WO2021130381A1 - Combination of a hdac inhibitor and vx molecules, and its use for the treatment of cystic fibrosis - Google Patents

Abstract

The present invention relates to the combination of a histone deacetylase (HDAC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) modulator for use in the treatment of CFTR mediated diseases such as cystic fibrosis (CF).

Description

COMBINATION OF A HD AC TNHTRITOR AND VX MOLECULES. AND ITS USE TOR THE TREATMENT OF CYSTIC FIBROSIS

TECHNICAL FIELD

The present invention provides new pharmacological tools for treating cystic fibrosis transmembrane conductance regulator (CFTR) mediated diseases, in particular cystic fibrosis (CF).

STATE OF THE ART

Cystic fibrosis (CF) is a recessive genetic disease that affects approximately 30,000 children and adults in the United States and approximately 30,000 children and adults in Europe.

In patients with CF, mutations in cystic fibrosis transmembrane conductance regulator (CFTR) endogenously expressed in respiratory epithelia leads to reduced apical anion secretion causing an imbalance in ion and fluid transport. The resulting decrease in anion transport contributes to enhanced mucus accumulation in the lung and the accompanying microbial infections that ultimately cause death in CF patients. In addition to respiratory disease, CF patients typically suffer from gastrointestinal problems and pancreatic insufficiency that, if left untreated, results in death. In addition, the majority of males with cystic fibrosis are infertile and fertility is decreased among females with cystic fibrosis. In contrast to the severe effects of two copies of the CF associated gene, individuals with a single copy of the CF associated gene exhibit increased resistance to cholera and to dehydration resulting from diarrhea - perhaps explaining the relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed a variety of disease causing mutations (Cutting, G. R. etal. (1990) Nature 346:366-369; Dean, M. etal. (1990) Cell 61:863:870; and Kerem, B-S. etal. (1989) Science 245:1073-1080; Kerem, B-S etal. (1990) Proc. Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1700 disease causing mutations in the CF gene have been identified (http://www.genet.sickkids.on.ca/cftr/app). The most prevalent mutation is a deletion of phenylalanine at position 508 of the CFTR amino acid sequence, and is commonly referred to as AF508-CFTR. This mutation occurs in the majority of the cases of CF and is associated with a severe disease. The deletion of residue 508 in AF508-CFTR prevents the nascent protein from folding correctly. This results in the inability of the mutant protein to exit the ER, and traffic to the plasma membrane. As a result, the number of channels present in the membrane is far less than observed in cells expressing wild-type CFTR. In addition to impaired trafficking, the mutation results in defective channel gating. Together, the reduced number of channels in the membrane and the defective gating lead to reduced anion transport across epithelia leading to defective ion and fluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709- 2727). Studies have shown, however, that the reduced numbers of AF508-CFTR in the membrane are functional, albeit less than wild-type CFTR (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk and Foskett (1995), J. Cell. Biochem. 270: 12347-50). In addition to AF508-CFTR, other disease causing mutations in CFTR that result in defective trafficking, synthesis, and/or channel gating could be up- or down- regulated to alter anion secretion and modify disease progression and/or severity.

In the last decade, drugs that target specific defects in the CFTR protein have been developed. As a group, these drugs are called modulators because they are intended to modulate the function of the CFTR protein so that it can serve its primary function, i.e. to create a channel for chloride to flow across the cell surface. Although modulators can’t yet completely restore proper chloride flow, they can improve the flow enough to relieve symptoms for people with CF.

There are three main types of CFTR modulators: the so-called potentiators, correctors and amplifiers.

The first type of CFTR modulator is called a “potentiator”. Potentiators help chloride flow through the CFTR protein channel at the cell surface. The CFTR protein is shaped like a tunnel that can be closed by a gate. Potentiators hold the gate open so chloride can flow through.

The drug ivacaftor (VX-770; Kalydeco^®) is a potentiator. This drug can help patients with gating and conduction mutations in CFTR. It also works on residual function and splice mutations where an insufficient amount of normal protein is present. In all of these mutations, some CFTR protein reaches the surface of the cell. However, either not enough protein reaches the cell surface, or the protein does not allow enough chloride to flow through. By holding the gate on the CFTR protein open, potentiators allow more chloride to flow through and reduce the symptoms of CF. The next type of CFTR modulator is called a “corrector”. Correctors help the CFTR protein to form the right 3D shape so that it is able to move or traffic to the cell surface. Almost half of people with CF have two copies of the F508del mutation, which prevents the CFTR protein from forming the right shape. The corrector drugs, e.g. lumacaftor (VX-809), tezacaftor (VX-661) or elexacaftor (VX-445), help the CFTR protein to form the right shape, traffic to the cell surface, and stay there longer. But, even with lumacaftor and tezacaftor, only about a third of the CFTR protein reaches the cell surface, so by itself it hardly reduces the symptoms of CF.

Additionally, the proteins that do reach the cell surface do not open sufficiently to allow chloride to pass out of the cell. But, if a corrector is used in combination with a potentiator to hold the gate on the CFTR protein open, enough chloride can then flow to reduce the symptoms of CF. The combinations of lumacaftor/ivacaftor (VX-809/VX-770; Orkambi^®) and tezacaftor/ivacaftor (VX-661/VX-770; Symdeko^®) are therefore used to treat especially people with two copies of the F508del mutation. Tezacaftor/ivacaftor also can be used to treat people with a single copy of one of 26 specified mutations, regardless of their other mutation. Tritherapies, e.g. combining VX-445/VX-661/VX-770, have also been proposed.

The last type of CFTR modulator is called an “amplifier.” Amplifiers increase the amount of CFTR protein that the cell makes. Many CFTR mutations produce insufficient CFTR protein. If the cell made more CFTR protein, potentiators and correctors would be able to allow even more chloride to flow across the cell membrane. However, amplifiers, which are being developed, are not yet available.

However, there is still a need for novel treatments of CFTR mediated diseases.

SUMMARY OF THE INVENTION

The inventors have shown that by combining a HD AC (histone deacetyl ase) inhibitor such as givinostat and CFTR modulators such as VX modulators, it is possible to treat CFTR mediated diseases such as CF, especially those due to AF508-CFTR. Notably, a HDAC inhibitor boosts the efficacy of CFTR modulators, possibly by its inhibitory action on HDAC 6. Definitions

The definitions below represent the meaning generally used in the context of the invention and should be taken into account unless another definition is explicitly stated.

In the frame of the invention, the articles “a” and “an” are used to refer to one or several {i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element, i.e. one or more than one elements.

The terms “around”, “about” or “approximately” as used therein when referring to a measurable value such as an amount, a temporal duration and the like should be understood as encompassing variations of ± 20% or ± 10%, preferably ± 5%, more preferably ± 1%, and still more preferably ± 0.1% from the specified value.

Intervals/ranges: throughout this disclosure, various aspects of the invention can be presented in the form of a value interval (range format). It should be understood that the description of values in the form of an interval is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

“Isolated” means altered or removed from its natural environment or state. For example, an isolated nucleic acid or peptide is a nucleic acid or peptide which has been extracted from the natural environment in which it is usually found whether this be in a plant or living animal for example. A nucleic acid or peptide for example which is naturally present in a living animal is not an isolated nucleic acid or peptide in the sense of the invention whereas the same nucleic acid or peptide partially or completely separated from other components present in its natural environment is itself “isolated” in the sense of the invention. An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non native environment such as, for example, a host cell.

In the context of the present invention, the following abbreviations for the commonly occurring nucleic acid bases are used: “A” refers to adenosine, “C” refers to cytosine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine. The term “abnormal” when used in the context of organisms, tissues, cells or components thereof, refers to those organisms, tissues, cells or components thereof that differ in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells or components thereof that display the “normal” (expected) respective characteristic. Characteristics, which are normal or expected for one cell or tissue type, might be abnormal for a different cell or tissue type.

The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ , amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is an animal, preferably a mammal, more preferably a human. It may also be a mouse, a rat, a pig, dog or non-human primate (NHP), such as the macaque monkey.

In the sense of the invention, a “disease” or “pathology” is a state of health of an animal in which its homeostasis is adversely affected and which, if the disease is not treated, continues to deteriorate. Conversely, in the sense of the invention, a “disorder” or “dysfunction” is a state of health in which the animal is able to maintain homeostasis but in which the state of health of the animal is less favourable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily result in deterioration in the state of health of the animal over time.

A disease or disorder is “alleviated” (“reduced”) or “ameliorated” (“improved”) if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by the subject, or both of these, is reduced. This also includes the disappearance of progression of the disease, i.e. halting progression of the disease or disorder. A disease or disorder is “cured” (“recovered”) if the severity of a symptom of the disease or disorder, the frequency with which such a symptom is experienced by the patient, or both, is eliminated.

In the context of the invention, a “therapeutic” treatment is a treatment administered to a subject who displays the symptoms (signs) of pathology, with the purpose of reducing or removing these symptoms. As used herein, the “treatment of a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by the subject. A treatment is said to be prophylactic when it is administered to prevent the development, spread or worsening of a disease, particularly if the subject does not have or does not yet have the symptoms of the disease and/or for which the disease has not been diagnosed. As used herein, “treating a disease or disorder” means reducing the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject. Disease and disorder are used interchangeably herein in the context of treatment.

In the sense of the invention, an “effective quantity” or an “effective amount” of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. The expression “therapeutically effective quantity” or “therapeutically effective amount” refers to a quantity which is sufficient or effective to prevent or treat (in other words delay or prevent the development, prevent the progression, inhibit, decrease or reverse) a disease or a disorder, including alleviating symptoms of this disease or disorder.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the use of a histone deacetylase (HDAC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) corrector for the treatment of a CFTR mediated disease, advantageously cystic fibrosis.

The present invention thus relates to the combination of a histone deacetylase (HDAC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) corrector for use in the treatment of a CFTR mediated disease, advantageously cystic fibrosis.

In other words, a histone deacetylase (HDAC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) corrector are used to prepare a medicament intended for the treatment of a CFTR mediated disease, advantageously cystic fibrosis.

The invention thus relates to a method of treating a CFTR mediated disease, advantageously cystic fibrosis, comprising administering to a subject in need thereof, at an efficient dose, a histone deacetylase (HDAC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) corrector.

The first active ingredient of a combination according to the invention is an inhibitor of histone deacetylases or HDAC inhibitor (HDACi).

As known in the art, the inhibitors of histone deacetylases (HDAC inhibitor) are compounds able to modify the epigenome. Their level of action relates to covalent modifications of histones. Preferably, such modifications concern: the acetylation of lysine residues, the methylation of lysine residues and of arginine, the phosphorylation of threonine and serine residues, the ubiquitination and the sumoylation of lysine residues.

Preferably, such compounds are able to modulate the histone acetylation level, especially by acting on the histone modification enzymes either directly at the level of their activity, or genetically at the level of their expression. The histone acetylation level results from the activity of two antagonistic enzymes: histone deacetylases (HD AC), resulting in a repressed chromatin, and hi stone-acetyl transferases (HAT) which allow the gene expression.

More preferably, such compounds are able to inhibit the activity of the enzymes involved in the deacetylation of the histones.

There are several classes of HD AC inhibitors according to their inhibition mode and to the class of HDACs they target.

Such compounds may be of any nature, for example proteins, peptides, antibodies, chemical molecules, or nucleic acids (antisense oligonucleotides, siRNA, shRNA, ribozymes, ...).

Histone deacetylation inhibitors comprise: o hydroxamic acids or salts thereof:

^■ trichostatin A (TSA);

^■ belinostat (PXD101);

^■ Panobinostat (LBH589);

^■ Givinostat (ITF2357);

^■ Resminostat (4SC-201);

^■ Abexinostat (PCI-24781);

^■ Quisinostat;

^■ Ricolinostat (ACY-1215);

^■ Citarinostat (ACY-241);

^■ Practilinostat ;

^■ CHR-3996 ;

^■ alpha compound 8;

^■ MC1568;

^■ Tubacin;

^■ Tubastatin;

^■ suberoylanilide hydroxamic acid (SAHA or vorinostat or MK063), having formula: o cyclic tetrapeptides and depsipeptides: trapoxin B; apicidin; o benzamides:

^■ entinostat (MS-275), having formula:

^■ CI-994;

^■ 106 ;

^■ 4SC-202;

^■ Tacedinaline (CI994) ;

^■ Mocetinostat (MGCD0103) o electrophilic ketones:

^■ trifluoromethyl ketones;

^■ a-cetoamides; o Aliphatic acid compounds:

^■ phenylbutyrate, having formula:

^■ valproic acid or sodium valproate;

^■ butyric acid o Dacinostat (LAQ824);

- the other molecules: o nicotinamide; o cyclic tetrapeptides such as Romidepsin; o dihydrocoumarin; o naphthopyranone; o 2-hydroxynaphaldehydes; o 10-hydroxy-2-decenoic acid (10HDA); o SB939; o CUDC-101; o CUDC-907 ; o AR-42; o CHR-2845; o 4SC-202; o CG200745; o Sulforaphane; o Kevetrin; o Apicidin; o Sodium butyrate; o (-)-Depudecin; o Sirtinol; o Cambinol ; o Other Sirtuins inhibitors such as Ex-527; o /V-Hydroxy- 1 ,3-dioxo- l//-benz[de]isoquinoline-2(3//)-hexanamide ₀r Scriptaid; o The hydroxamate derivative of butyric acid; o Isobutyramide; o CBHA (m-carboxycinnamic acid bishydoxyamide); o HC toxin; o M344 (4-dimethylamino-N-(6-hydroxycarbamoyl-hexyl)-benzamide); o Nullscript (4-(l,3-dioxo-lH,3H-benzo[de]isoquinolin-2-yl)-N- hy droxybutanami de) ; o PCI-34051, having formula:

The chemical formulas of a number of these inhibitors are described in document Kazantsev and Thompson (Nature Reviews Drug Discovery, 7(10), 2008, 854-866).

Further, inhibitors of the methylation of histones may be: SL11144, having formula:

- DZNep (3-Deazaneplanocin: inhibitor of S-adenosylhomocysteine hydrolase), having formula:

According to a preferred embodiment of the invention, the compound modifying the epigenome is a HD AC inhibitor.

According to the invention, the HDAC inhibitors are preferably those able to inhibit HDAC6. They can be pan-inhibitors, i.e. inhibitors of all types of HDACs. Alternatively, they can have a similar inhibitory action versus different HDACs including HDAC6, or a superior or even exclusive inhibitory action (selective inhibitor) versus HDAC6.

According to a specific embodiment, the HDAC inhibitor is givinostat (ITF2357), belinostat (PXD101), advantageously givinostat (ITF2357).

As an example of a HDAC6 inhibitor, tubacin can be used. Other examples are: ricolinostat (ACY-1215), tubastatin A or tubastatin A HC1, citarinostat (ACY-241), Nexturastat A, HPOB, SKLB-23bb, and WT161.

Further useful compounds are: TH34, scriptdroxinostat, BRD73954, CAY10603, ACY- 738.

Said HDAC inhibitors are usually administered orally or possibly intravenously.

The second active ingredient of a combination according to the invention is a CFTR modulator. According to a particular embodiment, the second active ingredient of a combination according to the invention is a CFTR corrector.

Advantageously, the CFTR corrector is chosen in the following group: lumacaftor (VX- 809), tezacaftor (VX-661), elexacaftor (VX-445), VX-455, VX-659, Cavosonstat (N91115) FDL169, Corr-4a, VRT-422 and VRT-325. More advantageously, the CFTR corrector is VX-809 (lumacaftor). According to a preferred embodiment and when the second active ingredient of the combination according to the invention is a CFTR corrector, it further comprises a CFTR potentiator. Said CFTR potentiator can be PG-01, VRT-532 or VX-770 (ivacaftor or Kalydeco^®), advantageously VX-770.

According to a specific embodiment, the combination of the invention comprises VX-809 and VX-770, advantageously givinostat, VX-809 and VX-770.

Said CFTR correctors and/or potentiators are usually administered orally, possibly in unitary compositions, advantageously tablets or capsules.

The HDAC inhibitor and the CFTR modulator, advantageously the CFTR corrector, and possibly the CFTR potentiator, may be administered simultaneously (e.g. in separate or unitary compositions) or sequentially in either order. In the latter case, the two or three compounds will be administered within a period and in an amount and manner that is sufficient to ensure that the advantageous or synergistic effect is achieved. It will be appreciated that the preferred method and order of administration and the respective dosage amounts and regimes for each component of the combination will depend on the particular HDAC inhibitor and CFTR modulator (corrector and/or potentiator) being administered, the route of administration of the combination, the disease being treated and the particular host being treated. The optimum method and order of administration and the dosage amounts and regime can be readily determined by those skilled in the art using conventional methods and in view of the information set out herein.

The present invention further relates to a product containing as first active ingredient a HDAC inhibitor, and as second active ingredient a CFTR modulator, advantageously a CFTR corrector possibly associated with a CFTR potentiator, as a combined preparation for simultaneous, separate or sequential use in the treatment of patients suffering from a CFTR mediated disease, advantageously cystic fibrosis.

Those skilled in the art could easily determine the effective amount from the results presented hereinafter. In general, it is contemplated that a therapeutically effective amount of a HDAC inhibitor and a CFTR modulator would be from 0.005 mg/kg to 100 mg/kg body weight, and in particular from 0.005 mg/kg to 10 mg/kg body weight. It may be appropriate to administer the required dose as two, three, four or more sub-doses at appropriate intervals throughout the day. Said sub-doses may be formulated as unit dosage forms, for example, containing 0.5 to 500 mg, and in particular 10 mg to 100 mg of active ingredient per unit dosage form. In view of their useful pharmacological properties, the components of the combination according to the invention, i.e. the HDAC inhibitor and the CFTR modulator, advantageously a CFTR corrector (and possibly potentiator), may be formulated into various pharmaceutical forms for administration purposes. The components may be formulated separately in individual pharmaceutical compositions or in a unitary pharmaceutical composition containing both or all components.

The present invention also concerns pharmaceutical compositions containing as active ingredients at least the two compounds as defined above, as well as the use of these compounds or this composition as a medicinal product or medicament. Advantageously, such compositions comprise a therapeutically effective amount of their combination, and a pharmaceutically acceptable carrier.

The present invention therefore also relates to a pharmaceutical composition comprising a HDAC inhibitor and a CFTR modulator, advantageously a CFTR corrector and possibly a CFTR potentiator, together with one or more pharmaceutically acceptable carriers or excipients.

In a specific embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. or European Pharmacopeia or other generally recognized pharmacopeia for use in animals, and humans. The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol and the like.

The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, sustained-release formulations and the like. Examples of suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject. To prepare pharmaceutical compositions for use in accordance with the invention, an effective amount of a particular compound, in base or acid addition salt form, as the active ingredient is combined in intimate admixture with a pharmaceutically acceptable carrier, which carrier may take a wide variety of forms depending on the form of preparation desired for administration. These pharmaceutical compositions are desirably in unitary dosage form suitable, preferably, for administration orally, rectally, percutaneously, or by parenteral injection.

For example, in preparing the compositions in oral dosage form, any of the usual pharmaceutical media may be employed, such as, for example, water, glycols, oils, alcohols and the like in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like in the case of powders, pills, capsules and tablets. Because of their ease in administration, tablets, possibly scored or effervescent tablets, and capsules represent the most advantageous oral dosage unit form, in which case solid pharmaceutical carriers are obviously employed. They can be taken with a little water before or during the main meal.

In a particular embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for e.g. intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to release pain at the site of the injection. The composition is preferably in a liquid form, advantageously a saline and/or glucose composition, more advantageously a phosphate buffered saline (PBS) composition or a Ringer-Lactate solution.

As already mentioned, the amount of the therapeutic agents of the invention, i.e. the compounds as disclosed above, which will be effective in the treatment of a disease can be determined by standard clinical techniques. In addition, in vivo and/or in vitro assays may optionally be employed to help predict optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, the weight and the seriousness of the disease, and should be decided according to the judgment of the practitioner and each patient’s circumstances.

However and according to a particular aspect, the fact that the CFTR modulator, advantageously a CFTR corrector, possibly associated with a CFTR potentiator, is used in combination with a HDAC inhibitor, preferably a HDAC6 inhibitor, allows decreasing the quantity of said CFTR modulators to be administered. According to a specific embodiment, the quantity or concentration of the CFTR modulator can be reduced by a factor 2, 3, 4, 5 or even 6, 7, 8, 9 or 10. In other words, the effective quantity of the CFTR corrector, and possibly of the CFTR potentiator, in the presence of the other compound is 2, 3, 4, 5 or even 6, 7, 8, 9 or 10 times inferior to its effective quantity in the absence of said other compound.

According to a preferred embodiment, when combined in the same composition, the CFTR modulator, advantageously a CFTR corrector, and possibly a CFTR potentiator, is present in an amount inferior to its amount in a composition not comprising a HDAC inhibitor.

Alternatively, it is the HDAC inhibitor which amount is reduced because of the combination with a CFTR modulator, advantageously a CFTR corrector, and possibly a CFTR potentiator.

Suitable administration should allow the delivery of a therapeutically effective amount of the therapeutic product to the target tissues, depending on the disease.

Available routes of administration are topical (local), enteral (system-wide effect, but delivered through the gastrointestinal (GI) tract), or parenteral (systemic action, but delivered by routes other than the GI tract). In some embodiments, the preferred route of administration is generally enteral which includes oral administration. According to other embodiments, it can be a parenteral administration, especially via intramuscular (i.e. into the muscle) or systemic administration (i.e. into the circulating system). In this context, the term “injection” (or “perfusion” or “infusion”) encompasses intravascular, in particular intravenous (IV), and intramuscular (IM) administration. Injections are usually performed using syringes or catheters.

According to one embodiment, the composition is administered orally, intramuscularly, intraperitoneally, subcutaneously, topically, locally, or intravascularly, advantageously orally.

According to a preferred embodiment, the combination or composition according to the invention is administered daily, for example once per day. The treatment can last several weeks, several months, several years or even for the whole life. As already stated, the patient is advantageously a human, particularly a new bom, a young child, a child, an adolescent or an adult. The therapeutic tool according to the invention, however, may be adapted and useful for the treatment of other animals, particularly pigs, mice, pets such as dogs, farm animals or macaque monkeys.

As already mentioned, the present invention relates to the treatment of CFTR mediated diseases. According to a specific embodiment, the CFTR mediated disease is cystic fibrosis (CF). Advantageously, the CFTR mediated diseases are of genetic origin. In other words, the treated diseases are linked to one or more mutations in the CFTR gene, in one copy thereof or in both.

According to one embodiment, the mutations responsible for the disease may be point mutations. However, the disease may be linked to mutations which are larger than points, for example, the deletion of a codon in the gene which codes a protein which is still at least partially active.

Of particular interest are the class II (misfolding and/or premature degradation) and class III (functional impairment) CFTR variants.

According to one embodiment, the CFTR mediated disease to be cured by the combination according to the invention is a disease for which a treatment with a CFTR modulator, advantageously a CFTR corrector, more advantageously a CFTR corrector and a CFTR potentiator, has a positive effect. In other words and advantageously, the patients of interest are those having CFTR responsive mutation(s), i.e. which respond to a treatment with a CFTR modulator, advantageously a CFTR corrector, more advantageously a CFTR corrector and a CFTR potentiator.

According to a specific embodiment, the CFTR mediated disease can be due to a CFTR mutated protein having a mutation chosen in the following group: P67L, G85E, E92K, S492F, DE508 (or F508del), R560T, L1077P, M1101K, N1303K and G551D.

According to another embodiment, the CFTR mutations of interest are those selected in the following group:

711+3A G;

2789-5G A;

3272-26A G;

3846+lOkbC T;

A455E; A1067T;

D110E;

D110H;

D579G;

D1152H;

D1270N;

E56K;

E193K;

E831X;

FI 052V; F1074L ; K1060T ; L206W ; P67L ; R74W; R117C; R347H; R352Q; R1070W; S945L; and S977F.

According to the invention, a preferred mutation is AF508 (or F508del), advantageously in case of a AF508 homozygote subject.

In the context of the invention, the provided solution relies on the use of the claimed combination to ensure a proper folding/trafficking and possibly gating of the CFTR protein, advantageously of the mutated CFTR protein, and thus to restore a normal phenotype.

According to one embodiment, the CFTR mediated disease to be cured by the combination of the invention is not due to mutation(s) leading to a truncated protein, i.e. due to premature stop codons.

According to one aspect, the combination according to the invention is associated with other treatments for the same disease, especially another compound for treating the same disease. According to one embodiment, the present invention concerns a composition, advantageously a pharmaceutical composition or a medicinal product containing a combination according to the invention and potentially other active molecules (other gene therapy proteins, chemical groups, peptides or proteins, etc.) for the treatment of the same disease or a different disease, advantageously of the same disease.

As known in the art and in relation to CF, another treatment of the same disease includes chest physiotherapy. As well, another compound for treating the same disease can be a mucolytic agent, an antibiotic agent, or digestive enzymes.

In relation to CFTR mediated diseases, a further compound able to prevent the cellular degradation of the protein can be administered simultaneously or at different times. In case of simultaneous administration, the different compounds can be associated in the same composition.

According to one embodiment, the combination or composition according to the invention comprises another CFTR modulator or another compound modifying epigenome, e.g. those listed above.

The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, fourth edition (Sambrook, 2012); “Oligonucleotide Synthesis” (Gait, 1984); “Culture of Animal Cells” (Freshney, 2010); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1997); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Short Protocols in Molecular Biology” (Ausubel, 2002); “Polymerase Chain Reaction: Principles, Applications and Troubleshooting”, (Babar, 2011); “Current Protocols in Immunology” (Coligan, 2002). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods.

EXAMPLES

The invention and its advantages are understood better from the examples shown below supporting the annexed figures. In particular, the present invention is illustrated with regard to the effect of various HDAC inhibitors (givinostat and belinostat) combined with the trafficking modulator VX-809 and/or the CFTR potentiator VX-770 in an in vitro model for cystic fibrosis (CF) linked to mutation F508del in the gene encoding the CFTR channel. These examples are not however in any way limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Givinostat in combination with VX-809 and VX-770 rescues F508del CFTR function.

(A-D) Measure of YFP fluorescence (A, B) and quantification of CFTR activity (n=3) (C, D) in HEK-F508del/YFP cells treated with givinostat (10 mM) and/or VX-809 (3 pM) for 24 hours with addition (B, D) or not (A, C) of VX-770 (10 pM) for 30 min.

Figure 2: Belinostat in combination with VX-809 and VX-770 rescues F508del CFTR function.

(A-D) Measure of YFP fluorescence (A, B) and quantification of CFTR activity (n=3) (C, D) in HEK-F508del/YFP cells treated with belinostat (10 pM) and/or VX-809 (3 pM) for 24 hours with addition addition (B, D) or not (A, C) of VX-770 (10 pM) for 30 min. Figure 3: Rescue of misfolded F508del-CFTR degradation by givinostat.

Western blot analysis (A) and quantification (n=3) of CFTR expression (B/ C-band) and (C/ B-band) following treatment of CFBE-F508del cells with different combinations of BTZ (5 nM), givinostat (10 pM) or VX-809 (3 pM) for 24 hours. LaminBl was used to evaluate the loading. Data are presented as fold-change relative to DMSO 0.1% treatment. BTZ = bortezomib. MATERIALS AND METHODS

Cell Culture, transduction and pharmacological treatments.

Human embryonic kidney (HEK293 MSR Grip Tite) cells stably coexpressing eYFP (H148Q/I152L) and F508del-CFTR-3HA (HEK-F508del/YFP) were grown in DMEM supplemented with 10% FBS, 50 pg/mL zeocin, 0.6 mg/mL G418, 10 pg/mL blasticidin. Human bronchial epithelial cell line CFBE41o- derived from a CF patient were modified to stably expressing F508del CFTR (F508del-CFTR) (K. Kunzelmann etal ., Am. ./. Respir. Cell Mol. Biol., 8(5), 522- 529, 1993; D. C. Gruenert et al, ./. Cyst. Fibros ., 3 Suppl 2, 191- 196, 2004 ; B. Illek et al, Cell. Physiol. Biochem., 22(1- 4), 57- 68, 2008). CFBE cells were grown in MEM (Life Technologies) supplemented with 10% FBS (Eurobio Scientific), 1% penicillin/streptomycin (P/S) (Life Technologies) and 1% L-glutamine (Life Technologies). Twenty-four hours after seeding, cells were treated with BTZ (Selleckchem), givinostat (Selleckchem), belinostat (Selleckchem), Lumacaftor (VX-809; Selleckchem), Ivacaftor (VX-770; Selleckchem) or the carrier 0.1% dimethyl sulfoxide (DMSO, VWR). Cells were analyzed after 24 hours of treatment.

Immunoblotting.

Twenty-four hours after treatments with the tested compounds or the carrier, the CFBE- F508del-CFTR cells were collected. Proteins were extracted by cell lysis buffer (RIP A Buffer, Thermo Scientific) and completed with Proteases Inhibitors (Complete PIC, Roche). Protein concentration was measured using the Pierce BCA Protein Assay Kit (ThermoScientific) and the absorbance at 562 nm was evaluated using a TriStar plate reader (Berthold). A total of 50 pg of protein was separated on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred onto nitrocellulose membrane (GE Healthcare) with a Novex Gel Transfert Device (ThermoScientific) following the manufacturer’s instructions. Membranes were then blocked in Tris-Buffer-Saline IX (TBS) containing 0.05% Tween-20 and 5% milk for 1 hour at room temperature. Incubation with primary antibodies diluted in blocking buffer was carried out at 4°C for 2 hours with the mouse anti-CFTR 1 :5000 and 1 : 1000 (596 and 570, Cystic Fibrosis Foundation distribution program) and the rabbit anti-LaminBl 1 : 15000 (abl6048, abcam).Washing was carried out 4 times for 8 minutes at room temperature with TBS + 0.1% Tween20 and the membranes were incubated with a donkey anti-mouse antibody IRDye-680 1:10000 (926-32222, Li- Cor) or a donkey anti-rabbit antibody IRDye-800 1:5000 (926-32213, Li-Cor) in blocking buffer. Washing was carried out 4 times for 8 minutes at room temperature with TBS + 0.1% Tween20. Proteins were revealed using the ECL Prime Western Blotting Detection Reagent kit (GE Healthcare) and images were acquired in a dark room using a G:Box (Syngene) equipped with GeneSys software following the manufacturer’s instructions.

YFP-based CFTR functional assay.

The assay was performed according to L.J. Galietta etal. ( FEBSLett ., 499(3), 220-4, 2001). Briefly, CFTR activity was measured in stably transfected HEK cells coexpressing the halide- sensitive yellow fluorescent protein YFP-H148Q/I152L and F508del CFTR mutant. Cells were seeded in poly-l-lysine coated 96- well black/clear bottom microplates. The CFTR functional assay was carried out 24 hours after individual compound treatments with 3 mM VX-809, 10 pM givinostat or belinostat, or combined treatments. Cells were incubated for 30 min with 50 pi of PBS containing CPT- AMPc (100 pM) before to be transferred to a TrisStar plate reader (Berthold). When indicated, before being treated with of PBS containing CPT- AMPc, cells were incubated with 10 pM VX- 770 for 30 min. Each condition was tested in triplicate. Cell fluorescence (excitation: 485 nm; emission: 535 nm) was continuously measured before and after addition of 100 pi of PBS- Nal. Cell fluorescence recordings were normalized to the initial average value measured and signal decay was fitted to an exponential function to derive the maximal slope. Maximal slopes were converted to rates of change in intracellular I- concentration (in mM/sec). The experiments were repeated three times.

Statistical analysis.

Data are presented as means ± SD. Statistical analysis was performed using the Student’s t test.

RESULTS

The impact of a HD AC inhibitor, givinostat, alone or in combination with FDA approved CF treatments Lumacaftor (VX-809) or Orkambi (VX-770 + VX-809) has been investigated. These drugs provide modest clinical benefit to a subset of patients and research of new molecules able to act in synergy with those treatments is considered as a promising avenue in CF field.

To do so, embryonic kidney (HEK293 MSR Grip Tite) cells stably coexpressing eYFP (H148Q/I152L) and F508del-CFTR-3HA (Trzcmska-Daneluti et al., Mol. Cell Proteomics, 2015) were used. The fluorescence of this yellow fluorescent protein (eYFP) variant can be quenched in response to iodide influx entering the cell through a functional CFTR channel.

Treatment with 10 mM givinostat (Figure 1) induced similar level of YFP quenching to that seen with 3 mM Lumacaftor (VX-809), a CFTR trafficking modulator. Importantly, additional effects on the channel activity were obtained using the combination of givinostat with Lumacaftor (VX-809) treatment (Figures 1A and 1C) and synergistic improvements were observed with Orkambi (VX-809/VX-770) treatment (Figures IB and ID).

Similar results have been obtained using 10 mM belinostat (Figure 2) instead of givinostat, especially a highly increased efficacy with the combination of belinostat with Orkambi (VX-809/VX-770) treatment. Results were confirmed by measuring CFTR protein levels (B- and C- band) following treatments with 10 mM givinostat, 3 mM Lumacaftor (VX-809), or combinational treatments by immunoblot analysis showing that givinostat induces a synergistic improvement in the trafficking efficiency of the F508del CFTR mutant (Figure 3). CONCLUSION

The present study identifies HDAC inhibitors, especially those having an action on HDAC6, as candidate molecules to improve the efficiency of classical treatment based on

CFTR corrector and/or potentiator.

Claims

1. A combination of a histone deacetylase (HD AC) inhibitor and a cystic fibrosis transmembrane conductance regulator (CFTR) corrector for use in the treatment of a CFTR mediated disease.

2. A combination for its use according to claim 1, wherein the disease is cystic fibrosis (CF).

3. A combination for its use according to claim 1 or 2, wherein the HD AC inhibitor inhibits HD AC 6.

4. A combination for its use according to any of claims 1 to 3, wherein the HD AC inhibitor is givinostat (ITF2357).

5. A combination for its use according to any of claims 1 to 4, wherein the HD AC inhibitor is a selective HDAC6 inhibitor.

6. A combination for its use according to claim 1, 2, 3 or 5, wherein the HD AC inhibitor is selected in the group consisting of: tubacin, ricolinostat (ACY-1215), tubastatin A or tubastatin AHC1, citarinostat (ACY-241), Nexturastat A, HPOB, SKLB-23bb, andWT161.

7. A combination for its use according to any of the preceding claims, wherein the CFTR corrector is VX-809 (lumacaftor).

8. A combination for its use according to any of the preceding claims, wherein the combination further comprises a CFTR potentiator.

9. A combination for its use according to claim 8, wherein the CFTR potentiator is VX-770 (ivacaftor).

10. A combination for its use according to claim 9, wherein the combination comprises VX-809 and VX-770.

11. A combination for its use according to claim 10, wherein the combination comprises givinostat, VX-809 and VX-770.

12. A combination for its use according to any of claims 1 to 11, wherein the combination is in the form of a pharmaceutical composition comprising the HD AC inhibitor, the CFTR corrector and possibly the CFTR potentiator.

13. A combination for its use according to any of claims 1 to 11, wherein the combination is for simultaneous, separate or sequential use.

14. A combination for its use according to any of the preceding claims, wherein the disease is due to AF508-CFTR.

15. A combination for its use according to any of the preceding claims, wherein the combination is administered orally, intramuscularly, intraperitoneally, subcutaneously, topically, locally, or intravascularly, advantageously orally.