JP7127817B2 - pulmonary hypertension drug - Google Patents

Description

The present invention relates to a medicament for preventing and/or treating pulmonary hypertension by preventing and/or ameliorating pulmonary vascular remodeling, and the use of said medicament.

Pulmonary hypertension (PH) is associated with pulmonary artery endothelial disorders (eg, persistent vasoconstriction, hyperplasia, proliferation and coagulopathy), smooth muscle proliferation (eg, smooth muscle proliferation and abnormal apoptosis, and Ca handling defects). ), which is an obscure disease accompanied by increased pulmonary artery pressure against a background of pulmonary vascular remodeling caused by inflammation, immune abnormalities, etc. (Non-Patent Document 1).

Currently, the main drugs used to treat pulmonary hypertension are cyclic GMP pathway-related vasodilators (such as riociguat), endothelin receptor antagonists (such as ambrisentan and bosentan), and prostacyclin preparations. Vasodilators (such as epoprostenol and beraprost). Although the prognosis of PH has been improved by combination therapy with these vasodilators, it is still a disease with a poor prognosis.
In particular, in collagen pulmonary hypertension (CTD-PH) including scleroderma pulmonary hypertension (SSc-PH), not only pulmonary artery remodeling but also pulmonary venous remodeling is complicated, and conventional vasodilators are effective. There are not a few patients who are inadequate and suffer fatal complications such as pulmonary edema due to pulmonary vein stenosis. In order to effectively treat pulmonary hypertension accompanied by remodeling of pulmonary veins, it is necessary to develop a therapeutic drug with a mechanism of action different from that of conventional vasodilators.

In recent years, tumorioid growth has been observed in pulmonary arteries in PH, and application of antitumor drugs to PH is expected (Non-Patent Document 1). Some vitamins also have anti-tumor effects. Vitamin A and its derivatives (retinoids) are classified as fat-soluble vitamins, and have various receptor-mediated (genomic pathway) and non-receptor-mediated actions (RARα, RARβ, RARγ, RXRα, RXRβ, and RXRγ). genomic pathway), and is used as a therapeutic drug for psoriasis and leukemia due to its cell growth inhibitory effect. In PH patients, blood retinoid levels are low, and it has been reported that all trans retinoic acid (ATRA), a ligand of RARα, has an inhibitory effect on the proliferation of pulmonary artery smooth muscle cells associated with serotonin load (non-patent Reference 2). ATRA has also been reported to have an inhibitory effect on smooth muscle proliferation in a rat model of monocrotaline-induced pulmonary hypertension (Non-Patent Document 3). On the other hand, no PH improving effect was observed in hypoxia-induced pulmonary hypertension rat models (Non-Patent Document 4). As can be seen from these reports, retinoids seem to have some relationship with pulmonary hypertension, but the details are still unknown.

As described above, administration of vasodilators is the main method in the current treatment of PH. Medicine is desperately needed.

EP2604263A1

Guignabert et al., CHEST 147:529-537 2015 Preston et al., Circulation 111:782-790 2005 Xin et al., Genetics and Molecular Research 14:14308-14313 2015 Zhang et al., Circ J 74:1696-1703 2010 Okita et al., J Gastroenterol 50:191-202 2011

In view of the above circumstances, the present invention aims to provide a novel therapeutic agent for pulmonary hypertension that does not have an acute vasodilatory effect as a main effect, and to provide a novel therapeutic method for hypertension.

The present inventors administered the acyclic retinoid peretinoin, which is a ligand for RARβ and RXRα, unlike the cyclic retinoid ATRA, to pulmonary arterial hypertension model mice (hypoxia-induced pulmonary hypertension model). The inventors have found an effect of improving pulmonary vascular remodeling without mediated by early vasodilatory action, and completed the present invention.
Peretinoin has already completed a Phase II trial in humans for suppressing recurrence of hepatocellular carcinoma, and it has been confirmed that no problematic side effects occur. In this respect, clinical application to humans is considered to be very easy (Patent Document 1 and Non-Patent Document 5).

That is, the present invention is the following (1) to (4).
(1) A pulmonary vascular remodeling improving drug containing an acyclic retinoid, a salt thereof, or a solvate thereof.
(2) The improving drug according to (1) above, wherein the acyclic retinoid is peretinoin.
(3) A medicament for preventing and/or treating pulmonary hypertension, containing an acyclic retinoid or a salt thereof, or a solvate thereof.
(4) The medicament according to (3) above, wherein the acyclic retinoid is peretinoin.

The medicament of the present invention exerts an excellent effect in treating pulmonary hypertension through prevention and improvement of progression of pulmonary vascular remodeling.

Unlike existing therapeutic agents for pulmonary hypertension, the medicament of the present invention improves the pathology of pulmonary hypertension without inducing vasodilation in the acute phase. It can also be applied to the pathology of pulmonary hypertension (for example, CTD-PH, etc.) that exacerbates the condition.

The medicaments of the present invention are less likely to cause problematic side effects in patients to whom they are administered.

Effect of peretinoin administration on cardiac dynamics exacerbated by pulmonary hypertension (1). The values of PAT (Pulmonary Acceleration Time) and PAT/ET (Ejection Time) of the control group, PH group and peretinoin-administered group are shown. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test) Effect of peretinoin administration on cardiodynamics exacerbated by pulmonary hypertension (2). The analysis results of the pressure-volume curve (PV loop) of the control group, PH group and peretinoin-administered group are shown. Effect of peretinoin administration on cardiodynamics exacerbated by pulmonary hypertension (3). RVSP (right ventricular systolic pressure), RV (right venticule)/IVS (interventricular septum) + LV (left venticule), HR (heart rate) and BW (body weight) of the control group, PH group and peretinoin-administered group show. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test) Effect of peretinoin administration on cardiac dynamics exacerbated by pulmonary hypertension (4). The values of CO (cardiac output), SV (stroke volume), RVEDV (right ventricular end-diastolic volume) and Ea (arterial elastance) of the control group, PH group and peretinoin-administered group are shown. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test) Effect of peretinoin administration on cardiac dynamics exacerbated by pulmonary hypertension (5). RVEF (right ventricular ejection fraction) values of a control group, a PH group and a peretinoin-administered group are shown. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test) Effect of peretinoin administration on acute hemodynamics. Epoprostenol and pretinoin were administered to C57B6/6J mice (points of administration of each drug are indicated by arrows), and changes in right ventricular pressure (upper panel) and cardiac output (lower panel) were measured. The results are shown.

Histological examination of the effect of peretinoin on pulmonary artery media that thickens with pulmonary hypertension (1). The results of EVG (Eastica Van Gieson) staining of tissue sections of pulmonary artery media of mice in the control group, PH group and peretinoin-administered group are shown. Histological examination of the effect of peretinoin on pulmonary artery media that thickens with pulmonary hypertension (2). It was calculated by analyzing the image in FIG. 7 with Image J. In the analysis, 20 pulmonary arteries with a diameter of 25 μm to 50 μm were randomly extracted from each mouse, and the medial well thickness, % wall thickness and medial thickness index shown below were analyzed. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). * p<0.05 (Mann-Whitney U-test) Medial well thickness: (outer diameter-inner diameter)/2 % wall thickness: (medial wall thickness/outer diameter) × 100 Medial thickness index: [(area ext-area int )/area ext]×100 Medial well thickness: Pulmonary artery medial thickness outer diameter: Pulmonary artery inner diameter % wall thickness: Medial thickness index: Medial thickness index: Indices of media thickness area ext : Area of the area surrounded by the external elastic lamina area int : Area of the area surrounded by the internal elastic lamina Histological examination of the effect of peretinoin on pulmonary artery media that thickens with pulmonary hypertension (3). The results of immunostaining tissue sections derived from the pulmonary arteries of mice in the control group, PH group, and peretinoin-administered group with an anti-actin antibody are shown. Examination of the effects of peretinoin on gene expression associated with pulmonary vascular remodeling (1). FIG. 4 shows the results of measuring the expression levels of IL-6, ET1, survivin140 and TGFβ by qRT-PCT using RNA prepared from lung tissues of the control group, PH group and peretinoin-administered group. Data are shown as mean±SEM (n=6 for each group). *p<0.05 (Mann-Whitney U-test) Examination of the effects of peretinoin on gene expression associated with pulmonary vascular remodeling (2). Fig. 2 shows the results of measuring the expression levels of αSMA and Fn (fibronectin) by qRT-PCT using RNA prepared from lung tissues of the control group, PH group and peretinoin-administered group. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test)

Comparison of the effects of ATRA and peretinoin on pulmonary vascular remodeling (1). The values of CO, RVSV, Ea and RV/IVS+LV in the control group, PH group, peretinoin-administered group and ATRA-administered group are shown. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6). *p<0.05 (Mann-Whitney U-test) Comparison of the effects of ATRA and peretinoin on pulmonary vascular remodeling (2). EF (ejection fraction) values of the control group, PH group, peretinoin-administered group and ATRA-administered group are shown. Data are shown as mean±SEM (control group: n=7, PH group: n=5, peretinoin-administered group: n=6, ATRA-administered group: n=6). *p<0.05 (Mann-Whitney U-test)

Investigation of the effect of peretinoin administration on remodeled pulmonary vessels (1). RV/IVS+LV, RVEDV, Ea of the PH group and the peretinoin-administered group (unlike Figures 1 to 13, administration of peretinoin to the model mouse was started after completion of the pulmonary hypertension model mouse. Same in Figure 15). and Pmax (synonymous with RVSP, maximum right ventricular pressure). Data are shown as mean±SEM (PH group: n=5, peretinoin-administered group: n=4). *p<0.05 (Mann-Whitney U-test) Investigation of the effect of peretinoin administration on remodeled pulmonary vessels (2). Emax (maximum elastance) and EF values of the PH group and the peretinoin-administered group are shown. Data are shown as mean±SEM (PH group: n=5, peretinoin-administered group: n=4). *p<0.05 (Mann-Whitney U-test) Histological examination of the effects of peretinoin administration on remodeled pulmonary artery media (1). Control group (Control (B6)), group 3 weeks after SU5416 administration (HySU 3 weeks), group 3 weeks after administration of SU5416 and peretinoin (HySU/ACR 3 weeks), group 5 weeks after administration of SU5416 (HySU 5 weeks) ) and SU5416 administered for 3 weeks, followed by administration of SU5416 and peretinoin for 2 weeks (HySU 3 weeks + HYSU/ACR 2 weeks). Histological examination of the effects of peretinoin administration on remodeled pulmonary artery media (2). The image in FIG. 16 was analyzed with Image J in the same manner as in FIG. 8 to calculate the medial well thickness. Data are shown as mean ± SEM (HySU 3 weeks: n = 5, HySU/ACR 3 weeks: n = 6, HySU 5 weeks: n = 4, HySU 3 weeks + HYSU/ACR 2 weeks: n = 4). . *p<0.05 (Mann-Whitney U-test) Confirmation of the effect of administration of peretinoin to pulmonary hypertension model mice by echo. Echo (upper) and HE-stained images (lower) of heart short-axis cross-sections of pulmonary hypertension model mice (PH group) and mice to which peretinoin was administered after completion of the pulmonary blood pressure model (peretinoin-administered group) are shown.

A first embodiment of the present invention is a pulmonary vascular remodeling-improving agent comprising an acyclic retinoid or a salt thereof, or a solvate thereof.
Here, "retinoid" refers to vitamin A and its derivatives, which are classified into cyclic retinoids and acyclic retinoids according to their structural characteristics. Cyclic retinoids include ATRA (all trans retinoic acid) described above, 9-cis retinoic acid (alitretinoin), and the like.
In embodiments of the present invention, "acyclic retinoid" means a retinoid that does not have a ring structure in its molecule. Examples of "acyclic retinoids" include, but are not limited to, peretinoin (CAS number: 81485-25-8), geranylgeranoic acid, dihydrogeranylgeranoic acid (2,3-dihydrogeranylgeranoic acid, 4,5-didehydro-10,11-dihydrogeranylgeranoic acid and 14,15-dihydrogeranylgeranoic acid), tetrahydrogeranylgeranoic acid and the like, preferably peretinoin.

Salts of acyclic retinoids may be used in embodiments of the present invention. Salts of acyclic retinoids may be pharmaceutically acceptable salts. For example, when an acidic group is present, alkali metal and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium, etc. ammonia, methylamine, dimethylamine, trimethylamine, dicyclohexylamine, tris(hydroxymethyl)aminomethane, N,N-bis(hydroxyethyl)piperazine, 2-amino-2-methyl-1-propanol, ethanolamine, N- Salts of amines such as methylglucamine and L-glucamine; and salts with basic amino acids such as lysine, δ-hydroxylysine and arginine. Salts of mineral acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc. when basic groups are present; methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, acetic acid, propionate, tartaric acid , fumaric acid, maleic acid, malic acid, oxalic acid, succinic acid, citric acid, benzoic acid, mandelic acid, cinnamic acid, lactic acid, glycolic acid, glucuronic acid, ascorbic acid, nicotinic acid, salicylic acid, etc. salts; or salts with acidic amino acids such as aspartic acid and glutamic acid.

Solvates of acyclic retinoids or salts thereof may also be used in embodiments of the present invention. Solvents that form solvates include, but are not limited to, water, pharmaceutically acceptable organic solvents such as ethanol, acetone, ethyl acetate and hexane, and the like.
Acyclic retinoids, salts thereof, and solvates thereof can be produced by known methods. It can be manufactured easily. Commercially available acyclic retinoids may also be used in the practice of the present invention.

“Pulmonary vascular remodeling” is defined as the remodeling of pulmonary vascular endothelial cells and pulmonary vascular smoothness due to various factors such as vascular endothelial cell damage, vascular spasm, apoptosis resistance of vascular endothelial cells, inflammatory cell infiltration and adventitial fibrosis. It means that muscle cells exhibit cell proliferative changes similar to tumors, causing hypertrophy, thickening or fibrosis of pulmonary vascular walls, resulting in narrowing and/or obstruction of the vascular lumen.
The "improving drug" in the present embodiment has an effect of suppressing the progress of pulmonary vascular remodeling and an effect of returning blood vessels in a remodeling state to their original state or a state close thereto (reverse remldeling). It is a drug that has

A second embodiment of the present invention is a medicament for preventing and/or treating pulmonary hypertension containing an acyclic retinoid or a salt thereof, or a solvate thereof.
The medicament according to the second embodiment of the present invention suppresses and/or restores pulmonary vascular remodeling, thereby having efficacy as a prophylactic and/or therapeutic agent for pulmonary hypertension. Therefore, pulmonary hypertension targeted by the pharmaceutical according to the present embodiment includes all diseases that are accompanied by pulmonary vascular remodeling and cause an increase in pulmonary artery pressure. Such pulmonary hypertension includes, but is not limited to, idiopathic pulmonary arterial hypertension, hereditary pulmonary arterial hypertension, drug- and toxin-induced pulmonary arterial hypertension, other diseases (connective tissue disease pulmonary arterial positive pulmonary hypertension, including pulmonary arterial hypertension, pulmonary veno-occlusive disease and/or pulmonary telangioma , neonatal persistent pulmonary hypertension, left ventricular systolic dysfunction, left ventricular diastolic dysfunction, valvular disease, congenital/acquired left heart inflow/outflow disorders and lungs associated with left heart disease such as congenital cardiomyopathy Hypertension, chronic obstructive pulmonary disease, interstitial lung disease, other respiratory diseases showing a mixture of restrictive and obstructive types, sleep-disordered breathing, alveolar hypoventilation disorder, chronic high-altitude exposure, growth disorder, etc. pulmonary hypertension due to hypoxemia and/or pulmonary hypertension due to hypoxemia, chronic thromboobstructive pulmonary hypertension, blood disorders (chronic hemolytic anemia, osteoplasty, splenectomy, etc.), systemic diseases (sarcoidosis, pulmonary histiocytosis, lymphangioleiomyomatosis, etc.), metabolic diseases (diabetes, Gaucher disease, thyroid disease), other diseases (tumor obstruction, mediastinitis fibrosis, chronic renal failure, segmental pulmonary hypertension) Examples include pulmonary hypertension due to complex factors of unknown cause.

A second embodiment of the present invention is a medicament containing an acyclic retinoid, a salt thereof, or a solvate thereof (hereinafter also referred to as "the medicament of the present invention"), and the preferred acyclic retinoid is peretinoin. be. The medicament of the present invention may be in the form of administering an acyclic retinoid, a salt thereof, or a solvate thereof as an active ingredient, but generally, in addition to these active ingredients, one or more It is desirable to administer in the form of a pharmaceutical composition containing pharmaceutical additives (hereinafter also referred to as "pharmaceutical composition of the present invention"). In addition, the pharmaceutical composition according to the embodiment of the present invention may be blended with other known drugs.

The medicament or pharmaceutical composition of the present invention may be in an oral or parenteral dosage form, and is not particularly limited. agents, ointments, creams, gels, patches, inhalants, injections, and the like. These formulations are prepared according to a conventional method. Liquid formulations may be dissolved or suspended in water or other suitable solvents at the time of use. Moreover, tablets and granules may be coated by a well-known method. Injections are prepared by dissolving the antibody of the present invention or functional fragments thereof in water. agents may be added.

A person skilled in the art can determine the type of pharmaceutical additive used in the production of the pharmaceutical or pharmaceutical composition of the present invention, the ratio of the pharmaceutical additive to the active ingredient, or the method of producing the pharmaceutical or pharmaceutical composition, depending on the form. It can be selected as appropriate. As additives for formulations, inorganic or organic substances, or solid or liquid substances can be used. It can be blended at 95.0% by weight, or between 1% and 90.0% by weight. Specifically, examples of pharmaceutical additives include lactose, glucose, mannitol, dextrin, cyclodextrin, starch, sucrose, magnesium aluminometasilicate, synthetic aluminum silicate, sodium carboxymethylcellulose, hydroxypropyl starch, and calcium carboxymethylcellulose. , ion exchange resin, methylcellulose, gelatin, gum arabic, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, polyvinyl alcohol, light anhydrous silicic acid, magnesium stearate, talc, tragacanth, bentonite, veegum, titanium oxide, sorbitan fatty acid ester, Sodium lauryl sulfate, glycerin, fatty acid glycerin ester, refined lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, liquid paraffin, white petrolatum, fluorocarbon, nonionic surfactant, propylene glycol or water.

In order to produce a solid preparation for oral administration, the active ingredient is mixed with excipients such as lactose, starch, crystalline cellulose, calcium lactate or silicic anhydride to form a powder, or if necessary A binder such as sucrose, hydroxypropyl cellulose or polyvinylpyrrolidone, and a disintegrant such as carboxymethyl cellulose or carboxymethyl cellulose calcium are added and wet or dry granulated to form granules. For producing tablets, these powders and granules may be tableted as they are or after adding a lubricant such as magnesium stearate or talc. These granules or tablets are coated with an enteric base such as hydroxypropyl methylcellulose phthalate, methacrylic acid-methyl methacrylate polymer to form an enteric preparation, or ethylcellulose, carnauba wax or hydrogenated oil to form a sustained release preparation. You can also To manufacture capsules, hard capsules are filled with powders or granules, or active ingredients are dissolved in glycerin, polyethylene glycol, sesame oil, olive oil, etc. and then coated with gelatin to form soft capsules. be able to.

For the production of injections, the active ingredient may be mixed with hydrochloric acid, sodium hydroxide, lactose, lactic acid, sodium, pH adjusters such as sodium monohydrogen phosphate or sodium dihydrogen phosphate, sodium chloride or glucose, if necessary. Dissolve in distilled water for injection with a tonicity agent, filter aseptically and fill in an ampoule, or further add mannitol, dextrin, cyclodextrin or gelatin and lyophilize in a vacuum to prepare a dissolution type injection for use. good too. Alternatively, lecithin, polysorbate 80, polyoxyethylene hydrogenated castor oil, or the like may be added to the active ingredient and emulsified in water to prepare an emulsion for injection.

For the preparation of rectal formulations, the active ingredient is dissolved by moistening with a suppository base such as cocoa butter, tri-, di- and monoglycerides of fatty acids or polyethylene glycol, poured into molds and allowed to cool, or the active ingredient is extruded. It may be dissolved in polyethylene glycol, soybean oil, or the like, and then coated with a gelatin film.

The dosage and administration frequency of the pharmaceutical or pharmaceutical composition according to the embodiment of the present invention are not particularly limited, and the purpose of treatment, prevention of deterioration / progression of the disease to be treated, type of disease, patient's weight and age, etc. It can be selected as appropriate according to the judgment of a doctor or pharmacist, depending on the conditions.
In general, the daily oral dosage for adults is about 0.01 to 1,000 mg (weight of active ingredient), and it can be administered once a day, divided into several times, or every few days. . When used as an injection, it is desirable to administer 0.001 to 100 mg (active ingredient weight) per day for adults continuously or intermittently.

A medicament or pharmaceutical composition according to an embodiment of the present invention is prepared as a sustained release formulation, such as an implant or microencapsulated delivery system, using a carrier capable of preventing immediate elimination from the body. can do. Biodegradable and biocompatible polymers can be used as such carriers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be readily prepared by those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. Liposomes are prepared through filters of suitable pore size to a suitable size for use as a lipid composition containing, but not limited to, phosphatidylcholine, cholesterol and PEG-derived phosphatidylethanol (PEG-PE), followed by reverse-phase evaporation. can be refined by

A drug or pharmaceutical composition according to an embodiment of the present invention may be provided in the form of a kit together with an instruction such as an administration method. The drug or pharmaceutical composition contained in the kit is manufactured from a material that effectively maintains the activity of the active ingredient for a long period of time, does not allow agents to adhere to the inside of the container, and does not alter the quality of the constituent ingredients. Supplied by container. For example, a sealed glass ampoule may contain a buffer or the like sealed in the presence of a neutral, non-reactive gas such as nitrogen gas.
In addition, the instructions for use of the kit may be printed on paper or the like, or may be stored and supplied on an electromagnetically readable medium such as a CD-ROM or DVD-ROM.

A third embodiment of the present invention is a method for preventing and/or treating pulmonary hypertension, comprising administering the medicament or pharmaceutical composition of the present invention to a patient.
The term "treatment" as used herein means preventing or alleviating the progression and exacerbation of pulmonary hypertension in patients who have already developed pulmonary hypertension. It is the action to take.
In addition, "prevention" means to prevent the onset of pulmonary hypertension in advance in a patient who is likely to develop pulmonary hypertension, and is a treatment aimed at preventing the onset of pulmonary hypertension in advance. .

Where this specification has been translated into English and contains the words "a", "an" and "the" in the singular, unless the context clearly indicates otherwise, the singular as well as the singular It also includes multiple items.
EXAMPLES The present invention will be further described below with reference to Examples, but these Examples are merely illustrations of embodiments of the present invention and do not limit the scope of the present invention.

1. Investigation of the inhibitory effect of peretinoin on the progression of pulmonary hypertension in mice 1-1. Pulmonary Hypertension Model Mouse The effect of peretinoin was examined using a pulmonary hypertension model mouse (pulmonary arterial hypertension model mouse) bred with a VEGFR antagonist under hypoxic conditions.
As a pulmonary hypertension model mouse, SU5416 (AdooQ-BioScience: #A1243) was administered intraperitoneally three times every week to 8-week-old male C57B6/6J mice at a dose of 20 mg/kg. The mice were raised in a hypoxic chamber (8.5% oxygen concentration) for 3 weeks. Three weeks after the first administration of SU5416, various measurements were performed. A PH (pulmonary hypertension) group of pulmonary hypertension model mice was fed with a normal diet, and a peretinoin-administered group was fed with a diet containing 0.06% by weight of peretinoin from the first administration of SU5416. In addition, the control group was fed with a normal diet in a normal breeding environment (21% oxygen concentration) without administration of SU5416.

1-2. echo measurement
Using vevo 2100 (Fuji Film Visual Sonic), the pulse Doppler method was used to measure the ejection waveform of the right ventricular outflow tract. ET (Ejection Time) was measured (Fig. 1).
Both PAT and PAT/ET were longer in the peretinoin-administered group than in the PH group. PAT/ET correlates with pulmonary artery pressure (shorter PAT/ET indicates higher pulmonary artery pressure), suggesting that peretinoin lowers pulmonary artery pressure.

1-3. Measurement of pulmonary artery pressure and right ventricular function using conductance catheter Conductance catheters (PVR 839 and PVR 1035; MPVS-Ultra pressure-volume system; Millar Instruments) were used to examine pulmonary artery pressure, right ventricular function, etc. in pulmonary hypertension model mice. was used to analyze the pressure-volume curve (PV loop). The measurement was performed according to the method described in Am J Physiol Heart Circ Physiol 301: H2198-H2206, 2011.
In the PV loop, the PH group showed an increase in right ventricular pressure and a decrease in cardiac output, but the peretinoin-administered group showed an improvement in the decrease in right ventricular pressure and cardiac output, which is quite similar to the curve in the control group. (Fig. 2).
RVSP (right ventricular systolic pressure: right ventricular systolic pressure) was approximately the same as the pulmonary artery systolic pressure, but was significantly reduced by peretinoin administration (Fig. 3, RVSP). RV/IVS+LV is the weight ratio of RV (right venticule) to IVS (interventricular septum) + LV (left ventricular), reflecting the weight of the right ventricle, and is high in right heart failure. However, administration of peretinoin significantly decreased this value (Fig. 3, RV/VS+LV).
There was no significant difference in HR (heart rate) and BW (body weight) among the control group, PH group and peretinoin administration group.
These results confirmed improvement in pulmonary artery systolic pressure and right ventricular weight.

In addition, peretinoin administration increased CO (cardiac output) and SV (stroke volume) (Figure 4, CO and SV) and Ea (arterial elastance: pulmonary vascular resistance). decreased. RVEF (right ventricular ejection fraction) was also increased by administration of peretinoin, and an improvement in contractility was observed (Fig. 5). RVEDV (right ventricular end-diastolic volume) was also reduced by peretinoin administration (Fig. 4, RVEDV).
These results confirmed improvement in cardiac output and right ventricular afterload.

We investigated changes in pulmonary vascular resistance. C57B6/6J (wild-type) mice were treated with epoprostenol and pretinoin, pulmonary vasodilators traditionally used for pulmonary hypertension (time points of administration of each drug are indicated by arrows). Changes in right ventricular pressure (Fig. 6, upper waveform) and cardiac output (Fig. 6, lower waveform) were examined. As a result, the right ventricular pressure decreased with the initiation of epoprostenol. After stopping epoprostenol and starting peretinoin, blood pressure returned to baseline. Right ventricular pressure is determined by vascular resistance and cardiac output, but since cardiac output was unchanged, it is thought that the pressure changed as a result of changes in vascular resistance due to the administration of each drug. . In fact, even when the vascular resistance (Ea) immediately after drug administration was calculated, it was confirmed that epoprostenol decreased vascular resistance, but peretinoin did not.
From the above results, administration of peretinoin does not induce pulmonary vasodilation in the acute phase, and thus it is considered possible to avoid the occurrence of side effects associated with administration of conventional pulmonary vasodilators.

1-4. Histological examination 1% formalin was injected into the trachea of deeply sedated mice by inhalation anesthesia, 20 cm pressure was applied to expand the lungs, fixed overnight in 10% formalin, and then paraffin-embedded tissues. The specimen was subjected to Elastica Van Gieson (EVG) staining (Fig. 7). Image J was used for analysis, and medial wall thickness (pulmonary artery media thickness), % wall thickness (ratio of media thickness to outer diameter of pulmonary artery), and medial thickness index (index of medial thickness: large thickness) was calculated (Fig. 8). From Figures 7 and 8, it was confirmed that peretinoin improved the thickening of the media of the pulmonary artery.
Next, tissue sections from the pulmonary arteries of mice in the control group, PH group and peretinoin-administered group were stained with α-Actin antibody (1A4): sc-32251 (santa cluz). Staining was performed using the VECTOR MOM Immunodetection Peroxidase Kit.
Fig. 9 confirmed a decrease in smooth muscle cells (αSMA-positive cells) in the media of the pulmonary artery, suggesting that administration of peretinoin inhibited proliferation of smooth muscle cells.

1-5. Effect on the expression of genes related to pulmonary vascular remodeling We investigated how the expression of genes involved in inflammation, adventitial fibrosis, and apoptosis resistance associated with pulmonary vascular remodeling would be affected by peretinoin administration. .
Using total RNA extracted from lung tissue as a template, qRT-PCR was performed to detect IL-6 (inflammatory cytokine gene), ET1 (fibrosis-related gene), survivin140 (apoptosis resistance gene) and TGFβ (pulmonary hypertension). , the expression of the gene whose expression is increased and which is one of the causes of abnormal growth) was examined. The primers used are shown below.
・Amplification of IL-6
Il6 forward: GATGGATGCTACCAAACTGGAT (sequence number 1)
Il6 Reverse: CCAGGTAGCTATGGTACTCCAGA (SEQ ID NO: 2)
・Amplification of ET1
ET-1 forward: CAAGGAGCTCCAGAAACAGC (sequence number 3)
ET-1 Reverse: TTCTGCACTCCATTCTCAGCTC (SEQ ID NO: 4)
・Amplification of Survivin140
Survivin140 forward: TGGACAGACAGAGAGCCAAG (SEQ ID NO: 5)
Survivin140 Reverse: AGTTCTTGAAGGTGGCGATG (SEQ ID NO: 6)
・Amplification of TGFβ
Tgfb1 forward: GCAACATGTGGAACTCTACCAGAA (sequence number 7)
Tgfb1 Reverse: GACGTCAAAAGACAGCCACTCA (SEQ ID NO: 8)
As a result, as shown in FIG. 10, IL6, ET1, survivin140 and TGFβ, whose expression was increased in the PH group, decreased to the level of each gene in the control group by administration of peretinoin.

We also examined the expression of αSMA and Fn (fibronectin) genes, which are components of the pulmonary vascular media and adventitia. The primers used for qRT-PCR are as follows.
・Amplification of αSMA
aSMA forward: GGCATCCACGAAACCACCTA (sequence number 9)
aSMA reverse: CACGAGTAACAAATCAAAGC (SEQ ID NO: 10)
・Amplification of Fn
Fn forward: ATGTGGACCCCTCCTGATAGT (sequence number 11)
Fn Reverse: GCCCAGTGATTTCAGCAAAGG (SEQ ID NO: 12)
As a result, it was confirmed that the expression of αSMA and Fn, which were elevated in the PH group, both decreased by administration of peretinoin (Fig. 11).
These results suggest that peretinoin improves pulmonary vascular inflammation, fibrosis, resistance to apoptosis, etc., and has the effect of suppressing remodeling of the pulmonary adventitia and media.

2. Comparison of cyclic retinoids with ATRA 2-1. Dosage of ATRA The effect of ATRA, a cyclic retinoid, on pulmonary vascular remodeling was investigated in comparison with peretinoin.
For comparative experiments, the above-mentioned pulmonary hypertension model mouse (pulmonary arterial hypertension model mouse) was used. According to a previous report (Kada et al., Arterioscler Thromb Vasc Biol. 27:1535-1541 2007), administration of peretinoin at 100 mg/kg and ATRA at 10 mg/kg had equivalent potencies as retinoids. ATRA was mixed in doses of 100 mg/kg and 10 mg/ml, respectively, and fed. When the mice were fed diet containing 100mg/kg/day of ATRA, all mice died within 2 weeks.

2-2. Investigation of the effect of ATRA administration on pulmonary vasculature and cardiodynamics CO, RVSP, Ea and RV/IVS+LV were measured using a conductance catheter. As a result, the peretinoin-administered group showed improvement in all functions, while the ATRA-administered group showed almost no effect (Fig. 12). In addition, no improvement in EF (ejection fraction) was observed in the ATRA-administered group (Fig. 13).
From the above results, it was found that administration of peretinoin showed improvement in all measured items, while administration of ATRA had almost no effect.

3. Investigation of reverse remodeling effect of peretinoin on remodeled pulmonary vessels 1 above. used a mouse model to demonstrate that administration of peretinoin at the same time as the progression of pulmonary remodelin effectively inhibited the progression of pulmonary hypertension.
Next, when peretinoin is administered to a model mouse in which pulmonary vascular remodeling has already occurred (that is, after creating a pulmonary hypertension model mouse, administering to the model mouse), the remodeled pulmonary vessels are improved. I considered whether it was possible.
3-1. pulmonary hypertension model mouse
Eight-week-old male C57B6/6J mice were fed SU5416 intraperitoneally at a dose of 20 mg/kg three times weekly in a hypoxic chamber (8.5% oxygen concentration) for 3 weeks. Hypertensive mice were created. During this period, the mice were fed with a normal diet containing no peretinoin. The mice with pulmonary hypertension were divided into two groups: a diet group containing 0.06% by weight of peretinoin and a continuous diet group. 5 weeks after administration), various measurements were performed. The control group was fed with a normal diet in a normal rearing environment (21% oxygen concentration) without administration of SU5416.

3-2. Measurement using a conductance catheter When peretinoin was administered to pulmonary hypertension model mice in which the pulmonary vessels were remodeled, RVEDV, Ea and Pmax (synonymous with RVSP, which is the maximum right ventricular pressure) showed significant differences. In addition, although there was no significant difference in RV/IVS+LV, a downward trend was shown (Fig. 14).
In addition, Emax (maximum elastance: maximum elastance at the end of ventricular systole, one of the indices of contraction), which increased compensatory due to increased pulmonary vascular resistance, significantly decreased, and EF, which had decreased, increased significantly. (Fig. 15).
From the above results, it was found that pulmonary hypertension improved even when peretinoin was administered in a state in which pulmonary vascular remodeling had already occurred.

3-3. Histological Examination To further confirm that peretinoin exhibits a reverse remodeling effect, a histological examination was performed. Specimen collection and staining were carried out by the method described in 1-4 above. Improvement of pulmonary artery media thickness was confirmed by EVG staining, and reverse remodeling was obtained by administration of peretinoin (“HYSU 3 weeks + Hysu/ACR 2 weeks” in FIGS. 16 and 17).

3-4. Echo measurement Echo (Fig. 18, upper) and HE-stained images (Fig. 18, lower) of cardiac short-axis cross-sections of pulmonary hypertension model mice (PH group) and mice to which peretinoin was administered after pulmonary blood pressure remodeling (peretinoin-administered group). )It was confirmed. From Figure 17, it was found that the enlarged right heart of the pulmonary hypertension model mouse (Figure 18A) was reduced by administration of peretinoin (Figure 18B).

From the above results, it was confirmed that peretinoin is also effective against pulmonary hypertension in which pulmonary vascular remodeling has already occurred.

The present invention provides prophylactic or therapeutic agents for pulmonary hypertension containing acyclic retinoids such as peretinoin. The medicament according to the present invention reduces side effects that may be caused by existing medicaments, and exhibits unprecedented therapeutic effects. Therefore, the present invention is highly expected to be used in the medical field.

Claims (2)

A pulmonary vascular remodeling improving drug containing peretinoin or a salt thereof, or a solvate thereof. A medicament for the prevention and/or treatment of pulmonary hypertension, containing peretinoin or a salt thereof, or a solvate thereof.

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