The present application is a divisional application of the application having an application date of 2012/03/20/2012, an application number of 201280012793.8, entitled "pharmaceutical composition comprising HDAC inhibitor and steroid and use thereof".
Summary of The Invention
The present invention relates to the unique discovery that the combination of an HDAC inhibitor and a steroid will improve survival in patients suffering from cancer, such as lymphoma. These compounds are pharmaceutically acceptable compounds and can be used at least as a pretreatment prior to other treatments. These substances may be used together or administered separately and as pharmaceutically acceptable compounds.
It has been found that steroids can be used to increase a patient's response to treatment with HDAC inhibitors and reduce side effects, such as somnolence.
In a first aspect, the present invention relates to a pharmaceutical composition comprising an HDAC inhibitor, an acid or salt thereof or a mixture thereof and a steroid or salt thereof.
In a second aspect, the present invention relates to the use of a composition as defined above for the treatment of cancer.
Thus, by providing such a pharmaceutical composition or the use of such a composition, the survival rate of patients suffering from cancer, e.g. lymphoma, is improved and it is for the first time possible to treat elderly with sustained efficacy, who need lower chemotherapeutic doses.
In a third aspect, the present invention relates to a kit comprising at least one pharmaceutical composition comprising an HDAC inhibitor, an acid or salt thereof or a mixture thereof and a steroid or a salt thereof.
In a fourth aspect, the present invention relates to a kit comprising at least one suitable container or suitable package containing an HDAC inhibitor, an acid or salt thereof or a mixture thereof and at least one suitable container or suitable package containing a steroid or a salt thereof.
By providing such a kit, it is easy to pre-treat a patient suffering from cancer before other treatments in the hospital.
In a fifth aspect the present invention relates to a method of assessing the effect of a substance on CHOP-sensitivity of a cell line, the method comprising the steps of
a. Providing a cell line selected from the group consisting of: : WSU-NHL, Karpas-422, ULA, SU-DHL-5 and SU-DHL-8
b. Adding a combination of cyclophosphamide, doxorubicin, vincristine and prednisone
c. Adding a substance to be evaluated to each cell line
d. And assessing the viability of these cells.
In a final aspect is a method of treating a patient suffering from cancer by administering a pharmaceutical composition comprising an HDAC inhibitor, an acid or salt thereof, or a mixture thereof, and a steroid or a salt thereof. The pharmaceutical composition may be used in combination with one or more other therapeutic methods, including chemotherapy, immunotherapy. The chemotherapy may be, for example, CHOP (cyclophosphamide, doxorubicin, vincristine and prednisone) and the immunotherapy may be the DC20 antibody.
The present application discloses the following:
item 1. A pharmaceutical composition comprising
a. An HDAC inhibitor, a pharmaceutically acceptable acid or salt thereof or mixtures thereof and
b. a steroid or a pharmaceutically acceptable salt thereof.
Item 2. the pharmaceutical composition of item 1, wherein the HDAC inhibitor is a hydroxamic acid/carbamic acid, a cyclic peptide, a fatty acid, or a benzamide compound.
Item 3. the composition of item 2, wherein the HDAC inhibitor is selected from the group consisting of: vorinostat, Romidepsin, valproic acid, Aspabistat, panobistat, belinsstat, entinostat and reminostat.
Item 4. the composition of item 3, wherein the HDAC inhibitor is selected from the group consisting of: valproic acid or semi-sodium valproate, sodium valproate or magnesium valproate.
Item 5. the composition of any one of items 1 to 4, wherein the steroid is selected from the group consisting of: prednisone, prednisolone, dexamethasone or betamethasone.
Item 6. the composition of item 5, wherein the steroid is prednisone.
Item 7. the composition of any one of the preceding items, wherein the HDAC inhibitor is valproic acid and the steroid is prednisone.
Item 8. the composition according to any of the preceding items, further comprising a pharmaceutically acceptable additive, diluent, carrier, excipient or buffer.
Item 9. the composition according to any one of the preceding items, selected from the group consisting of: granules, powders, tablets, coated tablets, microcapsules, microgranules or effervescent forms.
Item 10. the composition of any of the preceding items, wherein the effervescent form is selected from the group consisting of a tablet or a powder.
Item 11. an apparatus selected from the group consisting of: a vial, ampoule, sachet or infusion bag containing a composition according to any of items 1-10, or a device containing an HDAC inhibitor, a pharmaceutically acceptable acid or salt thereof or a mixture thereof, and a device containing a steroid or a pharmaceutically acceptable salt thereof.
Item 12. an HDAC inhibitor, a pharmaceutically acceptable acid or salt thereof or a mixture thereof and a steroid or a pharmaceutically acceptable salt thereof for the pretreatment of cancer.
Item 13 a HDAC inhibitor, a pharmaceutically acceptable acid or salt thereof or a mixture thereof and a steroid or a pharmaceutically acceptable salt thereof, for the pretreatment of sarcoma, malignant melanoma, skin cancer, estrogen receptor dependent and independent breast cancer, ovarian cancer, prostate cancer, renal cancer, colon and colorectal cancer, pancreatic cancer, head and neck cancer, small and non-small cell lung cancer, and blood cell cancer.
Item 14. an HDAC inhibitor, a pharmaceutically acceptable acid or salt thereof or a mixture thereof and a steroid or a pharmaceutically acceptable salt thereof, for use in the pretreatment of a disease selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, T-cell lymphoma, myeloma, and hodgkin's lymphoma.
Item 15. a kit comprising a pharmaceutical composition according to any one of items 1-10 provided in a suitable container and/or with suitable packaging and instructions for use.
Item 16. a kit comprising an HDAC inhibitor or a pharmaceutically acceptable acid or salt thereof or mixtures thereof and a steroid or a pharmaceutically acceptable salt thereof provided in a suitable container and/or with suitable packaging and instructions for use, e.g., how to administer the compounds.
Item 17. the kit of item 16, wherein the two active compounds are stored in separate containers and/or in different packages.
Item 18. the kit of items 15-17, wherein the kit further comprises an antibody, monoclonal antibody, or functional fragment thereof that binds to CD 20.
Item 19. the kit of any of items 15-18, wherein the container is a vial, ampoule, pouch, or infusion bag.
Item 20. a method of pretreating a human suffering from cancer by administering a pharmaceutical composition according to any one of items 1-10.
Detailed description of the invention
HDAC and steroids
In a first aspect, the present invention relates to a pharmaceutical composition comprising an HDAC inhibitor, an acid or salt thereof or a mixture thereof and a steroid or salt thereof. The HDAC inhibitor may be a hydroxamic acid/carbamic acid, cyclic peptide, aliphatic acid or benzamide compound, such as vorinostat, romidepsin, valproic acid, ispambostat (aspanobolistat), belinostat, entinostat and reminostat (reminiostat), some of which are trademarks. Alternatively the HDAC inhibitor may be valproic acid, for example sodium or magnesium valproate or a mixture thereof. Examples include Volinostat (marketed in the United states under the trademark Volinostat)) Romidepsin (marketed in the United states under the trademark ROMIDESSEN)) Panobinostat. One form of valproic acid is when mixed with sodium valproate, i.e. a mixture of acids and salts (half sodium valproate), marketed under different trade names Depakote, Depakote ER, Depakene, Depacon, Depakine, Valparin, Stavzor and Ergenyl. Sodium valproate is marketed in Sweden under the trademark Absensor, Depakinee, Orfiril. Valproic acid is marketed in Sweden under the trademarks Ergenyl and Depakine.
The steroid used in the present invention may be selected from glucocorticoids and includes prednisone, prednisolone, dexamethasone and betamethasone. In one example the HDAC inhibitor is valproic acid and the steroid is prednisone.
Other ingredients
The pharmaceutical composition as defined above may further comprise one or more other pharmaceutically acceptable pharmaceutical ingredients, such as a pharmaceutically acceptable diluent, carrier, excipient and buffer. "pharmaceutically acceptable" means a non-toxic compound that does not reduce the efficacy of the biological activity of the active ingredients. Such pharmaceutically acceptable additives, diluents, buffers, carriers or Excipients are known in the art (see Remington's Pharmaceutical sciences (leimington Pharmaceutical sciences), 18 th edition, A.R Gennaro (A.R gennalo), ed., Mack publishing company (mike publishing company) (1990) and handbook of Pharmaceutical Excipients (handbook of Pharmaceutical adjuvants), 3 rd edition, a.kibbe (a. gibb), ed., Pharmaceutical Press (2000)).
The term "buffer" is intended to mean an aqueous solution comprising an acid-base mixture, with the aim of stabilizing the pH. Examples of buffering agents are magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic salts; ringer's solution (Ringer's); ethanol; a pH buffer; polyesters, polycarbonates and/or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.
The term "diluent" is intended to mean an aqueous or non-aqueous solution, in order to dilute the compound in a pharmaceutical formulation. The diluent may be one or more of salt, water, polyethylene glycol, propylene glycol or ethanol.
The excipient may be one or more of carbohydrates, surfactants, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrins, which are added to the composition, for example, to facilitate lyophilization. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylhydroxyethylcellulose, alginates, carageenan, hyaluronic acid and their derivatives, polyacrylic acid, polysulfonates, polyethylene glycol/polyethylene oxide, polyethylene oxide/polypropylene oxide copolymers, polyvinyl alcohol/polyvinyl acetate of different degrees of hydrolysis, and polyvinylpyrrolidone, all of different molecular weights, which are added to the composition, for example to control viscosity, to obtain bioadhesive adhesion, or to protect the lipids from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-and triglycerides, ceramides, sphingolipids and glycolipids, all of different acyl chain lengths and saturations, egg yolk lecithin, soy lecithin, hydrogenated egg yolk and soy lecithin, which are added to the composition for similar reasons as polymers. Examples of minerals are mica, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition in order to obtain some benefit, such as reduced liquid accumulation or favourable pigment properties.
Examples of suitable aqueous and anhydrous carriers which may be employed in the pharmaceutical compositions of the present invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. The prevention of the action of microorganisms on the target compounds can be ensured by the inclusion of different antibiotics and antifungal drugs, such as parabens, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
Administration mode
The pharmaceutical formulations according to the invention can be used topically or systemically. Routes of administration include topical, ocular, nasal, pulmonary, buccal, parenteral (intravenous, subcutaneous, and intramuscular), oral, vaginal, and rectal. Oral administration is most commonly used.
The pharmaceutical composition can be administered to a patient in a pharmaceutically effective amount. "pharmaceutically effective dose" refers to a dose sufficient to produce the desired effect associated with the condition for which it is administered. The exact dosage will depend on the mode of administration, the nature and severity of the disease. Depending on the general health, sex, age and weight of the patient, different doses will be required. The administered dose may be given in a single dose or several smaller doses may be given and may also be given by multiple sub-divided doses given at specific intervals. Depending on the mode of administration, these active compounds or substances can also be administered together or separately.
Suitable preparation forms are, for example, granules, powders, tablets, coated tablets, (micro) capsules, micro-granules, effervescent powders or granules, suppositories, ampoules, injections and also extended-release formulations of the active substance, in which formulations excipients, diluents or carriers are usually used as described above. Other formulations may be those that produce different release profiles of the active ingredient, as will be known to those skilled in the art. Examples include sustained release, sustained action, extended release, delayed or timed release, controlled release, modified release, or continuous release. The advantage of sustained release tablets or capsules is that they can be taken a few times less than immediate release formulations of the same drug and they maintain a more stable level of the drug in the bloodstream. Today the most powerful time release drugs are formulated so that the active ingredient is embedded in a matrix of one or more insoluble substances (different substances: some acrylates and even chitin; these substances are usually patented) so that the dissolved drug has to find its way out through the pores in the matrix. Some drugs are encapsulated in polymer-based tablets that have a laser-drilled hole on one side of the tablet and a porous membrane on the other side. Gastric acid passes through the porous membrane, thus propelling the drug through the laser-drilled hole. At the same time, the entire drug dose is released into the systemic system, while the polymer container remains intact and is later expelled through the normal digestive system. In some formulations, the drug is dissolved into a matrix that physically swells to form a gel, allowing the drug to leave the outer surface of the gel. Microencapsulation is also considered to be a more complete technique to produce complex dissolution profiles. By coating an active pharmaceutical ingredient around an inert core and laminating with insoluble materials to form a microsphere, it is possible to obtain more consistent and reproducible dissolution rates. All of which are known to those skilled in the art.
An example of one formulation form is an effervescent product.
Effervescence is the reaction of an acid and a base (in water) to produce carbon dioxide. Examples of acids used in this reaction are citric acid, tartaric acid, malic acid, fumaric acid, adipic acid, citrate, succinic acid and mixtures thereof. Citric acid is most commonly used and it imparts a citrus-like taste to the product. Examples of bases used in this effervescent reaction are sodium carbonate, potassium carbonate, sodium bicarbonate, potassium bicarbonate, calcium bicarbonate, magnesium carbonate, sodium polyethylene glycol carbonate, carboxylysine and mixtures thereof. Sodium bicarbonate is most commonly used in effervescent formulations.
These compounds may be mixed together in the effervescent product or alternatively each may be used alone. One example is when two active substances are wrapped separately from each other, the resulting effervescent product gives different release profiles of the two active substances/ingredients.
The effervescent product may be in powder form or in tablet form.
If the product is a tablet, it may contain at least one additive selected from the group consisting of binders, lubricants, emulsifiers, surfactants (such as polysorbate 80 and sodium lauryl sulfate), flavors, fragrances (examples of taste-imparting ingredients) (such as orange, lemon, bergamot, grapefruit, banana, apricot and strawberry) and coloring agents (including natural or synthetic), vitamins, sweeteners (examples of taste-imparting ingredients) (potassium acesulfame, sodium saccharin, aspartame, stevia and sucralose), nutritional additives (such as antioxidants, peptides), and mixtures thereof.
Substances which add taste, colour or antioxidant properties to the effervescent composition may be plant polyphenols from natural sources, such as blueberries, cranberries, grapes and tea leaves.
Additionally the tablet may contain various lubricants suitable for use in the composition including water dispersible lubricants, water soluble lubricants, water insoluble lubricants and combinations thereof. Examples of useful water-soluble lubricants include sodium benzoate, polyethylene glycol, L-leucine, adipic acid, and combinations thereof.
Tablets may also contain water-insoluble lubricants including, for example, stearates (e.g., magnesium, calcium and zinc stearates), oils (e.g., mineral, hydrogenated and partially hydrogenated vegetable, and cottonseed oils), and combinations thereof. The effervescent agent may also contain vitamins and minerals.
The manufacturing process for producing effervescent products involves critical steps that need to be carefully treated during formulation and manufacturing processes, which are well known to those skilled in the art. The production of effervescent products must be carried out in a very low humidity area. The best way to produce an effervescent product is to work in an environment with strict control of humidity.
The process of producing tablets, known as "tableting" or "compression", requires the addition of pharmaceutically acceptable excipients known to those skilled in the art as powders, for example by mixing, granulating and tableting. It is a very common practice to add lubricants after granulation in tablet production; the most commonly used substance is magnesium stearate. Substances such as magnesium stearate can cause problems during the manufacture of effervescent tablets because they are insoluble in water and therefore produce a film on the upper layer of water after dissolution of the tablet. A strategy to overcome this problem is to use other lubricants that are soluble in water; for example a spray-dried mixture of L-leucine and polyethylene glycol. Alternatively, the absence of any lubricant has the advantage of avoiding the blending step, but also has the disadvantage of having special needs in production.
Amounts and dosages
The HDAC inhibitor may be administered in varying amounts depending on the HDAC inhibitor to be used. As is known to those skilled in the art. This also applies to steroids. The active ingredients may be administered together or separately. However, the following are some examples of amounts that may be used.
Examples of administration include administration of 20 to 200mg of prednisone or prednisolone per day, such as 50-200, 100, 150, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg. Betamethasone is administered in an amount of 4 to 32mg per day, for example 10-25, 10-20, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 or 32 mg. Dexamethasone can be administered in an amount of 10-80mg per day, for example 20-70, 10, 20, 30, 40, 50, 60, 70 or 80 mg. Prednisone may be administered in a single dose or, if desired, in multiple doses.
The HDAC inhibitor, e.g., valproic acid, may be administered orally or intravenously in a range of from about 500mg to about 15000mg per day, e.g., from about 4000mg to about 15000mg per day, e.g., from about 400mg to about 3000mg per day. For example, the oral dosage may be about 800, about 1600, about 2400, about 3000, about 6000, about 9000, about 15000mg per day. It will be appreciated that the total amount per day may be administered in a single dose or in multiple doses, for example two, three, four or five times per day.
The HDAC inhibitor, e.g., belinostat, can be administered orally in a range of from about 6mg to about 3000mg per day, e.g., from about 40mg to about 3000mg per day, e.g., from about 400mg to about 3000mg per day. For example, the oral dose may be about 4, about 40, about 400, about 800, about 1600, about 2400, about 2800, or about 3000mg per day. It will be appreciated that the total amount per day may be administered in a single dose or in multiple doses, for example two, three or four times per day.
The HDAC inhibitor or a pharmaceutically acceptable salt thereof, the steroid or a pharmaceutically acceptable salt thereof may be administered separately or in combination at least once daily (e.g., at the morning hours, about 5 to 8 am). However, any substance may be administered 1, 2, 3, 4 or even up to 5 times per day. Examples of administrations include: HDAC inhibitors were administered three times and prednisone once per day. Generally, steroids are administered 1-2 times per day depending on the formulation properties and the release properties of the active substance. The HDAC inhibitor and the steroid may be administered at least 24-72 hours, e.g. 30-60, 40-50 or 48 hours, prior to said immunization and/or chemotherapy, and the steroid and HDAC inhibitor may be administered simultaneously or sequentially. However, it may be administered prior to the treatment with chemotherapy and or immunotherapy, but it may also be administered during or after the treatment, but it is mandatory to administer before.
Reagent kit
In one aspect, the invention relates to a kit comprising a pharmaceutical composition in a suitable container or package, wherein the composition is as described above.
In another aspect the invention relates to a kit comprising an HDAC inhibitor, or a pharmaceutically acceptable acid or salt thereof or a mixture thereof and a steroid, or a pharmaceutically acceptable salt thereof or a mixture thereof.
The kit may comprise separate containers for the HDAC inhibitor and the steroid. The kit may further comprise an antibody, monoclonal antibody or functional fragment thereof that binds to CD 20. The kit may also contain instructions for use, such as written instructions on how and when to administer the compounds or compositions.
For example, the kit may comprise one or more vesicant(s) (blisters) comprising a plurality of tablets or granules. The active compounds can be mixed in tablets or granules or dispersed into different tablets or granules and dispersed in tablets or granulated by a barrier (e.g. coated). The compounds or pharmaceutical compositions may also be held in the kit in the same or separate distinct sachets.
The kit may contain other parts, for example including appropriate solutions for dilution (e.g. physiological saline solution, glucose solution, etc.), reagents (e.g. to adjust pH), and means (e.g. bags, tubes, syringes, needles, transfer devices) for assembly and use (e.g. for preparation of the formulation and subsequent administration).
The written instructions may also include a list of indications for which the formulation (e.g., HDAC inhibitor therein) is suitable to treat and how to administer the compound or compounds.
Methods of treatment and uses
The present invention also relates to a method of treatment wherein a composition or compound as defined above is used to treat cancer in a human, for example to treat a disease selected from the group consisting of: sarcomas, malignant melanomas, skin cancers, estrogen receptor dependent and independent breast cancers, ovarian cancers, prostate cancers, renal cancers, colon and colorectal cancers, pancreatic cancers, head and neck cancers, small cell and non-small cell lung cancers, and blood cell cancers. Examples include treating a disease selected from the group consisting of: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia, myeloma, T-cell lymphoma and hodgkin's lymphoma.
The term "treatment" in the context of treating a disorder generally pertains to the treatment and therapy of a human in which some desired therapeutic effect is obtained.
The term treatment also includes other types of treatments and therapies in which, for example, two or more treatments are combined, e.g., simultaneously or sequentially, e.g., a steroid and an HDAC inhibitor are administered simultaneously or sequentially. For example, the compounds described herein may be used alone or in combination and may, for example, be used with other agents, such as cytotoxic agents and the like. Examples include chemotherapy (administration of an active agent, such as the disclosed compounds or CHOP). CHOP is a combination of cyclophosphamide, doxorubicin, vincristine and prednisone, which may be administered in amounts of 750 +/-10% mg/m250 +/-10% mg/m of cyclophosphamide21,4 +/-10% mg/m of doxorubicin2Vincristine and 50 +/-10% mg/m2Prednisone of (2). Immunotherapy, comprising an antibody, a monoclonal antibody or a functional fragment thereof, e.g., rituximab, ofatumumab, GA101, tussimumab (tositumumab), ibritumomab tiuxetan, ocraluzumab, vetuzumab, epratuzumab, FTBA05, AME-133V or R603. All of the above mentioned antibodies bind to CD20 present on B-cells. The amount of antibody administered may be 375 +/-10% mg/m2. Other examples are prodrugs; surgery, radiation, and gene therapy.
Antibodies are included in the therapeutic agents of the methods of the invention, including functional fragments thereof. "antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including products from Fab or other immunoglobulin expression libraries. The term antibody as used in the present invention is intended to include both intact molecules as well as fragments thereof, such as Fab and F (ab'). sub.2, Fv and SCA fragments, which are capable of binding to an epitope determinant on a protein of interest. A single chain antibody ("SCA") is a genetically engineered single chain molecule comprising a variable region of a light chain and a variable region of a heavy chain linked by a suitable flexible polypeptide linker.
The present invention relates to humans. These methods are therefore suitable for human therapy.
The following examples are intended to illustrate, but not to limit, the invention, either explicitly or implicitly, in any way, form, or type.
Examples of the invention
Biological evaluation
Based on five DLBCL cell lines with different sensitivities to CD20 antibody-CHOP induced cell death, the inventors of the present invention have established a cell line in vitro model of CD20 antibody-CHOP resistance (CD20 antibody is rituximab) (iceberg,lind en, Linderoth, Jerkeman and Drott; exp Cell Research (Experimental Cell Research); may 1; 317(8):1179-91). In this model, treatment with valproic acid at pharmacological concentrations showed a strong sensitive effect on CHOP-mediated cell death. This was associated with an enhancement of acetylation of histone H3, as determined by western blot analysis. Also, pre-treatment of DLBCL cell lines with a combination of valproic acid and prednisolone for 48 hours further increased their sensitivity to CHOP-induced cell death. In addition, pretreatment with hydroxamic acid/carbamate HDAC inhibitors trichostatin a and belinostat also sensitized DLBCL cell lines to CHOP-induced cell death, the effect being enhanced by the addition of prednisolone. Moreover, the steroid dexamethasone also enhanced the anti-inflammatory activity of the compoundThe sensitizing effect induced by valproic acid on CHOP-induced cell death. Taken together, the results indicate that the combination of HDAC inhibitor and steroid sensitizes B-cell lymphoma cell lines to CHOP-induced cell death. This suggests that combination therapy with HDAC inhibitors and steroids will increase the response of chemotherapy or immunochemotherapy in the treatment of lymphoma.
Example 1
Valproic acid (VPA) sensitizes DLBCL cell lines to CHOP treatment.
The DLBCL cell lines WSU-NHL, Karpas-422, ULA, SU-DHL-5 and SU-DHL-8 cells were treated with three different concentrations (0.1 mM, 2mM and 10mM, respectively) of valproic acid (VPA) alone or in combination with CHOP for 72 hours. The CHOP protocol used in all examples included 10. mu.M cyclophosphamide monohydrate, 20nM doxorubicin hydrochloride, 2nM vincristine sulfate and 20. mu.g/ml prednisolone. (the amount of the Ageberg,lindden, Linderoth, Jerkeman, and Drott; exp CellResearch (research on experimental cells); may 1; 317(8):1179-91).
Cell viability was assessed after 72 hours by trypan blue exclusion and normalized to untreated control cells for 0 day (seeding). Data are expressed as mean ± SEM, n ═ 3.
As shown in table 1, the addition of valproic acid increased cell death in response to CHOP therapy in all lymphoma cell lines, indicating that valproic acid in combination with CHOP is beneficial for lymphoma patients.
Table 1A: ULA cells
TABLE 1B: karpas-422 cells
Table 1C: WSU-NHL cells
Table 1D: SU-DHL-8 cells
Table 1E: SU-DHL-5 cells
Example 2
Valproic acid (VPA) at physiologically relevant concentrations sensitizes DLBCL cell lines to CHOP treatment.
DLBCL cell lines SU-DHL-8 (Table 2A) and WSU-NHL (Table 2B) were treated with 0.5mM or 1.5mM VPA alone or in combination with CHOP for 72 hours. The concentration of 0.5mM VPA was chosen because it is the standard serum concentration during the treatment of epileptic patients with continuous VPA. The concentration of 1.5mM VPA was chosen because it is the highest tolerable serum concentration that the inventors noticed during 5 days of treatment with VPA when taken as isosexes.
Cell viability was assessed after 0 hours (day 1), 24 hours (day 2), 48 hours (day 3) and 72 hours (day 4) by trypan blue exclusion and normalized to untreated control cells, respectively. The data are expressed as mean values, n being 3.
The effect of treatment on viability was tested against the effect of CHOP treatment alone. Student's unpaired t-test was used to assess significant differences. All tests were bi-directional. The effect was considered statistically significant, P <0.05 (. sup.)) and P <0.01 (. sup.)).
Viability (% of control cells on day 1) is shown in table 2A (SU-DHL-8) and table 2B (WSU-NHL, n ═ 3), respectively.
As shown in table 2, VPA at physiologically relevant concentrations enhanced cell death in lymphoma cell lines in response to CHOP treatment, showing that a physiologically relevant dose of valproic acid in combination with CHOP may be beneficial for lymphoma patients.
TABLE 2A
TABLE 2B
Example 3
Valproic acid (VPA) does not interfere with rituximab-mediated cellular cytotoxicity.
To evaluate the effect of VPA on rituximab-induced antibody-dependent cellular cytotoxicity (ADCC) of CD20 antibody.
WSU-NHL cells (tables 3A and 3B) or SU-DHL-8 cells (tables 3C and 3D) were labeled with PKH26, either left untreated or incubated with 1.5mM VPA for 24 hours, after which 0, 0.1, or 10. mu.g/ml rituximab was added.
NK cells were added such that the ratio of effector to target cells was 10:1, after which the cells were incubated for a further 20 hours. Dead target cells were identified as double positive for PKH26 and 7-AAD and read as an indication for the assay. The data shown demonstrate the percentage of dead cells, representing two independent experiments. The data show that VPA does not affect rituximab-induced ADCC, consistent with the use of VPA with the CD20 antibody in lymphoma patients.
Table 3A: WSU-NHL cells
Table 3B: WSU-NHL cells
Table 3C: SU-DHL-8 cells
Table 3D: SU-DHL-8 cells
Example 4: pretreatment with valproic acid (VPA) alone sensitizes DLBCL cell lines to CHOP treatment. Independently of VPAPretreatment with the combination of VPA and prednisolone significantly increased cell death compared to pretreatment.
Pretreatment with VPA prior to CHOP could theoretically increase the response to CHOP therapy by inducing DNA damage. However, VPA treatment can lead to symptoms such as somnolence in patients. These symptoms can be alleviated by the concurrent treatment with prednisolone. To investigate the effect of pretreatment with VPA and prednisolone prior to CHOP, WSU-NHL cells were treated with different combinations of VPA, prednisolone (P, 20 μ g/ml) and CHOP as shown below. Viability was assessed by trypan blue exclusion at the indicated time points (n ═ 3). Significant differences were assessed using Student's unpaired t-test. All tests were bi-directional. The effect was considered statistically significant, P <0.05, P <0.01, P < 0.001.
As shown in table 4, pretreatment with VPA alone sensitizes lymphoma cell lines to CHOP treatment. Moreover, the addition of prednisolone further significantly increased CHOP-induced cell death compared to VPA pretreatment alone. These data show that pretreatment with a combination of VPA and prednisolone prior to CHOP therapy would be beneficial for lymphoma patients.
Example 5: pretreatment with the combination of VPA and the steroid dexamethasone significantly improved over pretreatment with VPA aloneCHOP-induced cell death.
To investigate whether other steroids than prednisolone could sensitize VPA and CHOP induced cell death, WSU-NHL cells were treated with different combinations of VPA, the steroid dexamethasone (D, 1 μ M) and CHOP as shown below. Viability was assessed by trypan blue exclusion at the indicated time points (n ═ 3). Significant differences were assessed using Student's unpaired t-test. All tests were bi-directional. The effect was considered statistically significant, P <0.05, P <0.01, P < 0.001.
As shown in table 5, pretreatment with dexamethasone further increased VPA-CHOP-induced cell death compared to pretreatment with VPA alone. These data show that pretreatment with a combination of VPA and other steroids (e.g., dexamethasone) prior to CHOP therapy is beneficial for lymphoma patients.
Example 6: pre-treatment with HDAC inhibitor trichostatin A or belinostat sensitizes DLBCL cell lines to CHOP treatmentFeeling is felt. With trichostatin A orCompared with single pretreatment of belinostat, the combination of trichostatin A and prednisolone is used for pretreatmentTreatment significantly increased CHOP-induced cell death.
To investigate whether pretreatment of HDAC inhibitors, distinct from other HDAC inhibitor subgroups, could also sensitize CHOP-induced cell death, WSU-NHL cells were treated with different combinations of hydroxamic acid/carbamic acid HDAC inhibitors trichostatin a (tsa) and belongstat, prednisolone (P, 20 μ g/ml) and CHOP as shown below. Viability was assessed by trypan blue exclusion at the indicated time points (n ═ 3). Significant differences were assessed using Student's unpaired t-test. All tests were bi-directional. The effect was considered statistically significant, P <0.05, P <0.01, P < 0.001.
As shown in table 6, pretreatment with trichostatin a alone sensitizes lymphoma cell lines to CHOP treatment. Moreover, the addition of prednisolone significantly increased CHOP-induced cell death compared to pre-treatment with VPA alone. These data show that pretreatment with a combination of HDAC inhibitor, also from the hydroxamic acid/carbamate group, and prednisolone, prior to CHOP therapy is beneficial for lymphoma patients.
Belinstat showed similar results (data not shown).