WO2010115874A1 - Methods for the treatment and the diagnosis ofpulmonary arterial hypertension - Google Patents

Abstract

The present invention relates to methods for treating pulmonary arterial hypertension by administrating apelin/APJ targeting drugs and for diagnosing pulmonary arterial hypertension in a subject by measuring apelin levels in biological sample from said subject.

Description

METHODS FOR THE TREATMENT AND THE DIAGNOSIS OF PULMONARY ARTERIAL HYPERTENSION

FIELD OF THE INVENTION

The present invention relates to methods for the treatment and the diagnosis for pulmonary arterial hypertension using apelin/APJ targeting drugs.

BACKGROUND OF THE INVENTION Pulmonary arterial hypertension (PAH) is defined as pulmonary vascular disease affecting the pulmonary arterioles resulting in an elevation in pulmonary artery pressure and pulmonary vascular resistance but with normal or only mildly elevated left-sided filling pressures. PAH is caused by a constellation of diseases that affect the pulmonary vasculature. The mechanisms are still poorly understood. Particularly, a mutation in the bone morphogenetic protein type 2 receptor (BMPR2, a TGF-b receptor) has been identified as a cause of familial primary pulmonary hypertension (PPH) (Lane KB et Al, 2000; Deng Z et Al, 2000). It is estimated that 6% of cases of PPH are familial, and that the rest are "sporadic". Interestingly, the BMP pathway is reduced in all forms of PAH (Lingling Du et AL, 2003; Atkinson et AL, 2002; Teichert-Kuliszewska, 2006). The incidence of PPH is estimated to be approximately 1 case per 1 million of population. PAH is a progressive disease associated with a high mortality. Subjects with PAH may develop right ventricular (RV) failure. The extent of RV failure predicts outcome (Mc Laughlin VV, 2002).

The evaluation and diagnosis of PAH is reviewed by McLaughlin and Rich (2004) and McGoon et al (2004). A clinical history, such as symptoms of shortness of breath, a family history of PAH, presence of risk factors and findings on physical examination, chest x-ray and electrocardiogram may lead to the suspicion of PAH. The next step in the evaluation will usually include an echocardiogram. To confirm the diagnosis of PAH a cardiac catheterization to directly measure the pressures on the right side of the heart and in the pulmonary vasculature is mandatory. The upper limit of normal for mean pulmonary artery pressure in an adult human is 19 mm Hg. A commonly used definition of mean pulmonary artery pressure is one-third the value of the systolic pulmonary artery pressure plus two-thirds of the diastolic pulmonary artery pressure. PAH is defined as a mean pulmonary artery pressure greater than or equal to 25 mm Hg with a PCWP less than or equal to 15-16 mm Hg, and a pulmonary vascular resistance (PVR) greater than or equal to 240 dynes sec/cm. Until recently, the only effective long-term therapy for PAH in conjunction with anticoagulant therapy was continuous intravenous administration of prostacyclin, also known as epoprostenol (PGI2) (Barst R et Al, 1996; Mc Laughlin V et Al, 1998). Recently, the nonselective endothelin receptor antagonist, bosentan, has shown efficacy for the treatment of PAH (Rubin LJ et Al, 2002). As the first orally bioavailable agent with efficacy in the treatment of PAH, bosentan represents a significant advance, but still additional treatment like PGI2 is required for a subset of subjects. Phosphodiesterase type V inhibitors (Sildenafil), analogues of PGI2 (Treprostinil) and other prostacyclin (Iloprost) are also used for treatment of PAH. Current treatments are ineffective after 2 years and drive to pulmonary transplant. Thus, there is a real need for new therapeutical strategies.

APJ (encoded by the AP J/ APLNR gene) is a cell surface receptor belonging to the G protein-coupled receptor family and has seven transmembrane domains (O'Dowd BF et Al, 1993). APJ is related to the angiotensin II receptor and has been described as being a coreceptor involved in the mediation of HIV-I neuropathogenesis. A natural ligand of APJ was identified and named apelin (APJ endogenous ligand). The apelin polypeptide is initially produced as a 77 amino acid protein (preproapelin) that is cleaved to produce cleavage products of 36 amino acids, 17 amino acids, and 13 amino acids. The peptide consisting of the C-terminal 13 amino acids of the apelin polypeptide is necessary and sufficient for the ability of an apelin polypeptide to interact with APJ. (US2006045880)

Contrasting results have been obtained and published on the role of apelin during experimental models of vascular lesions or pulmonary hypertension, leading to difficulties in interpreting the role of apelin. Different studies demonstrate that depending on the conditions and the models, apelin is able either to decrease or to increase vascular remodeling and media hypertrophy. As examples, the apelin gene inactivation in mice increases the vascular response of the pulmonary vessels to hypoxia (Suparna C. et al, 2009), but in contrast, inactivation of the apelin receptor APJ decreases atherosclerotic remodeling secondary to high cholesterol diet in apoE deficient mice (Hashimoto T. et al, 2005). These paradoxical results show a complex and important role of apelin in pulmonary hypertension, which is still misunderstood.

SUMMARY OF THE INVENTION The present invention relates to an APJ antagonist for use in the treatment of pulmonary arterial hypertension. The invention also relates to an inhibitor of APJ gene expression or an inhibitor of apelin gene expression for use in the treatment of pulmonary arterial hypertension. The invention also relates to pharmaceutical compositions thereof. The present invention further relates to a method for diagnosing pulmonary arterial hypertension in a subject, wherein the concentration of apelin is measured in a biological sample obtained from said subject.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The term "APJ" has its general meaning in the art and refers to a G protein coupled receptor. The term may include naturally occurring "APJ" and variants and modified forms thereof. The APJ can be from any source, but typically is a mammalian (e.g., human and non- human primate) APJ, particularly a human APJ. The term "apelin" has its general meaning in the art and refers to the natural ligand of

APJ receptor. The term may include naturally occurring "apelin" and variants and modified forms thereof. The apelin can be from any source, but typically is a mammalian (e.g., human and non-human primate) apelin, particularly a human apelin.

Inhibitor of expression of APJ or apelin

The term "expression" when used in the context of expression of a gene or nucleic acid refers to the conversion of the information, contained in a gene, into a gene product. A gene product can be the direct transcriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisense RNA, ribozyme, structural RNA or any other type of RNA) or a protein produced by translation of a mRNA and maturation. An "inhibitor of expression" refers to a natural or synthetic compound that reduces or suppresses the expression of a gene.

Consequently an "inhibitor of gene expression" refers to a natural or synthetic compound that has a biological effect to inhibit or significantly reduce the expression of a gene. Accordingly, an inhibitor of APJ gene expression refers to such compound that inhibits or reduces the expression of APJ gene; an inhibitor of apelin gene expression refers to such compound that inhibits or reduces the expression of apelin gene.

APJ antagonist

The terms "APJ receptor antagonist" or "APJ antagonist" includes any entity that, upon administration to a patient, results in inhibition or down-regulation of a biological activity associated with activation of the APJ receptor by apelin in the patient, including any of the downstream biological effects otherwise resulting from the binding to APJ receptor with apelin. Such APJ receptor antagonists include any agent that can block APJ receptor activation or any of the downstream biological effects of APJ receptor activation. For example, such a APJ receptor antagonist (e.g. a small organic molecule, an antibody directed against CXCR2) can act by occupying the ligand binding site or a portion thereof of the APJ receptor, thereby making APJ receptor inaccessible to its natural ligand, apelin, so that its normal biological activity is prevented or reduced. The term APJ receptor antagonist includes also any agent able to interact with the natural ligand of APJ, namely apelin. Said agent may be an antibody directed against apelin which can block the interaction between apelin and APJ or which can block the activity of apelin ("neutralizing antibody"). Subject

As used herein, the term "subject" denotes a mammal, such as a rodent, a feline, a canine, and a primate. Preferably a subject related to the invention is a human. Control

As used herein, the term "control" denotes a subject which is not affected by pulmonary arterial hypertension and has normal mean pulmonary arterial pressure (e.g. inferior to 19mmHg).

Biological sample The term "biological sample" is used herein in its broadest sense. A biological sample is generally obtained from a subject. A sample may be of any biological tissue or fluid with which bio marker of the present invention may be assayed. Frequently, a sample will be a "clinical sample", i.e., a sample derived from a patient. Such samples include, but are not limited to, bodily fluids which may or may not contain cells, e.g., blood (e.g., whole blood, serum or plasma), urine, synovial fluid, saliva, and joint fluid; tissue or fine needle biopsy samples, such as from bone or cartilage, and archival samples with known diagnosis, treatment and/or outcome history. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. The term "biological sample" also encompasses any material derived by processing a biological sample. Derived materials include, but are not limited to, cells (or their progeny) isolated from the sample or proteins extracted from the sample. Processing of a biological sample may involve one or more of: filtration, distillation, extraction, concentration, inactivation of interfering components, addition of reagents, and the like. Therapeutic methods and uses

A first object of the present invention relates to an inhibitor of apelin or APJ gene expression for use in the treatment of pulmonary arterial hypertension.

The invention also relates to an APJ antagonist for use in the treatment of pulmonary arterial hypertension.

Inhibitors of apelin or APJ gene expression

According to the invention, inhibitors of apelin or APJ gene expression, for use in the present invention, may be based on anti-sense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of apelin or APJ mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of apelin or APJs, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence encoding apelin or APJ can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Small interfering RNAs (siRNAs) can also function as inhibitors of apelin or APJ gene expression for use in the present invention. Apelin or APJ gene expression can be reduced by introducing in cells from a subject a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that apelin or APJ gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequences are known (e.g. see Tuschl T et Al, 1999; Elbashir SM et Al, 2001; Hannon GJ, 2002; McManus MT et Al, 2002; Brummelkamp TR et Al, 2002; U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836CD26CD26. shRNAs (short hairpin RNA) can also function as inhibitors of apelin or APJ gene expression for use in the present invention.

Ribozymes can also function as inhibitors of apelin or APJ gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleo lytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of apelin or APJ mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable.

Both antisense oligonucleotides and ribozymes useful as inhibitors of apelin or APJ gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half- life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone. APJ Antagonists

In one embodiment, such a molecule could be an antibody directed against apelin or APJ, which is able to block the interaction between these both proteins. Antibodies capable of specific binding to apelin or APJ may be derived from a number of species including, but not limited to, rodent (mouse, rat, rabbit, guinea pig, hamster, and the like), porcine, bovine, equine or primate and the like. Antibodies from primate (monkey, baboon, chimpanzee, etc.) origin have the highest degree of similarity to human sequences and are therefore expected to be less immunogenic. Procedures for raising "polyclonal antibodies" are well known in the art. For example, polyclonal antibodies can be obtained from serum of an animal immunized against apelin or APJ, which may be produced by genetic engineering for example according to standard methods well-known by one skilled in the art. (Harlow et al., 1988), which is hereby incorporated in the references. Laboratory methods for preparing monoclonal antibodies are also well known in the art (see, for example, Harlow et al, 1988). Monoclonal antibodies (mAbs) may be prepared by immunizing a mammal such as mouse, rat, primate and the like, with purified apelin or APJ protein. The antibody-producing cells from the immunized mammal are isolated and fused with myeloma or heteromyeloma cells to produce hybrid cells (hybridoma). The hybridoma cells producing the monoclonal antibodies are utilized as a source of the desired monoclonal antibody. This standard method of hybridoma culture is described in (Kohler and Milstein, 1975).

In still another embodiment, the APJ antagonist may be an aptamer in such a way that said aptamer inhibits the association of APJ with its natural ligand, apelin.

Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence.

A further object of the invention relates to a method of treating pulmonary arterial hypertension comprising administering to a subject in need thereof a therapeutically effective amount of an APJ antagonist or an inhibitor of apelin or APJ gene expression according to the invention.

In the context of the invention, the term "treating" or "treatment", as used herein, means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or one or more symptoms of such a disorder or condition.

By a "therapeutically effective amount" of an inhibitor is meant a sufficient amount of the inhibitor to treat disease, at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of inhibitor will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the severity of the disorder, activity of substance employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject, the duration of the treatment; drugs used in combination or coincidental with the specific substance employed, and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. The substance may be used in combination with any other therapeutic strategy for treating the disorders or conditions as above described.

Inhibitors and antagonists according to the invention could be an antibody of APJ (WO2006023893) or one of inhibitors described in the WO2006023893 patent application such as wortmannin, LYZ94002, GF109203X or PD098059.

Screening methods

Inhibitors and antagonists of the invention can be further identified by screening methods described in the state of the art. The screening methods of the invention can be carried out according to known methods.

The screening method may measure the binding of a candidate compound to APJ, or to cells or membranes bearing APJ, or a fusion protein thereof by means of a label directly or indirectly associated with the candidate compound. Alternatively, a screening method may involve measuring or, qualitatively or quantitatively, detecting the competition of binding of a candidate compound to the receptor with a labelled competitor (e.g., inhibitor or substrate).

For example, APJ cDNA may be inserted into an expression vector that contains necessary elements for the transcription and translation of the inserted coding sequence. Following vector/host systems may be utilized such as Baculovirus/Sf9 Insect Cells Retrovirus/Mammalian cell lines like HepB3, LLC-PKl, MDCKII, CHO, HEK293 Expression vector/Mammalian cell lines like HepB3, LLC-PKl, MDCKII, CHO, HEK293. Such vectors may be then used to transfect cells so that said cells express recombinant APJ at their membrane. It is also possible to use cell lines expressing endogenous APJ protein, such as Human Dermal Microvascular endothelial cells (HMEC-I) (Xu Y et al, 1994).

Pharmaceutical compositions

A further object of the invention relates to a pharmaceutical composition comprising an effective amount of an inhibitor or an antagonist according to the invention and pharmaceutically acceptable excipients or carriers.

Any therapeutic agent of the invention as above described may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the subject, etc.

The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

Preferably, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions.

The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

To prepare pharmaceutical compositions, an effective amount of an inhibitor or an antagonist according to the invention may be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The inhibitor or the antagonist according to the invention can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free 30 carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof.

Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed.

For parenteral administration in an aqueous solution, for example, the solution may be suitably buffered and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently used.

Compositions of the present invention may comprise a further therapeutic active agent.

The present invention also relates to a kit comprising an inhibitor or an antagonist according to the invention and a further therapeutic active agent.

Diagnostic methods

A further object of the invention relates to a method for diagnosing pulmonary arterial hypertension in a subject, wherein the concentration of apelin is measured in a biological sample obtained from said subject and compared with control samples.

According to the invention, a high concentration of apelin in said biological sample is indicative of a pulmonary arterial hypertension or of a pathological process leading to pulmonary hypertension.

Typically, the biological sample used for diagnosing a pulmonary arterial hypertension according to the method of the invention by assessing the level of apelin can result from serum samples or plasma, from biological samples containing macrophages (the level in macrophages measured) or from a biopsy, and more specifically from a lung biopsy. Preferably, the measure of serum levels of apelin in macrophages can constitute an attractive less invasive alternative than the analysis of tissue samples. Typically, the expression of apelin is measured in a lung biopsy or in macrophages.

The expression of apelin can be measured at the level of the mRNA or at the level of the protein as follows:

Determination of the expression level of apelin by quantifying mRNAs: total RNAs can be easily extracted from a biological sample. The biological sample may be treated prior to its use, e.g. in order to render nucleic acids or proteins available.

Techniques of cell or protein lysis, concentration or dilution of nucleic acids, are known by the skilled person.

Determination of the expression level of apelin can be performed by a variety of techniques. Generally, the expression level as determined is a relative expression level.

More preferably, the determination comprises contacting the sample with selective reagents such as probes, primers or ligands, and thereby detecting the presence, or measuring the amount, of or nucleic acids of interest originally in the sample. Contacting may be performed in any suitable device, such as a plate, microtiter dish, test tube, well, glass, column... In specific embodiments, the contacting is performed on a substrate coated with the reagent, such as a nucleic acid array or a specific ligand array. The substrate may be a solid or semi-solid substrate such as any suitable support comprising glass, plastic, nylon, paper, metal, polymers and the like. The substrate may be of various forms and sizes, such as a slide, a membrane, a bead, a column, a gel, etc. Level of mRNAs

Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the subject) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA may be then detected by hybridization (e. g., Northern blot analysis).

Alternatively, the extracted mRNA may be subjected to coupled reverse transcription and amplification, such as reverse transcription and amplification by polymerase chain reaction (RT-PCR), using specific oligonucleotide primers that enable amplification of a region in the nucleic acid of apelin may be used. Quantitative or semiquantitative RT-PCR is preferred. Real-time quantitative or semiquantitative RT-PCR is particularly advantageous. Extracted mRNA may be reverse transcribed and amplified, after which amplified sequences may be detected by hybridization with a suitable probe or by direct sequencing, or any other appropriate method known in the art. Other methods of Amplification include ligase chain reaction (LCR), transcription mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

In another embodiment, the expression level may be determined by DNA microarray analysis. Such DNA microarray or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of 5 complexes between 20 target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semiquantified.

Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art [for a review see e.g. (Hoheisel, 2006)). Level of proteins Determination of the expression level of apelin by quantifying proteins: other methods exist for determining the expression level of apelin.

Such methods comprise contacting a biological sample with a binding partner capable of selectively interacting with the apelin present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal. The presence of the apelin peptide can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme- labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, immunocytochemistry, immunohistochemistry, etc. The reactions generally include revealing labels such as fluorescent, chemiluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith. More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A biological sample containing or suspected of containing the marker protein is then added to the coated wells.

After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate washed and the presence of the secondary binding molecule detected using methods well known in the art.

FIGURES:

Figure 1. BMPs down-regulate apelin in microvascular endothelial cells. (A)

HMEC-I were stimulated with IOng/mL BMP4, 7 or 9 for 4, 7 or 24h. The levels of apelin mRNA were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in BMP -treated cells to those of untreated cells for each time point, both, normalized to RPL32 levels. Results are mean ± S.E. values of three independent experiments. *, p<0.05 relative to untreated cells (B) Secreted apelin level in cultured medium from HMEC-I cells treated or not with BMP4 (IOng/mL) for 72h was determined by a competitive radioimmunoassay. The relative secreted apelin concentration is calculated as the ratio of secreted apelin level in BMP -treated cells to those of untreated cells. Results are mean ± S.E. values of three independent experiments, * p<0.05 relative to untreated conditions. (C) HLMECs were stimulated with 50ng/mL BMP4, BMP7 or 9 for 24h. The relative apelin expression levels were calculated as the ratio of apelin expression in BMP -treated cells to those of untreated cells, both, normalized to RPL32 levels. Results are mean ± S.E. values of three independent experiments, * p<0.05 relative to untreated cells. Figure 2. Apelin down-regulation by BMP4 is mediated by BMPR2 and SMAD pathway. (A) HMEC-I were transfected with a non specific (control siRNA), a BMPR2 specific siRNA (BMPR2 siRNA) or transfection agent only (Mock). After transfection, cells were starved overnight, stimulated with BMP4 (IOng/mL) for 24h and then total RNA was extracted. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of mock untreated cells, both, normalized to RPL32 levels. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to mock untreated cells. (B) HMEC-I were infected with the adenoviruses coding for beta-galactosidase (LacZ), a dominant negative form of ALKl (dnALKl) and a dominant negative form of ALK6 (dnALKβ). After infection, cells were starved overnight and then stimulated with BMP4 (lOng/mL) for 7h. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of uninfected, untreated cells, both, normalized to GAPDH levels. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to uninfected, untreated cells. (C) HMEC-I were transfected with a non specific (control siRNA) or a ALK-I specific siRNA (ALKl siRNA). After transfection, cells were starved overnight, stimulated with BMP4, BMP7 or BMP9 (IOng/mL) for 24h and then total RNA was extracted. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of untreated cells, both, normalized to RPL32 levels. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to untreated cells. (D) HMEC-I were infected with the adenoviruses coding for beta-galactosidase (LacZ) or SMAD7 (Ad.SMAD7). After infection, cells were stimulated with BMP4 (IOng/mL) for 4 or 7h. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of uninfected, untreated cells, both, normalized to GAPDH levels. Results are mean± S.E. values of 2 independent experiments.

Figure 3. Apelin down-regulation by BMPs is dependent of a transcriptional and direct mechanism. (A) HMEC-I cells were pre-treated or not with Actinomycin D (ActD) for 15min before being stimulated with BMP 4,7 or 9 (10 ng/mL) for 5h. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of control cells, both, normalized to RPL32 levels. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to control cells. (B) HMEC-I cells were pre-treated or not with Cycloheximide (CHX) for 15min before being stimulated with BMP 4, 7 or 9 (10 ng/mL) for 5h. Apelin mRNA levels were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of control cells, both, normalized to RPL32 levels. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to control cells. #, p< 0.05 relative to CHX untreated cells. (C) and (D) HMEC-I cells were treated with IOng/mL BMP4 and BMP7 for 5h. Nuclear RNAs were isolated and analysed by PCR (C) or real-time PCR (D) using primers that hybridize to intron 1 and exon 2 and detect only the apelin pre- mRNA. Apelin pre-mRNA expression was normalized to GAPDH mRNA expression. Results are mean± S.E. values of 3 independent experiments. *, p<0.05 relative to control cells.

Figure 4. BMPs inhibit hypoxia-induced endothelial cell proliferation through the inhibition of apelin expression. (A) BrdU incorporation in MEECs treated or not with BMP4, 7 or 9 at 50 ng/niL or 200 ng/niL and exposed or not to hypoxia (1% O₂) for 24h. Results represent means ± S.E. of 3 independent experiments. *, p<0.05 relative to normoxia.(B) . BrdU incorporation in MEECs infected with a control adenovirus (AdGFP) or an adenovirus coding for the human apelin (AdAPE), treated or not with BMP 4, 7 or 9 (50ng/mL) and exposed or not to hypoxia (1%O2). Results represent means ± S.E. of 3 independent experiments. *, p<0.05 relative to normoxia. (C) MEECs were treated with BMP4 or BMP9 (50ng/mL) and exposed or not to hypoxia (1% O₂) 24h. The levels of apelin mRNA were determined by real-time RT-PCR analysis. The relative apelin expression levels were calculated as the ratio of apelin expression in experimental cells to those of normoxic untreated cells, both normalized to RPL32 levels. Results are mean ± S.E. values of three independent experiments. *, p<0.05 relative to normoxic untreated cells.

Figure 5. Apelin expression in human monocytes and macrophages from PAH patients (A) PCR analysis of apelin mRNA in human monocytes and macrophages isolated from the blood of healthy subjects. HMEC-I are used as a positive control of the PCR (B) Apelin mRNA expression in human macrophages differentiated from monocytes isolated from control subjects, from patients with idiopathic PAH (iPAH) and from patients with

BMPR2-linked PAH (hPAH). The relative apelin expression levels were calculated as the ratio of apelin expression in patients to those of control subjects, both, normalized to RPL32 levels. Results are mean ± S.E. values of 10 individuals. *, p<0.05 relative to control subjects.

Figure 6. Systolic Pulmonary arterial pressure in rats injected with adenovirus Apelin or GFP+/-monocrotalin (MCT). Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). A polyvinyl catheter was introduced into the right jugular vein, and then pushed through the right ventricle (RV) into the pulmonary artery. A polyethylene catheter was inserted into the right carotid artery.

Figure 7. Fulton index in rats injected with adenovirus apelin or GFP Ad +/- monocrotalin (MCT). Fulton index is the ratio of the weight of the right ventricle to the weight of the left ventricle and septum (RW[LV + S]).

EXAMPLE: Example 1

Materials and methods

Recombinant adenoviruses construction. The adenovirus vector containing the complete coding sequence for the human apelin (Ad.APLN) was constructed according to He et al, 1998. Adenoviruses expressing human SMAD7, were a gift from Peter ten Dijke, dominant negative forms of ALK3 and ALK6 were a gift from A. Moustakas.

Circulating Monocyte Isolation and phagocytic differentiation. Mononuclear cells were isolated from venous blood of healthy donors (n=10, age 37±5yrs) and patients with idiopathic PAH (iPAH, n=10, aged 58±4yrs), or BMPR2-linked PAH, (mPAH, n=10, age 44±5 yrs). Circulating monocytes and differentiated macrophages were obtained as previously described in Maouche et al, 2008. This study was approved by the ethical committee of the Pitie-Salpetriere Hospital and informed consent was obtained from all participants.

Apelin assay. HMEC-I were treated with BMP4 (IOng/mL) for 72h. Supernatant was snap frozen and kept at -80⁰C until use. Secreted Apelin-36 was measured using the Apelin- 36 Radioimmunoassay Kit according to manufacturer's recommendation (Phoenix Pharmaceuticals, Belmont, CA).

Transcription assay of the apelin gene. Gene transcription was measured by quantifying pre-mRNA as previously described in Lu et al, 2007. Apelin pre-mRNA was measured by real-time PCR. Apelin pre-mRNA expression was normalized to GAPDH expression measured by real-time PCR

Results

BMPs down-regulate apelin in microvascular endothelial cells. DNA microarray experiments were performed to identify BMP target genes in HMEC-I cells stimulated for 7h or 24h by IOng/mL of BMP4. Among the modulated genes, including IDl, ID2 and ID3, which were markedly up-regulated (data not shown), we observed a strong down-regulation of apelin expression by BMP4 at both 7h and 24h of stimulation.

We confirmed, by real-time PCR, the down-regulation of apelin expression in HMEC- 1 cells treated by IOng/mL of BMP4 (Fig. IA). By competitive radioimmunoassay, we showed that secreted apelin levels were slightly but significantly decreased in the culture medium of cells treated with BMP4 compared to control cells (Fig. IB).

Apelin mRNA levels were also decreased by other members of BMP growth factors family, BMP7 and BMP9, with a similar to BMP4 pattern (Fig. IA). We found that 4h after stimulation and for at least 24h, these BMPs inhibited by 60% and by up to 96% the mRNA expression of apelin. In order to check whether this modulation also occurred in pulmonary vascular endothelial cells, apelin expression was measured in primary human lung microvascular endothelial cells (HLMEC) after 24h of BMP treatment (50ng/mL). BMP4 and BMP7 inhibited apelin expression by 50% whereas BMP9 inhibited apelin expression for more then 96 % (Fig. 1C). Thus we decided to further investigate the modulation of apelin by BMPs, as apelin could be an important player in the vascular tone modulation and vascular remodelling of IPAH.

Apelin down-regulation by BMPs is mediated by BMPR2 and the SMAD pathway. We knocked-down the expression of BMPR2 by using a BMPR2 -targeting siRNA. BMP down-regulation of apelin expression is abolished in cells transfected with BMPR2- directed siRNA compared with those transfected with non-specific siRNA (Fig.2A), demonstrating that the down-regulation of apelin expression by BMP is mediated by BMPR2. HMEC-I were infected with adenoviruses coding for dominant negative (dn) forms of ALK3 and ALK6. Over-expression of dominant negative forms of both type I receptors, ALK3 and ALK6 suppressed the BMP-induced down regulation of apelin expression. These results demonstrate the implication of either of these type I receptors in transduction by interaction with BMPR2, and that this interaction is interrupted by either dnALK3 or dnALKβ. Because BMP9 was shown to be a ligand for the type I receptor ALKl (David et al, 2007), we tested its possible implication. We knocked-down the expression of ALKl by transfecting a ALK-I- targeting siRNA. We still observed BMP down-regulaion of apelin expression in cells transfected with ALKl -directed siRNA compared with those transfected with non-specific siRNA (Fig.2C) demonstrating that ALKl is not involved.

BMPs activate intracellular pathways including the SMAD pathway. We inhibited that pathway by adenoviral overexpression of the SMAD pathway inhibitor SMAD7 and showed by real-time PCR analyses that SMAD7 overexpression abolished the BMP down-regulation of apelin expression and even increased the basal levels of apelin expression (Fig. 2D). This shows that BMP4 inhibits apelin expression via the SMAD pathway and that even in unstimulated conditions, some basal SMAD activity, possibly due to BMP ligands in the culture medium, inhibits apelin expression.

Molecular mechanism of apelin down-regulation by BMPs. In order to determine which step was involved in the BMP-induced apelin down-regulation, we inhibited DNA transcription or mRNA translation by pre-treating cells with Actinomycin D or cycloheximide, respectively, before BMP stimulation. Actinomycin D (Fig. 3A), but not cycloheximide (Fig. 3B), abolished the inhibition by BMP4, BMP7 and BMP9 of apelin mRNA expression, showing that apelin inhibition by BMPs is transcriptional and does not seem to require synthesis of other proteins. We analysed the transcriptional activity of the apelin gene by measuring pre-mRNA expression by real-time PCR using primers that hybridize to sequences found in introns and, therefore, specifically recognize pre-mRNA (primers could also bind to genomic DNA but this was removed during RNA processing). After 5h of BMP treatment, pre-mRNA levels were strongly reduced by BMP4 or BMP7 stimulation, showing that apelin transcription is inhibited by BMP stimulation (Fig. 3C and 3D). We characterized the apelin promoter activity in response to BMP by transfecting HMEC-I with luciferase reporter constructs under the control of fragments of the human apelin promoter. None of the constructs tested, including one containing 2500bp of the proximal promoter, responded to BMP4 stimulation (supplementary Fig. 1). We also tested the hypothesis that the transcriptional down regulation of apelin by BMPs is due to the RNA degradation by a microRNA. However by inhibiting the processing enzyme DICER with a DICER-targeting siRNA, we did not see any differences between BMP -treated and untreated cells (Supplementary Fig. 2).

Thus BMP downregulation of apelin expression is due to a transcriptional direct mechanism. However regulatory elements involved were not identified and further investigations will be necessary to fully characterize the molecular mechanism. Enforced expression of Apelin restored hypoxia-induced endothelial cell proliferation inhibited by BMPs. We performed BrdU incorporation assay in endothelial cells treated with BMP4, BMP7 and BMP9, exposed or not to hypoxia (1% 02). When endothelial cells were exposed to hypoxia for 24h, BrdU incorporation was significantly increased. This effect was partially abolished in the presence of BMPs (Fig 4A). We recently demonstrated that apelin participates to hypoxia-induced endothelial cell proliferation (Eyries et al, 2008). We hypothesized that down-regulation of apelin expression by BMPs inhibits the hypoxia-induced cell proliferation. Indeed, when we over-expressed apelin by using an adenovirus vector we observed an increased BrDU incorporation rate showing that inhibition of hypoxia-induced endothelial cell proliferation is no more detectable (Fig. 4B). Determination of apelin expression levels revealed that hypoxia-induced apelin expression are reduced when cells are treated with BMPs (Fig. 4C).

Alltogether, these results demonstrated that BMPs inhibit hypoxia-induced endothelial cell proliferation by the inhibition of apelin expression. Apelin expression in human macrophages. When monocytes are differentiated into macrophages by MCSF (macrophage colony stimulating factor) treatment, apelin mRNA expression was observed (Fig. 5A). Apelin expression in macrophages differentiated from monocytes is MCSF independent since we used other differentiating methods and apelin was always expressed in macrophages and absent in monocytes (data not shown). Thus we measured the levels of apelin mRNA in macrophages from patients with idiopathic or

BMPR2-linked heritable PH compared to control subjects. We observed a significant 1.5-fold

(p<0.05) increase in apelin mRNA levels in iPAH patients and a 3-fold increase (p<0.005) in hPAH patients compared to control subjects (Fig 5 B).

Example 2: Experimental Pulmonary Hypertension in rats by Apelin-Ad and monocrotalin injection.

Adult male Wistar rats weighing 200-250 g were used (Charles River). Pulmonary hypertension (HP) was induced in rats by a single s.c. injection of monocrotalin (MCT, 60 mg/kg).

Delivery of Adenovirus Vectors to the Lungs. To investigate the consequence of Apelin overexpression on MCT-inducing pulmonary hypertension, rats were randomly divided into 2 groups (16 animals in each group), of which two were treated with Ad-apelin and the others given the Ad-Null. Then for each treatment the animals were separated into two groups, one group given s.c. injection of monocrotalin and the other received vehicle. Ad.apelin, or Ad.Null as the control (10⁸ pfu) was diluted before use with sterile saline, pH 7.4, in a final volume of 150 μl. Rats were anesthetized with intraperitoneal ketamine and xylazine before intratracheal instillation of 150 μl/rat.

Assessment of Pulmonary Hypertension. Rats were anesthetized with sodium pentobarbital (60 mg/kg, i.p.). A polyvinyl catheter was introduced into the right jugular vein, and then pushed through the right ventricle (RV) into the pulmonary artery. A polyethylene catheter was inserted into the right carotid artery. The heart was dissected and weighed for calculation of the RV hypertrophy index (RV/[LV + S]).

Results. These results show that apelin enforced expression in lungs, owing to injection of an apelin encoding adenovirus in lung airways is able to induce pulmonary arterial hypertension in rats. Apelin enforced expression also aggravates PAH induced by monocrotalin, a well established model of PAH.

Because apelin mRNA is strongly decreased by BMPs, a signaling pathway which is weakened in idiopathic and heritable PAH secondary to a BMPR2 mutation, these results provide a link between BMP target genes and PAH development, and give a clue for a potential drug target.

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Claims

1. An APJ antagonist for use in the treatment of pulmonary arterial hypertension.

2. An Inhibitor of APJ gene expression for use in the treatment of pulmonary arterial hypertension.

3. An Inhibitor of apelin gene expression for use in the treatment of pulmonary arterial hypertension.

4. Pharmaceutical compositions comprising an antagonist or an inhibitor according to any one of claims 1-3 for the treatment of pulmonary arterial hypertension.

5. A method for diagnosing pulmonary arterial hypertension in a subject, wherein the concentration of apelin is measured in a biological sample obtained from said subject.

6. The method according to the claim 4 wherein said method comprises the steps consisting of:

• obtaining a biological sample from the subject; and

• detecting the concentration of apelin in said biological sample, and

• comparing the concentration of the apelin in this biological sample to a control;

• wherein a higher apelin concentration than control is indicative of a pulmonary arterial hypertension.