
In a preferred embodiment of this aspect of the invention, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy.
In another more preferred embodiment, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In an even more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
The authors of the present invention describe for the first time the use of indazole compounds of the present invention for the prevention, relief, improvement, and/or treatment of cancer, preferably hematological cancer, more preferably acute myeloid leukemia or monoclonal gammopathy, and even more preferably, among monoclonal gammopathies, those selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. Particularly, the monoclonal gammopathy is multiple myeloma. Multiple myeloma (MM) is a neoplasm which is characterized by clonal proliferation of malignant plasma cells in the bone marrow and is associated with the presence of monoclonal component or protein M in blood and/or serum.
The therapeutic improvements brought about by immunomodulatory drugs such as lenalidomide and proteosome inhibitors such as bortezomib, among other agents, have allowed a significant progress in the control of this disease. However, MM is still considered incurable. In order to achieve an effective therapy against this disease, the authors of the present invention have developed and tested indazole compounds of synthetic origin which have an anti-tumor effect.
Specifically, it is demonstrated that the different compounds under studied induce selective apoptosis in myeloma cell lines and plasma cells in the first stage of malignancy in MM patients, without affecting the viability of the normal cells of healthy donors, including hematopoietic stem cells. This antiproliferative effect is mediated by the activation of caspases, mainly caspase 2, and partially prevented by a pan-caspase inhibitor. Indazole compound-induced apoptosis correlated with an increase in the expression of Bax and Bak and a decrease in Bcl-xL and Mcl-1. Furthermore, treatment with indazole compounds induced a biphasic Akt/PKB response and significantly increased the levels of ceramide in MM cells. Surprisingly, blocking the synthesis of ceramide prevented indazole compound-induced apoptosis, which indicates that ceramides play a key role in the pro-apoptotic effect of said compounds in MM cells. Furthermore, blocking the cannabinoid receptor CB2 also inhibited indazole compound-induced apoptosis. The compound WIN-55 increased the anti-myeloma activity of dexamethasone and melphalan, overcoming cell resistance to melphalan in vitro. Finally, the administration of the cannabinoid WIN-55 to a plasmacytoma mouse model significantly suppressed tumor growth in vivo.
The authors of the present invention have observed a dramatic anti-leukemia effect with specific CB2 agonists which was completely reverted when the cells were co-incubated with antagonists of CB2 receptors, an intriguing data compared to other authors who suggest that CB2 may be a proto-oncogene that is involved in leukemogenesis because, when it is overexpressed in myeloid precursors, Cb2 induces blockade of neutrophilic development and stimulates the migration of Cb2-expressing cells in vitro.
In the present invention, the inventors show a new family of CB2 cannabinoid-specific derivatives which reduce the viability of AML cells. Furthermore, this effect was highly selective, given that the viability of normal healthy cells, including hematopoietic stem cells, remained unaffected. Furthermore, the data demonstrates that a synthetic agonist of the cannabinoid receptor, WIN 55,212-2, potently and in a dose-dependent manner induced by the apoptotic cell death of AML cells.
All in all, the data suggests that these indazole compounds can be considered therapeutic agents in the treatment of cancer, preferably a hematological cancer, more preferably in the treatment of acute myeloid leukemia or a monoclonal gammopathy, and specifically, among monoclonal gammopathies, multiple myeloma.
The present invention therefore relates to the use of indazole derivatives in the production of a medicinal product for the prevention, relief, improvement, and/or treatment of a monoclonal gammopathy, preferably multiple myeloma.
Therefore, a first aspect of the invention relates to the use of a compound, hereinafter compound of the invention, of general formula (I):
where
In a preferred embodiment of this aspect, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy.
In a preferred embodiment of this aspect of the invention:
In another preferred embodiment:
In an even more preferred embodiment, the compound of the invention is selected from the list consisting of:
In another preferred embodiment of the first aspect of the invention, the monoclonal gammopathy is selected from the list consisting of multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In a more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
In this specification, when mention is made to the term “pharmaceutically and/or physiologically acceptable salts or solvates,” it refers to any pharmaceutically acceptable salt, ester, solvate, or any other compound which, when administered, is capable of providing (directly or indirectly) a compound such as those described herein. Nevertheless, it will observed that non-pharmaceutically acceptable salts also fall within the scope of the invention, because they may be useful for the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs, and derivatives may be carried out by means of methods known in the state of the art.
For example, the pharmaceutically acceptable salts of the compounds provided herein are synthesized from the compound of the invention by means of conventional chemical methods. Such salts are generally prepared, for example, by reacting free acid or base forms of these compounds with a stoichiometric amount of the suitable base or acid in water, or in an organic solvent, or in a mixture of both. Generally, non-aqueous media such as ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Examples of acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulfate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulfonate, and p-toluenesulfonate.
Examples of base addition salts include inorganic salts such as, for example, sodium, potassium, calcium, ammonium, magnesium, aluminum, and lithium, and organic base salts such as, for example, ethylenediamine, ethanolamine, N,N-dialkylenethanolamine, glucamine, and basic amino acid salts.
The compounds of the present invention represented by formula (I) may include isomers, depending on the presence of multiple bonds, including optical isomers or enantiomers, depending on the presence of chiral centers. The individual isomers, enantiomers, or diastereoisomers and the mixtures thereof fall within the scope of the present invention, i.e., the term isomer also refers to any mixture of isomers, such as diastereomers, racemic isomers, etc., even the optically active isomers thereof or the mixtures thereof in different proportions. The individual enantiomers or diastereoisomers, as well as the mixtures thereof, can be separated by means of conventional techniques.
Likewise, the prodrugs of the compounds of formula (I) fall within the scope of this invention. As it is used herein, the term “prodrug” includes any derivative of a compound of formula (I), for example and in a non-limiting manner: esters (including carboxylic acid esters, amino acid esters, phosphate esters, sulfonate esters of metal salts, etc.), carbamates, amides, etc., which when administered to an individual can be transformed directly or indirectly into said compound of formula (I) in the mentioned individual. Advantageously, said derivative is a compound which increases the bioavailability of the compound of formula (I) when administered to an individual, or it enhances the release of the compound of formula (I) in a biological compartment. The nature of said derivative is not critical provided that it can be administered to an individual and can provide the compound of formula (I) in a biological compartment of an individual. The preparation of said prodrug can be carried out by means of conventional methods known by those skilled in the art.
As it is used herein, the term “derivative” includes both pharmaceutically acceptable compounds, i.e., derivatives of the compound of formula (I) which can be used in the production of a medicinal product or food compositions, and non-pharmaceutically acceptable derivatives because they may be useful in the preparation of pharmaceutically acceptable derivatives.
The compounds of the invention can be in a crystalline form as free compounds or solvates. In this sense, as it is used herein the term “solvate” includes both pharmaceutically acceptable solvates, i.e., solvates of the compound of formula (I) which can be used in the production of a medicinal product, and non-pharmaceutically acceptable solvates, which may be useful in the preparation of pharmaceutically acceptable salts or solvates. The nature of the pharmaceutically acceptable solvate is not critical provided that it is pharmaceutically acceptable. In a particular embodiment, the solvate is a hydrate. The solvates can be obtained by conventional solvation methods known by those skilled in the art.
For their application in therapy, the compounds of formula (I), the salts, prodrugs, or solvates thereof, will preferably be in a pharmaceutically acceptable or substantially pure form, i.e., having a pharmaceutically acceptable level of purity, excluding normal pharmaceutical additives such as diluents and carriers, and not including material considered toxic at normal dosage levels. The levels of purity for the active ingredient are preferably greater than 50%, more preferably greater than 70%, and even more preferably greater than 90%. In a preferred embodiment, they are greater than 95% of the compound of formula (I), or of the salts, solvates, or prodrugs thereof.
Particularly preferred derivatives or prodrugs are those which increase the bioavailability of the compounds of the invention when they are administered to the subject (for example, allowing an orally administered compound to be absorbed more quickly, accelerating its passage to the blood) or improve the supply of the compound to a biological compartment (for example, the brain or lymphatic system) with respect to the initial compound.
A second aspect of the present invention relates to the use of a composition, hereinafter composition of the invention, comprising or consisting of a compound of the invention, in the production of a medicinal product for the prevention, relief, improvement, and/or treatment of cancer. Alternatively, it relates to the composition of the invention for use in the prevention, relief, improvement, and/or treatment of cancer.
In a preferred embodiment of this aspect, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy.
In a preferred embodiment of this aspect of the invention, the composition further comprises one or more pharmaceutically acceptable excipients or vehicles. Preferably, the composition of the invention is a pharmaceutical composition comprising as the only active ingredient a compound of the invention, although it may comprise one or more pharmaceutically acceptable excipients and/or vehicles. In another preferred embodiment, the composition further comprises another active ingredient. In a more preferred embodiment, the other active ingredient is selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony-stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any of the combinations thereof. In an even more preferred embodiment, the other active ingredient is dexamethasone. In another even more preferred embodiment, the other active ingredient is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalidomide or thalidomide.
In another preferred embodiment of this second aspect of the invention, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In a more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
The pharmaceutically acceptable adjuvants and vehicles which can be used in said compositions are adjuvants and vehicles known by those skilled in the art and commonly used in the production of therapeutic compositions.
In the sense used herein, the expression “therapeutically effective amount” refers to the amount of the agent or compound capable of developing the therapeutic action determined by the pharmacological properties thereof, which is calculated to produce the desired effect, and will generally be determined, among other causes, by the actual characteristics of the compounds, including the patient's age, condition, the severity of the abnormality or disorder, and the route and frequency of administration.
The compounds described in the present invention, the salts, prodrugs, and/or solvates thereof, as well as the pharmaceutical compositions containing them, can be used together with other additional drugs or active ingredients to provide a combination therapy. Said additional drugs can be part of the same pharmaceutical composition, or alternatively can be provided in the form of a separate composition for simultaneous or non-simultaneous administration with the pharmaceutical composition comprising a compound of formula (I), or a salt, prodrug, or solvate thereof.
As it is used herein, the term “active ingredient,” “active substance,” “pharmaceutically active substance”, or “pharmaceutically active ingredient” means any component that potentially provides pharmacological activity or another different effect in the diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of humans or other animals. The term includes those components that promote a chemical change in the production of the drug and are present therein in a modified form envisaged for providing the specific activity or effect.
Another aspect of the invention relates to a dosage form, hereinafter dosage form of the invention, comprising the compound of the invention or the composition of the invention.
In this specification, “dosage form” is understood as the mixture of one or more active ingredients with or without additives having physical characteristics for suitable dosing, preservation, administration, and bioavailability.
In another preferred embodiment of the present invention, the pharmaceutical compositions and dosage forms of the invention are suitable for oral administration in solid or liquid form. The possible forms for oral administration are tablets, capsules, syrups, or solutions and they may contain conventional excipients known in the pharmaceutical field, such as binding agents (e.g., syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone), fillers (e.g., lactose, sugar, cornstarch, calcium phosphate, sorbitol, or glycine), disintegrants (e.g., starch, polyvinylpyrrolidone, or microcrystalline cellulose) or a pharmaceutically acceptable surfactant such as sodium lauryl sulfate. Other dosage forms can be colloidal systems which include, among others, nanoemulsions, nanocapsules, and polymer nanoparticles.
The compositions for oral administration can be prepared as a mixture and dispersion using conventional Galenic pharmacy methods. The tablets can be coated following the methods known in the pharmaceutical industry.
The pharmaceutical compositions and dosage forms can be adapted as sterile solutions, suspensions, or lyophilisates of the products of the invention for parenteral administration using the suitable dose. Suitable excipients, such as pH-buffering agents or surfactants, can be used.
The formulations mentioned above can be prepared using conventional methods, such as those described in the pharmacopoeias of different countries and in other reference texts.
As it is used herein, the term “medicinal product” refers to any substance used for the prevention, diagnosis, alleviation, treatment, or curing of diseases in humans and animals.
The administration of the compounds, pharmaceutical compositions, or dosage forms of the present invention can be performed by means of any suitable method, such as intravenous infusion and through the oral, topical, or parenteral routes. Oral administration is preferred given the convenience it offers to the patients and given the chronic character of the diseases to be treated.
The administered amount of a compound of the present invention will depend on the relative efficacy of the chosen compound, the severity of the disease to be treated, and the patient's weight. However, the compounds of this invention will be administered one or more times a day, for example 1, 2, 3, or 4 times a day, with a total dosage between 0.1 and 1000 mg/kg/day. It is important to take into account that variations in the dose, as well as modifications in the route of administration, may have to be introduced depending on the patient's age and condition.
The compounds and compositions of the present invention can be used together with other medicinal products in combined therapies. The other drugs can be part of the same composition or of another different composition, for administration at the same time or at different times.
A third aspect of the invention relates to a combined preparation, hereinafter combined preparation of the invention, comprising or consisting of:
a) component A which is a compound (compound of the invention) or a composition (composition of the invention) as defined in the present invention, and
b) component B which is an active ingredient selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony-stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any of the combinations thereof.
In an even more preferred embodiment, the active ingredient of (b) is dexamethasone. In another even more preferred embodiment, the active ingredient of (b) is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalidomide or thalidomide.
In another preferred embodiment, the combined preparation of the invention further comprises pharmaceutically acceptable excipients. In another preferred embodiment, the combined preparation of the invention comprises, as active ingredients, only those mentioned above, although it may comprise other pharmaceutically acceptable excipients and vehicles.
A fourth aspect relates to the use of the combined preparation of the invention, wherein components (a) and (b) are administered simultaneously, separately, or sequentially for the prevention, relief, improvement, and/or treatment of a disease. Alternatively, it relates to the combined preparation of the invention for simultaneous, separate, or sequential use in therapy.
A preferred embodiment of this aspect relates to the use of the combined preparation of the invention in the production of a medicinal product for simultaneous, separate, or sequential use in the treatment of cancer. Alternatively, it relates to the combined preparation of the invention for simultaneous, separate, or sequential use for the treatment of cancer.
In a preferred embodiment of this aspect of the invention, the cancer is a hematological cancer. More preferably, the hematological cancer is acute myeloid leukemia or monoclonal gammopathy. In a more preferred embodiment of this aspect of the invention, the cancer is acute myeloid leukemia. In another more preferred embodiment of this aspect of the invention, the cancer is a monoclonal gammopathy.
In another preferred embodiment of this aspect, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In a more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
As it is understood herein, the term “treatment” refers to fighting the effects resulting from a disease or pathological condition of interest in a subject (preferably mammal, and more preferably a human) which includes:
(i) inhibiting the disease or pathological condition, i.e., stopping its development;
(ii) alleviating the disease or pathological condition, i.e., causing the regression of the disease or pathological condition or its symptomatology; and
(iii) stabilizing the disease or pathological condition.
As it is understood herein, the term “prevention” consists of preventing the onset of the disease, i.e., preventing the disease or pathological condition from occurring in a subject (preferably mammal, and more preferably a human), particularly when said subject has a predisposition to the pathological condition.
The compounds of the invention can be in a crystalline form as free compounds or solvates, and both forms are expected to fall within the scope of the present invention. Solvation methods are generally known in the art. Suitable solvates are pharmaceutically acceptable solvates. In a particular embodiment, the solvate is a hydrate.
The compounds of the invention or the salts or solvates thereof are preferably in a pharmaceutically acceptable form or a substantially pure form. Pharmaceutically acceptable form is understood, inter alia, as having a pharmaceutically acceptable level of purity, excluding normal pharmaceutical additives such as diluents and excipients, and without including any material considered toxic at normal dosage levels. The levels of purity for the compound of the invention are preferably above 50%, more preferably above 70%, and even more preferably above 90%.
In a preferred embodiment, it is above 95% of the compound of the invention or of the salts, solvates, or prodrugs thereof.
The compounds of the present invention may include enantiomers depending on the presence of chiral centers, or isomers depending on the presence of multiple bonds (for example, Z, E). The individual isomers, enantiomers, or diastereomers and mixtures thereof fall within the scope of the present invention. A compound drawn with explicit stereochemistry is for the purpose of depicting the racemic structure with the relative stereochemistry, as well as the enantiomers in different degrees of purity. In any case, the enantiomers and diastereoisomers of the compounds that are depicted with a particular stereochemistry are also part of the compounds of the invention.
Said compositions can have one or more indazole agents. Said indazole agents may be combined in the same or different proportions, and may be part of the same formulation, or may be formulated in different formulations for sequential, joint, or simultaneous administration.
The pharmaceutical compositions of the invention are administered topically, transdermally, orally, nasally, intramuscularly, intravenously, intraperitoneally, subcutaneously, enterally, or parenterally. Illustrative examples of topical or transdermal administration include, but are not limited to, iontophoresis, sonophoresis, electroporation, mechanical pressure, osmotic pressure gradient, occlusive cure, microinjections, needle-free injections by means of pressure, microelectric patches, and any combination thereof. Illustrative examples of dosage forms for oral administration include pills, capsules, pellets, solutions, suspensions, etc., and may contain conventional excipients, such as binders, diluents, disintegrants, lubricants, humectants, etc., and can be prepared using conventional methods. The pharmaceutical compositions can also be adapted for parenteral administration in the form of, for example, sterile lyophilized solutions, suspensions, or products, in the suitable dosage form; in this case, said pharmaceutical compositions will include suitable excipients, such as buffers, surface active agents, etc. In any case, the excipients will be chosen depending on the selected pharmaceutical dosage form. A review of the different pharmaceutical dosage forms of drugs and the preparation thereof can be found in the book entitled “Tratado de Farmacia Galénica,” by C. Fauli i Trillo, 10th edition, 1993, Luzán 5, S. A. de Ediciones.
Both the compositions of the present invention and the combined preparation can be formulated for administration to an animal, and more preferably to a mammal, including humans, in a variety of forms known in the state of the art. In that sense, they can be, without limitation, in sterile aqueous solution or biological fluids, such as serum. The aqueous solutions may or may not be buffered and have additional active or inactive components. The additional components include ionic force-modulating salts, preservatives including, but without limitation, antimicrobial agents, antioxidants, chelating agents, and the like, and nutrients including glucose, dextrose, vitamins, and minerals. Alternatively, the compositions can be prepared for administration in solid forma. The compositions can be combined with several inert vehicles or excipients, including, but without limitation, binders such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients such as starch or lactose; dispersing agents such as alginic acid or cornstarch; lubricants such as magnesium stearate, glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharine; or aromatizing agents such as mint or methyl salicylate.
Such compositions or combined preparations and/or the formulations thereof can be administered to an animal, including a mammal, and therefore humans, in a variety of forms including, but without limitation, intraperitoneally, intravenously, intramuscularly, subcutaneously, intrathecally, intraventricularly, orally, enterally, parenterally, intranasally, or dermally.
The dosage for obtaining a therapeutically effective amount depends on a variety of factors, such as the age, weight, sex, or tolerance of the mammal, for example. In the sense used herein, the expression “therapeutically effective amount” refers to the amount of indazole agent or agents which produce the desired effect, and will generally be determined, among other causes, by the actual characteristics of said prodrugs, derivatives, or analogs and the therapeutic effect to be achieved. The “adjuvants” and “pharmaceutically acceptable vehicles” which can be used in said compositions are vehicles known by those skilled in the art.
It must be stressed that the term “combined preparation” or also “juxtaposition” in this specification means that the components of the combined preparation do not have to be present as a combination, for example in a composition, to be available for the separate or sequential application thereof. Therefore, the expression “juxtaposed” means that it is not necessarily a real combination in view of the physical separation of the components.
Another aspect relates to a method for the treatment of a monoclonal gammopathy, comprising the administration of a compound of the invention or any of the pharmaceutically acceptable salts, esters, tautomers, solvates, and hydrates thereof, or any of the combinations thereof, as defined above.
In a preferred embodiment of this aspect of the invention, the composition further comprises one or more pharmaceutically acceptable excipients or vehicles. Preferably, the composition of the invention is a pharmaceutical composition comprising a compound of the invention as the only active ingredient, although it may comprise one or more pharmaceutically acceptable excipients and/or vehicles. In another preferred embodiment, the composition further comprises another active ingredient. In a more preferred embodiment, the other active ingredient is selected from the list consisting of prednisone, dexamethasone, doxorubicin, plerixafor, cyclophosphamide, granulocyte colony-stimulating factor, melphalan, thalidomide, lenalidomide, pomalidomide, bortezomib, carfilzomib, ixazomib, daratumumab, isatuximab, MOR202, elotuzumab, autologous stem cells (sASCT), allogeneic stem cells, or any of the combinations thereof. In an even more preferred embodiment, the other active ingredient is dexamethasone. In another even more preferred embodiment, the other active ingredient is melphalan. In another preferred embodiment, the other active ingredient is bortezomib. In another preferred embodiment, the other active ingredient is lenalidomide or thalidomide.
In another preferred embodiment of this second aspect of the invention, the monoclonal gammopathy is selected from multiple myeloma, plasma cell leukemia, Waldenstrom's macroglobulinemia, amyloidosis, or any of the combinations thereof. In a more preferred embodiment, the monoclonal gammopathy is multiple myeloma.
The preparation of indazole ether derivatives to be used according to the invention is described in European Journal of Medicinal Chemistry 2014, 73, 56-72 (EJMC-2014) and in patent PCT/ES2010/000400.
The compounds were prepared in several steps according to the methods described in EJMC-2014. The first step consists of protecting the nitrogen in position 1 of the indazole derivatives by means of ethyl chloroformate reaction. The second step consists of introducing the R2 group. The third step consists of deprotecting the nitrogen in position 1 and introducing the substituent R3 by means of reaction with the corresponding halides, where R1, R2, and R3 have the aforementioned meaning.
In the patent (PCT/ES2010/000400), these indazole derivatives are claimed for the treatment, prevention, or improvement of glaucoma, bronchial asthma, and chronic bronchitis, allergies such as contact dermatitis or allergy conjunctivitis, arthritis, pain, diseases associated with organ transplants, motor disorders associated with Tourette syndrome, Parkinson's disease, or Huntington's chorea, multiple sclerosis, emesis, and other toxic or undesirable effects associated with anti-cancer chemotherapy and appetite therapy.
The article (EJMC-2014) describes these indazole ether derivatives as potential drugs for the treatment of Alzheimer's disease.
Throughout the description and claims, the word “comprises” and variants thereof do not seek to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will be inferred in part from the description and in part from putting the invention into practice. The following examples and drawings are provided by way of illustration and do not seek to be limiting of the present invention.
The examples show that the compounds of the invention exert a pro-apoptotic effect on MM cells, without affecting the viability of healthy cells, by selectively interacting with CB2 receptors, triggering pro-apoptotic activity through the caspase-2 pathway, increasing pro-apoptotic regulators and reducing anti-apoptotic regulators, increasing the de novo synthesis of ceramide, and reducing mitochondrial membrane potential.
Furthermore, these new compounds inhibit tumor growth in vivo, and increase susceptibility to anti-myeloma drugs such as dexamethasone and melphalan.
Therefore, this invention represents a very promising therapy for multiple myeloma and related diseases.
Any research that involves animal or human samples was approved by the Clinical Research Ethics Committee (Comite Etico de Investigacion Clinica—CEIC) of Hospital Universitario Virgen del Rocio, and was carried out in accordance with the Declaration of Helsinki.
In vitro studies were carried out using six different human MM cell lines, U266, RPMI8226, MM1S, MM1R, U266-LR7, and RPMI-LR5. For ex vivo assays, human primary cells from healthy donors and MM patients were used (Table 1). Human MM cell lines U266, RPMI8226, and MM1.S were acquired from ATCC and lines U266-LR7, RPMI-LR5, and MM1.R were kindly provided by Dr. Enrique Ocio (Hospital Universitario de Salamanca, Spain). The primary cells were obtained from bone marrow (BM) aspirates or peripheral blood (PB) samples, and the peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque centrifugation and washed twice in phosphate buffered saline (PBS) containing 1% BSA. Hematopoietic stem cells and B and T lymphocytes were isolated from healthy PB donors by positive immunomagnetic separation using human MACS CD34+, CD19+, and CD3+ microbeads, respectively. MM plasma cells were obtained from the bone marrow (BM) of patients with an infiltration of cells of more than 30% (Table 1). The MM plasma cells were identified using CD138+, and were then distinguished from the other cell populations by flow cytometry using a suitable combination of antibodies: anti-human CD64-FITC, CD34-PE, CD56-APC, CD38-APC-H7, and CD45-Pacific Blue antibodies (BD Biosciences, San Jose, Calif.). All the cell lines were cultured in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin, as recommended by the supplier. For human primary cells, the concentration of FBS was up to 20%.
Cannabinoid agonists WIN-55,212-2 mesylate were acquired from Tocris Bioscience. Indazole agonists PGN-6, -17, -34, and -72, and selective CB2 antagonists PGN-8, -37, and -70 were synthesized and kindly provided by Dr. Nuria Campillo of the Centro de Investigaciones Biológicas, Madrid. Fumonisin B1 (FB1) was obtained from Enzo Life Sciences, Z-VAD (OMe)-FMK (pan-caspase inhibitor) was obtained from Abcam, and TMRE (tetramethylrhodamine methyl ester perchlorate) was obtained from Santa Cruz Biotechnology (Santa Cruz, Calif.). The anti-myeloma agents, dexamethasone and melphalan, were provided by the pharmacy department of Hospital Universitario Virgen del Rocio.
The extracts for Western blot were taken after 0, 2, 6, 18, and 24 hours. The cells were lysed according to Gilbert et al., 2002. (J Immunol Methods. 2002, 271:185-201), by adding 2% ASB-14 (Calbiochem, Beeston, United Kingdom) to the isotonic lysis buffer. The protein concentration was determined by Pierce® Microplate BCA Protein Assay kit-Reducing Agent Compatible (Pierce, Rockford, Ill.). The samples were subjected to SDS/PAGE in AnykD precast gels (Bio Rad, Hercules, Calif.) and transferred to PVDF membranes using Trans-Blot® Turbo™ System (Bio-Rad). The membranes were incubated over night at 4° C. with primary antibody in 0.05% Tween 20-Tris buffered saline (TTBS) then with the suitable secondary antibody, and they were subjected to chemiluminescence detection. As a control condition, the cells were treated with DMSO (<0.15%) in RPMI 1640 medium from Gibco (Gaithersburg, Md.).
The antibodies for caspase-2, -8, -9, active caspase-3, -p-Akt (phospho T308), p-Erk (T202+Y204), -p-p38MAPK (phospho T180+Y182), -p-JNK (phospho T183+Y185), -SPT, and the CB1 and CB2 receptors were from Abcam, anti-MCL-1 and -Bcl-xL were from Santa Cruz Biotechnology, anti-PARP was from Cell Signaling Technology, anti-Bax and -Bak were from BD Biosciences. Anti-beta-tubulin was from Sigma-Aldrich. All the horseradish peroxidase (HRP)-conjugated secondary antibodies used were from Jackson ImmunoResearch and produced in a donkey to prevent the possible cross-reactivity when several tests were performed.
The MM cell lines and primary cells were exposed to different doses of indazole compounds, and viability was evaluated at 18, 48, and 72 hours. Pretreatments with the ceramide synthesis inhibitor (FB1), pan-caspase (ZVAD-FMK), and CB antagonists (PGN-8, PGN-37, and PGN-70) were performed for 30 minutes, and then the cells were incubated with indazole compound WIN-55 up to 18 hours. Cell viability was determined using the MTT assay Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) according to the manufacturer's instructions.
The cell viability of the MM cell lines and primary cells from healthy PB donors was also evaluated by flow cytometry using 7-AAD/Annexin V. Patient bone marrow (BM) cells were analyzed using 7-AAD with a combination of monoclonal antibodies against myeloma-associated antigens (anti-CD56-APC, anti-CD45-Pacific Blue, and anti-CD38-APC-H7 [BD Biosciences]) and antibodies for distinguishing the granulomonocytic population (anti-CD64-FITC) and lymphocytic population (anti-CD45-Pacific Blue). The cells were acquired by means of FACSCanto II flow cytometer (BD Biosciences) and analyzed using Infinicyt™ Software (Cytognos, Spain).
The evaluation of the synergy of the indazole compound WIN-55 with other anti-myeloma agents, such as dexamethasone and melphalan, was done by evaluating cell viability by means of an MTT assay. The strength of the combination was quantified with Calcusyn software (Biosoft, Ferguson, Mo.), which is based on the Chou Talalay method, and it calculates a combination index (CI) with the following interpretation: CI>1: antagonist effect, CI=1: additive effect and CI<1: synergistic effect.
Cell lines U266 were exposed to 50 μM of WIN-55 for 15, 30, 45, and 60 minutes. The loss of mitochondrial membrane potential (Aym) was calculated using TRME (tetramethylrhodamine-ethyl-ester-perchlorate) according to the manufacturer's instructions and CCCP (2-[2-(3-chlorophenyl)hydrazinylidene] propanedinitrile) was used as a control to induce the loss of Aym.
The cells treated for 6 hours with indazole compounds were collected and placed on a slide. Immunofluorescence staining was performed as described previously by Vielhaber et al. 2001 (Glycobiology. 2001, 11:451-7) using anti-ceramide as the primary antibody. The ceramide antibody was obtained from Sigma-Aldrich, and the Alexa-488-conjugated secondary antibody was obtained from Abcam. As a control condition, the cells were treated with DMSO (<0.15%) in RPMI 1640 medium from Gibco (Gaithersburg, Md.).
The “NOD/scid/IL-2R gammae null” (NGS) mice were acquired from Charles River (France). The tumor xenografts were induced by the subcutaneous injection of 5×106 U266 cells mixed with 100 μl of Matrigel (BD Biosciences) in 8-week old mice. When the tumors became palpable (>0.5 cm), the mice were randomly assigned in the following groups (10 mice per group), which received i.p.: 1) 5 mg/kg WIN-55 every 24 hours, 2) 5 mg/kg WIN-55 every 48 hours, and 3) a vehicle. Two groups were left tumor-free and served as a negative control, receiving treatment every 24 or 48 hours, respectively. The tumor growth was evaluated daily by measuring the two bisecting diameters of the tumor with a digital Vernier gauge or caliper. The volume was calculated using the following formula: volume=length×(width) e2×0.4 mm3. The animals were sacrificed when the length or width of the tumor reached 2 cm.
Unless otherwise indicated, an experiment representative of at least three independent experiments is shown. For all the statistical analyses, the data was analyzed by means of Student's T-test with the SPSS software with a statistical significance P≤0.05. The means and standard deviations were also determined. The synergy was calculated using R as described in the Calcusyn software; a combination index (CI)<1 indicates synergy, and >1 indicates antagonism.
First, the effect of different indazole compounds of the invention on the proliferation and cell viability of several MM cell lines was evaluated by means of the MTT assay and flow cytometry. It was found that incubation with WIN-55, which is a non-selective indazole compound, significantly reduced the cell viability of all the MM cell lines compared with untreated control cells at 18 hours (
Furthermore, all the MM cell lines were treated with six different indazole compounds, PGN-6, -17, -34, and -72, which were characterized by their higher selectivity for the CB2 receptor compared with WIN-55. As shown in
The effect of the indazole compounds was more broadly examined ex vivo in myeloma plasma cells (MPCs) of six MM patients by flow cytometry using WIN-55. After treatment, the MPCs (CD38+) showed a significant decrease in cell viability from 70 to 85% at 20 and 50 μM, respectively (
These results indicate that the indazole compounds of the invention have a very selective pro-apoptotic effect on the myelomatous cells, whereas the viability of the healthy cells, including the hematopoietic precursor cells, is not affected.
17.24
17.21
13.64
12.05
11.66
13.72
In order to evaluate if the antiproliferative effect of WIN-55 is mediated by apoptotic mechanisms, the expression of PARP, caspases, and other pro/anti-apoptotic proteins (Bcl-2 family) in the most resistant MM cell line, U266, was analyzed. Treatment with the compounds of the invention induced a decrease in the expression of the full form of PARP in a time-dependent manner, with a concomitant increase in the expression of the 89 kDa fragment (CL_85 kDa), which could be detected 2 hours after exposure (
For the purpose of exploring which signaling pathways are mainly involved in the apoptosis induced by compounds of the invention, various parameters were evaluated:
Expression Profile of Phosphorylated JNK-, Erk1/2-, p38-MAPK-, and Akt-
Treatment with the compounds of the invention slightly overexpressed p-JNK and p-ERK1/2, whereas it moderately reduced the expression of p-p38-MAPK over incubation time. Oddly enough, WIN-55 induced a significant overexpression of p-Akt (phospho-T308) at early points in time, whereas at later times expression levels showed a decrease (
The de novo synthesis of ceramides is involved in the apoptosis induced by the compounds of the invention. Therefore, the expression of ceramides was evaluated by means of immunofluorescence in MM cells exposed to WIN-55, and a considerable increase in the expression of ceramides in U266 cells treated with the compound of the invention compared with untreated cells (
The tested compound of the invention attenuates the response to endoplasmic reticulum stress in myelomatous cells and induces an early loss of mitochondrial membrane potential. Given that myelomatous cells have a highly developed endoplasmic reticulum, they are prone to said organelle suffering stress. As a result, the effect of WIN-55 on the expression of certain endoplasmic reticulum stress marker proteins in U226 cells is evaluated. In contrast to what was expected, there was a slight but sustained decrease in the expression of endoplasmic reticulum stress marker proteins CHOP, ATF-4, p-IRE1, and XBP-1s (“spliced form”) compared with the control, and a slight increase in the level of XBP-1u (“unspliced” form), which indicates the lack of reticulum stress saturation, in cells treated with the compound/compounds of the invention compared with the control cells. This observation suggests that the tested compound of the invention reduces, or at least does not overload/saturate, the Unfolded Protein Response (UPR), which is activated under conditions of endoplasmatic reticulum stress in MM cells (
It is important to point out that the loss of mitochondrial membrane potential is a point of no return in apoptosis. As a result, changes in Aym in MM cells treated with the compound/compounds of the invention were analyzed. A considerable drop in Aym in U266 cells after 15 minutes of incubation was observed, and it continued to slightly drop over time (
In order to know if the pro-apoptotic effect of WIN-55 is mediated through the CB2 receptor, the effect of WIN-55 after treatment with three different selective CB2 antagonists, i.e., PGN-8, -37, and -70, was evaluated. As shown in
The expression of CB2 in different MM cell lines, as well as in normal hematopoietic cells from healthy individuals, was then evaluated. CB2 partially shows a 40 kDa band, consistent with the weight of the CB2 monomer, and another 30 kDa band which corresponds to the truncated form (
Anti-myeloma therapies consist of combinations of drugs with different mechanisms of action. For this reason, the effect of the compound of the invention WIN-55 on a dual combination with dexamethasone and melphalan, not only in cell lines U266 and RPMI, but also in their corresponding cell lines resistant to melphalan, U266-LR7 and RPMI-LR5, was analyzed. The combination index (CI) obtained from the analysis of cell viability data indicates that WIN-55 had a synergistic effect with both dexamethasone (DEX) and melphalan (MPH) (see
Finally, the antitumor effect of the compounds of the invention was analyzed in vivo, using a human MM xenograft model in immunodeficient NOD/SCID (NSG) mice. The MM cell line that is most resistant in vitro was used for this purpose. A considerable and progressive loss of tumor volume was observed after the administration of compounds of the invention compared with the corresponding ones treated with a vehicle (
Materials and Methods
Cell Cultures
The cell lines of acute myeloid leukemia KG-1a and HL60 (American Type Culture Collection) were cultured in IMDM medium supplemented with 2 mM of L-glutamine, 100 mg/mL of penicillin, 100 μg/ml of streptomycin, and 15% fetal bovine serum (FBS).
U937, NB-4, KG-1, and MOLM-13 (DMSZ, Braunschweig, Germany) were cultured in complete RPMI 1640 medium (with 2 mM of L-glutamine, 100 mg of penicillin, 100 μg/ml of streptomycin, and with 10% FBS), supplemented with 10 mM HEPES, 1×NaPuvuvato, and 1× non-essential amino acids.
All the cells were cultured in a humidified atmosphere of CO2/air (5%/95%).
The human primary cells were obtained from the bone marrow (BM) of AML patients and peripheral blood (PB) of healthy donors. Hematopoietic progenitor cells (CD34+) were isolated from samples of leukapharesis, and lymphocytes B (CD19+) and T (CD3+) from the buffy coats by means of positive immunomagnetic separation in the professional AutoMACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions.
This study was approved by the Clinical Research Ethics Committee (Comite Etico de Investigacion Clinica—CEIC) of Hospital Universitario Virgen del Rocio, and informed consents were obtained from all the patients and donors in accordance with the Declaration of Helsinki.
PGN Family Cannabinoid Synthesis
Cannabinoids PGN-6, -17, -34, -128, and -153, and selective CB2 antagonists PGN-8, -37, and -70 were synthesized in Dr. Paez's laboratory at the Centro de Investigaciones Biologicas, Madrid. PGN-6, -17, and -34 were synthesized following the method published by Gonzalez-Naranjo et al. 2014. Eur J Med Chem 73, 56-72.
Cannabinoids PGN-43, -128, and -153 were synthesized following the method published in patent document PCT/ES2016/070906.
Compounds and Treatments
WIN-55,212-2((R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinomethyl)-pyrrolo[1,2,3]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone mesylate were acquired from Tocris Bioscience (Bristol, UK), and cannabinoid agonists PGN6, PGN17, PGN43, PGN34, PGN128, and PGN153 and selective CB2 antagonists PGN-8, -37, and -70 were synthesized in Dr. Paez's laboratory at the Centro de Investigaciones Biol6gicas of Madrid.
They were added at the indicated concentrations to the culture medium for different incubation periods. The control cells were cultured with the relevant amounts of DMSO.
Myriocin (ISP-1), the serine-palmitoyltransferase (SPT) enzyme inhibitor, was obtained from Enzo Life Sciences (Lausen, Switzerland) and Z-VAD (OMe)-FMK (caspase inhibitor) was obtained from Abcam.
Cytarabine was supplied by the Pharmacy Department of the Hospital Universitario Virgen del Rocio.
Cell Viability and Apoptosis Assays
The cell lines and primary cells were cultured in 96-well plates (5×10e5 cells per well) with the addition of the indicated concentrations of WIN 55,212-2 or PGN cannabinoids in DMSO or with the solvent only in triplicate at 18, 48, and 72 hours. Caspase inhibitor Z-VAD (OMe)-FMK or the CB2 receptor antagonist PGN-8, -37, and -70, together with the cannabinoid WIN-55,212-2, were also added. Cell viability was determined by means of the WST-1 assay [2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium] according to the manufacturer's instructions (Dojindo Molecular Technologies). Optical densities were measured at 450 nm using a Multiskan™ Go Microplate plate reader (Thermo Fisher Scientific, Waltham, Mass., USA).
Apoptosis was evaluated by means of the annexin V/7AAD staining assay kit, according to the instructions provided by the manufacturer (R&D Systems Inc), and analyzed in a FACSCanto II flow cytometer (Becton Dickinson).
Mitochondrial Damage and Protein Expression Analysis
The controls that are untreated and the cells (106 cells per assay) treated with WIN-55,212-2 (50 μM) for 15 and 30 minutes at 37° C. were stained with 5 μM MitoSOX probe [Molecular Probes (Invitrogen)] to detect the mitochondrial superoxide. The MitoSOX signal was detected by flow cytometry.
The detailed methodology for analyzing mitochondrial membrane potential (APm), Western Blot analysis, and immunocytofluorescence analysis has been described elsewhere 29
Quantification of Ceramides
Cell lines HL60, KG-1a, and U937 (20×106 cells per condition) were treated with WIN-55,212-2 (50 μM) throughout different periods of time and the lipids were extracted with a 2:1 chloroform/methanol solution.
The culture cell lysate chloroform extracts were treated with 20 μl of internal standard (a solution of 826 μg of ceramide C17:0 in 25 ml of methanol) and were dried under a constant nitrogen stream at room temperature. The ceramides were reconstituted with 350 μl of an equimolecular mixture of methanol/formic acid (99:1, which contained 5 mM ammonium formate) and 2-propanol/formic acid (99:1).
Reconstituted samples (20 μl) were analyzed using a liquid chromatography system from Agilent (1200 series) featuring a binary pump (G1312A) connected to an API 2000 triple quadrupole mass spectrometer (Applied Biosystems) using an electrospray ionization interface in positive ionization mode (ESI+). The ceramides were separated in a Zorbax Eclipse XDB—C18 column (150×4.6 mm, 5 m) from Agilent. The working buffer was a 70:30 mixture of methanol/formic acid (99:1, which contained 5 mM ammonium formate) and 2-propanol/formic acid (99:1). The mobile phase was supplied at 0.5 ml/min in isocratic mode. This method provided the effective separation of the eight ceramides analyzed and the IS. The mass spectrometry was acquired by means of multiple reaction monitoring (MRM). The nebulizing gas (synthetic air), the gas of the curtain (nitrogen), and the gas of the heater (synthetic air) were set at 45, 25, and 45 (arbitrary units), respectively. The collision gas (nitrogen) was set at 3 (arbitrary units). The temperature of the gas of the heater was set at 500° C. and the electrospray capillary voltage at 5.5 kV.
The eight ceramides studied were quantified using calibration curves in which ceramide 17:0 was used as an internal standard. The ceramide content was directly proportional to the ceramide/internal standard ratio (r>0.99, p≤0.01). The relative standard deviations (RSD) were <10%.
Murine Model of AML
The experiments with animals described in this study were carried out according to the accepted standards of animal care and Spanish regulations for the wellbeing of animals used in experimental neoplasm studies, and the study was approved by the institutional committee on animal care.
The NOD/scid/IL-2R gammae null (NSG) mice were acquired from Charles River Laboratories International (L'Arbresle, France) and received food and water ad libitum, under specific pathogen-free conditions. When the mice were 8-12 weeks old, AML was induced by intravenous inoculation of the HL60 cell line, and the mice were monitored to confirm progression of the disease by means of studying weight loss and detecting human CD45+ cells in bone marrow BM aspirates and flow cytometry analysis. Once the presence of leukemia cells was confirmed, treatment with a vehicle, WIN-55 212 cannabinoid was administered at a dose of 5 mg/kg/day or cytarabine (ARA-C) at 50 mg/kg for 5 days.
The effect of the cannabinoid on normal hematopoiesis was also tested by means of the treatment of healthy BALB/c mice with WIN-55,212-2 at a dose of 5 mg/kg/day for 7 and 28 days. The bone marrow and the peripheral blood population were analyzed by means of flow cytometry and blood counts.
Statistical Analysis
For all the statistical analyses, SPSS software version 15.0 (Statistical Package for the Social Sciences, SPSS, Chicago, Ill., USA) was used and the statistical significance was defined as P<0.05. The error bars represent the standard error of the mean (SEM). The data was analyzed using the Student's T-test.
Results
WIN-55, 212-2 and the PGN Cannabinoid Family are Cytotoxic for Leukemia Cell Lines
Now it will be analyzed whether the exposure to cannabinoids had any effect on the viability of the tumor cells in vitro. To that end, the human AML U937, HL60, KG-1a, NB-4, MOLM-13, and KG-1 cell lines were cultured in serum-free medium and exposed to various concentrations of WIN-55,212-2 (range of 100 nM to 50 μM) for 18 hours, and cell viability was measured by means of WST-1 assays (Table 1). The three most sensitive cell lines are U937, HL60, and KG-1a.
The effect of WIN-55,212-2 and the PGN cannabinoid family on these three cell lines at 18, 48, and 72 hours was then analyzed by WST-1 assays and flow cytometry (supporting information of
The effects of the selective CB2 antagonists on cytotoxicity induced by cannabinoids were finally examined. The HL60 and KG1-a cell lines were exposed to WIN-55,212-2 (10 μM) in the presence or absence of CB2 antagonist PGN-8, -37 and -70, and cell viability was determined by means of the WST-1 assay 18 hours later (
WIN-55.212-2 Induces the Cleavage of Caspases, which is Blocked Using a Pan-Caspase Inhibitor
Activation of the caspase cascade is commonly associated with the induction of the apoptosis. Therefore, to elucidate the role of caspases in cannabinoid-induced apoptosis, the caspase activation pattern was examined after treatment with WIN-55,212-2. For this purpose, the HL60 cells were treated with this cannabinoid at a concentration of 50 μM or a vehicle for 2, 6, 18, and 24 hours. The cells were then collected and the expression of the various caspases was determined by means of Western blot analysis (
To further investigate the importance of caspases in the anti-leukemia action of cannabinoids, the capacity of the pan-caspase inhibitor Z-VAD(OMe)-FMK to rescue cells from cell death induced by cannabinoids was evaluated. The cells were preincubated for 60 minutes with the pan-caspase inhibitor, then WIN-55,212-2 was added and the incubation continued for 18 hours. As shown in
WIN-55,212-2 Induces Early Mitochondrial Damage and ER Stress
Since the cleavage of caspase 9 is a very early event in cannabinoid-induced apoptosis, and this change indicates activation of the intrinsic apoptosis pathway, the effect of WIN-55,212-2 on mitochondria, which are critically involved in triggering apoptosis through this pathway, was studied. For the purpose of further investigating the participation of the mitochondrial pathway in cannabinoid-induced apoptosis, the effect of WIN-55,212-2 treatment on AWm in the HL60 cell line was studied.
Exposure of the HL60 cells to 50 μM for 15 or 30 minutes led to a significant reduction in the mitochondrial membrane potential, as shown in
Furthermore, ROS have been associated with the activation of the intrinsic apoptotic pathway. In parallel, the production of ROS was studied using the fluorochrome MitoSox targeting mitochondria in AML and HSC cells. The results showed that the exposure of HL60 cells to cannabinoids led to a significant increase in ROS production levels at 15 minutes or more. In contrast, this experiment was reproduced in hematopoietic stem cells, but ROS levels remained unchanged (
Finally, ER stress in HL60 cells was studied by Western blot analysis. The cannabinoid increased the expression of crucial factors in the response of the unfolded protein (EPU) to ER stress, such as p-IRE1, p-PERK, and CHOP (
The Accumulation of Ceramides is Involved in Cannabinoid-Induced Apoptosis
Given that it has been reported that the new synthesized ceramide is involved in cannabinoid-induced apoptosis, it was investigated if the cannabinoid derivatives also act through a similar pathway in leukemia cells. Experiments were conducted with selective ceramide synthesis inhibitors, such as myriocin and fumonisin B1 (inhibitors of serine palmitoyltransferase (SPT) and ceramide synthase, respectively) (
The pharmacological blockade of ceramide synthesis with fumonisin B1 only partially prevented the decrease in the viability of HL60 cells after exposure to cannabinoids, evaluated by means of WST-1 assays at 18 hours (
Furthermore, significant differences in the amounts of certain subtypes of ceramides in untreated leukemia cells compared to those that are treated were observed by means of immunohistochemistry and HPLC/MS-MS in HL60 cells (
Oddly enough, ceramides 16:0, 18:0, and 18:1 followed a constant growth pattern up to 24 hours, whereas the ceramides having a longer chain reached their maximum level at 6 hours and then returned to their baseline levels.
Signaling Pathways Targeted by Cannabinoids in AML Cells
It has been demonstrated that AKT, ERK, JNK, and p38 MAPK regulation plays an important role in the survival or induction of apoptosis in a series of cell types. Therefore, it was examined if exposure to WIN-55,212-2 had any effect on the levels of the phosphorylated forms of these signaling molecules. For this purpose, the HL60 cells were exposed to a vehicle or to 50 μM of WIN-55,212-2 for 2, 6, 18, and 24 hours. The cells were then marked with antibodies specific for p-AKT, p-ERK, p-JNK, and p-p38 MAPK and studied by means of Western blot analysis (
The Western blot assays demonstrated that treatment with cannabinoids slightly over-regulates p-JNK and p-Erk1/2, whereas it slightly under-regulates p-p38-MAPK and p-AKT over time.
Expression of the pro-apoptotic regulator Bax is also studied by immunocytofluorescence analysis. It has been reported that this protein is involved in ceramide-induced apoptosis.
Antitumor Effect of the Cannabinoids on Murine Models of AML In Vivo
For the purpose of evaluating the effect of cannabinoids in vivo on normal HSC, BALB/c mice were treated with 5 mg/kg/day of WIN-55,212-2 for 7 and 28 days, and the different subpopulations of HSC identified by flow cytometry were evaluated. It has been confirmed that the cannabinoids do not affect the viability of the different hematopoietic progenitor populations and increase the number of cells under some conditions (
Furthermore, peripheral blood populations were analyzed by blood count studies and an increase in blood platelet count was observed in treated mice (
Finally, in order to study the effects of cannabinoid on the growth of human AML cells in vivo, HL60 cell xenograft models were used. For this purpose, NSG mice were treated with 5 mg/kg/day of WIN-55,212-2, 50 mg/kg of ARA-C for 5 days, or placebo once bone marrow infiltration with leukemia cells, evaluated by flow cytometry, is confirmed.
The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| P201630539 | Apr 2016 | ES | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/ES2017/070261 | 4/27/2017 | WO | 00 |
