Patent Application
20100280041
The present invention relates to chlorophenylpiperazine salt derivatives, 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine.HCl (hereinafter referred to as “Pulmodil”) and 7-[2-[4-(2-chlorophenyl)piperazinyl]-ethyl]-1,3-dimethylxanthine. Citric acid (hereinafter referred to as “Pulmodil-1”), obtained by a recrystallization method, and particularly relates to the activities of inhibiting a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction thereof.
Pulmonary arterial hypertension (PAH) caused by the dysfunction of pulmonary artery is a progressive and lethal disease. Raised pulmonary arterial pressure and the remodeling of the pulmonary vessels may cause right ventricular hypertrophy or failure, which may increase the risk and the death rate of the patients. Before becoming a lethal disease, the dysfunction of pulmonary artery has many early symptoms, such as injury, propagation or hypercontractility of endothelium of vascular smooth muscle, a transfer of inflammatory cells, etc.
Endothelium of pulmonary vessels is a specific place where various vasoactive mediators changing tensions of oxygen and carbon dioxide in the blood, blood pressure and flow, etc are produced. One of a diastolic mediator, Nitric oxide (NO), is generated by the endothelial NO synthase (eNOS) and effective in relaxing smooth muscle, inhibiting the activations of Neutrophil and platelet and improving the proliferation of the smooth muscle. NO is capable of stimulating its target enzyme, soluble guanylyl cyclase (sGC), for increasing the secondary messenger, cyclic guanosine monophosphate (cGMP), in tissues. Consequently, the increased cGMP will activate protein kinase G (PKG), and the PKG further phosphorylates several proteins related to the regulations of calcium ions in cells for achieving the function of relaxing vessels.
In many complicated mechanisms related to a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction, it is indicated that the activation of rho-kinase is associated with the contraction of smooth muscle cells, actin cytoskeleton organization, cell division and gene expression. It is also proved by the rat and mice animal models and human patients with pulmonary artery endothelium dysfunction that the activation of rho-kinase is involved in the pathological characteristics of a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction, and the activated rho-kinase would increase the Ca2+ sensitivity of the vascular smooth muscle cell, and thus cause the vasoconstriction.
Since rho-kinase related study is increased recently, inhibiting the expression of rho-kinase has been developed to a method in treating vasoconstriction. For example, the developed rho-kinase inhibitor is used to relieve the pathological changes caused by the vascular tension.
The present invention provides a kind of compounds, such as Pulmodil and Pulmodil-1, effective in inhibiting the expression of rho-kinase, activating the expression of eNOS, and reducing the right ventricular hypertrophy caused by the hypoxia-induced PAH. According to the mechanism for the compound “Pulmodil”, it could be applied to the inhibition of a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, a vascular obstruction and a cardiovascular disease. During the research process, the inventor found a simple synthetic method for the production of Pulmodil or Pulmodil-1. Pulmodil could be obtained from merely two reactants. Pulmodil has not only a good solubility, but also benefits of low toxicity and facile to be used in a therapeutical supplements. Therefore, the compound “Pulmodil” provided in the present invention is safe for a user in need thereof.
Hence, because of the defects in the prior arts, the inventors provide an observing device and method to effectively overcome the demerits existing in the prior arts.
A chlorophenylpiperazine salt derivative, i.e. Pulmodil, which is obtained by reacting 2-chloroethyl theophylline with 2-chlorophenyl piperazine and then recrystallizing the intermediate therefrom, is provided in the present invention. Pulmodil has the activities of relieving a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction, and the benefits of good solubility, low toxicity and safety.
It is an aspect of the present invention, a pharmaceutical composition for treating one of a cardiovascular disease and a pulmonary artery disease is provided. The pharmaceutical composition comprises one of a first compound having a Formula I:
and a second compound having a Formula II:
Preferably, the pulmonary artery disease comprises one selected from a group consisting of a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction.
Preferably, the HCl of Formula I and the acid of Formula II are derived from at least one of a xanthine and a piperazine.
Preferably, the acid is one of an organic acid and an inorganic acid.
Preferably, the organic acid comprises one selected from a group consisting of a citric acid, a maleinic acid, a fumaric acid, a tartaric acid, an oleic acid, a stearic acid, a benzenesulphonic acid, an ethyl benzenesulphonic acid, a benzoic acid, a succinic acid, a mesylic acid, a dimesylic acid, an acetic acid, a propionic acid, a pentanoic acid and an aspartic acid.
Preferably, the inorganic acid comprises one selected from a group consisting of a hydrochloride, a sulfuric acid, a phosphoric acid, a boric acid and a dihydrochloride.
Preferably, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier and an excipient.
Preferably, the second compound is a 7-[2-[4-(2-chlorophenyl)piperazinyl]-ethyl]-1,3-dimethylxanthine. Citric acid.
In accordance with another aspect of the present invention, a method for relieving a symptom including one selected from a group consisting of a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction in a mammalian subject in need thereof is provided. The method comprises steps of administering to the mammalian subject a pharmaceutically effective amount of a pharmaceutical composition including one of a first compound having a Formula I:
and a second compound having a Formula II:
Preferably, the mammalian subject is a human.
Preferably, the administration comprises one selected from a group consisting of an oral administration, an intravenous injection, a subcutaneous injection, an intraperitoneal injection, an intramuscular injection and a sublingual administration.
Preferably, the pharmaceutical composition further comprises at least one of a pharmaceutically acceptable carrier and an excipient.
Preferably, the second compound is a 7-[2-[4-(2-chlorophenyl)piperazinyl]-ethyl]-1,3-dimethylxanthine. Citric acid.
In accordance with another aspect of the present invention, a method for treating a cardiovascular disease in a mammalian subject in need thereof is provided. The method comprises the steps of administering to the mammalian subject a pharmaceutically effective amount of a substrate including one of a first compound having a Formula I and a second compound having a Formula II.
The above objects and advantages of the present invention will become more readily apparent to those ordinarily skilled in the art after reviewing the following detailed descriptions and accompanying drawings, in which:
The present invention will now be described more specifically with reference to the following embodiments. It is to be noted that the following descriptions of preferred embodiments of this invention are presented herein for the purposes of illustration and description only; it is not intended to be exhaustive or to be limited to the precise form disclosed.
A chlorophenylpiperazine salt derivative is provided in the present invention. The detailed descriptions of the preparation method, physical and chemical properties and the bioactivities of the chlorophenylpiperazine salt derivative are illustrated as follows.
The first preferred embodiment of the present invention is Pulmodul. Method 1: 2-Chloroethyl theophylline, 2-chlorophenyl piperazine and sodium hydroxide (NaOH) (or sodium hydrogen carbonate, NaHCO3) are dissolved in hydrous ethanol solution based on the molecular weight percentage and heated under reflux for three hours. After cooled overnight, the supernatant is decanted for proceeding the vacuum concentration and dry process, and then, one-fold volume of ethanol and three-fold volume of 2N hydrochloric acid (HCl) are added therein to dissolve at 50° C. to 60° C. as a saturated solution with pH 1.2. The saturated solution is sequentially decolorized with activated charcoal, filtered, deposited overnight and filtered to obtain a white crystal, i.e. Pulmodil. Pulmodil has a chemical formula as 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine.HCl, and a melting point of 249° C. to 252° C. The reaction formula is illustrated as follows. The chemical structure of Pulmodil is shown in
Method 2: 2-Chloroethyl theophylline and 2-chlorophenyl piperazine are dissolved in hydrous ethanol solution based on the molecular weight percentage and heated under reflux for three hours. After cooled overnight, the supernatant is decanted for proceeding the vacuum concentration and dry process, and then, one-fold volume of ethanol and three-fold volume of 2N hydrochloric acid (HCl) are added therein to dissolve at 50° C. to 60° C. as a saturated solution with pH 1.2. The saturated solution is sequentially decolorized with activated charcoal, filtered, deposited overnight and filtered to obtain a white crystal, i.e. Pulmodil. Pulmodil has a chemical formula as 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine. HCl, which has a melting point of 249° C. to 252° C. The reaction formula is illustrated as follows.
The second preferred embodiment of the present invention is Pulmodul-1. Method 1: 2-Chloroethyl theophylline, 2-chlorophenyl piperazine and NaOH (or NaHCO3) are dissolved in hydrous ethanol solution based on the molecular weight percentage and heated under reflux for three hours. After cooled overnight, the supernatant is decanted for proceeding the vacuum concentration and dry process, and then, ethanol and citric acid at a ratio of 1:1 (mole/mole) are added therein to dissolve at 50° C. to 60° C. as a saturated solution with pH 4.0. The saturated solution is sequentially decolorized with activated charcoal, filtered and deposited overnight to obtain a white crystal, i.e. Pulmodil-1. Pulmodil-1 has a chemical formula as 7-[2-[4-(2-chlorobenzene)-piperazinyl]ethyl]-1,3-dimethylxanthine.citric acid, and the reaction formula is illustrated as follows.
Method 2: 2-Chloroethyl theophylline, 2-chlorophenyl piperazine are dissolved in hydrous ethanol solution based on the molecular weight percentage and heated under reflux for three hours. After cooled overnight, the supernatant is decanted for proceeding the vacuum concentration and dry process, and then, ethanol and citric acid at a ratio of 1:1 (mole/mole) are added therein to dissolve at 50° C. to 60° C. as a saturated solution with pH 4.0. The saturated solution is sequentially decolorized with activated charcoal, filtered and deposited overnight to obtain a white crystal, i.e. Pulmodil-1. Pulmodil-1 has a chemical formula as 7-[2-[4-(2-chlorobenzene)piperazinyl]ethyl]-1,3-dimethylxanthine.citric acid, and the reaction formula is illustrated as follows.
The above mentioned two chlorophenylpiperazine salt derivatives, Pulmodil and Pulmodil-1, are merely preferred embodiments of the present invention, wherein the hydrochloride of Pulmodil could be substituted by other inorganic acid, such as a sulfuric acid, a phosphoric acid, a boric acid or a dihydrochloride, and the citric acid of Pulmodil-1 could be substituted by other organic acid, such as a maleinic acid, a fumaric acid, a tartaric acid, an oleic acid, a stearic acid, a benzenesulphonic acid, an ethyl benzenesulphonic acid, a benzoic acid, a succinic acid, a mesylic acid, a dimesylic acid, an acetic acid, a propionic acid, a pentanoic acid or an aspartic acid. During the synthetic process, various inorganic acid or organic acid could be used to produce different chlorophenylpiperazine salt derivatives. For example, when the inorganic acid is sulfuric acid, the derivative, 7-[2-[4-(2-chlorobenzene)-piperazinyl]ethyl]-1,3-dimethylxanthine.Sulfuric acid, could be obtained; and when the organic acid is maleinic acid, the derivative, 7-[2-[4-(2-chlorobenzene)-piperazinyl]ethyl]-1,3-dimethylxanthine.Maleinic acid, could be obtained. The rest may be deduced by analogy.
Regarding the chemical structure, Pulmodil has a main structure and an HCl molecule, and Pulmodil-1 has the same main structure and a citric acid. Although the main structure of Pulmodil can be obtained by the reaction of 7-ethylbromotheophylline with 1-(2-chlorophenyl)-piperazine, Pulmodil can be obtained by the synthetic method disclosed in the present invention without the preparing steps, such as filtration and recrystallization, etc., of the main structure compound (hereinafter referred to as “MSC”). It is apparent that the method disclosed in the present invention directly completes the synthesis of Pulmodil or Pulmodil-1 without a prior step of synthesizing a compound with the main structure. Further, since one reactant of the abovementioned reaction is 1-(2-chlorophenyl)-piperazine, instead of 1-(2-bromophenyl)-piperazine, the method disclosed in the present invention will not have a risk involving the contamination of bromine. In addition to the different synthetic methods between Pulmodil/Pulmodil-1 and the MSC, the physiochemical and physiological differences therebetween are described in detail as follows.
(1) Melting Point:
The melting point of MSC is ranged between 168° C. and 172° C., which is significantly lower than the melting point of Pulmodil (249° C. to 252° C.).
(2) HPLC Analysis:
For proving that Pulmodil is a single molecule but not dissociated into two parts, MSC and HCl, which perform functions, respectively, when functioning in on organism, purity of Pulmodil is determined by HPLC. First, Pulmodil and the standard solutions with different concentrations are prepared, wherein the standard is a compound having a chemical formula, C15H14ClNO2S.HCl, with the molecular weight of 344.26. Pulmodil is dissolved in a solution containing 25% acetonitrile and 0.1% formic acid. After a serial dilution, a series concentrations (0, 0.1, 0.2, 0.5, 1, 2, 5, 10 and 20 ng/ml) of the standard solutions are used in HPLC to calculate the low-concentration standard curve, and a series concentrations (0, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 ng/ml) thereof are used in HPLC to calculate the high-concentration standard curve. The standards are dissolved in a solution containing 50% acetonitrile, and then diluted with 100% acetonitrile. The concentration of the diluted Pulmodil is 18 ng/ml in the low-concentration standard curve and 450 ng/ml in the high-concentration standard curve, respectively. The column for HPLC is Luna C18 column (2.0 mm×50 mm, 5 mm, Phenomenex), in which the mobile phase includes 23% acetonitrile and 1.0% formic acid, and the flow rate is about 0.2 ml/min.
The parameters of tandem mass spectrometry are set as follows: capillary: 3.2 kV, cone: 40 V; source temperature: 80° C.; desolvation temperature: 400° C.; collision of 20 V; and multiplier: 500 V.
Pulmodil represents parent ion of 403.12 m/z (mass-to-charge ratio) and daughter ion of 222.95 m/z by the analysis of the tandem mass spectrometry, and standard represents parent ion of 308 m/z and daughter ion of 197.96 m/z thereby.
Please refer to Tables 1 and 2, which respectively represent the analytic results of Pulmodil solutions with low- and high-concentrations in HPLC. The result of peak-covered area is calculated according to the retention time of the sample, and the area percentage represents the purity of the material in the sample. It could be known from Tables 1 and 2 that Pulmodil has a purity of 100% in the samples. It shows that Pulmodil is still a single compound after being dissolved.
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(3) Solubility Test:
Solubilities of three compounds, i.e. Pulmodil, Pulmodil-1 and MSC, disclosed in the present invention are evaluated and the results are shown in Table 3. It is found that Pulmodil-1 and MSC are necessary to be dissolved by using a surfactant. As shown in Table 3, MSC can be dissolved in a solution containing propylene glycol and Pulmodil-1 can be dissolved in the solution containing polyethylene glycol (PEG). However, a drug prepared from a compound with too much organic solvent is dangerous. Pulmodil-1 and MSC solutions are acidic and not adequate for the general physiological environment. Comparing with Pulmodil-1 and MSC, Pulmodil could be dissolved in 5% (w/v) glucose solution, has better solubility and is easily prepared. Pulmodil is safe in usage since glucose solution is a popular medical supplement. Furthermore, the pH value of the dissolved Pulmodil is about 5.8 to 6.4, which closes to the general physiological environment.
In pharmaceutics, the water-soluble Pulmodil disclosed in the present application is facile to be formulated. Furthermore, Pulmodil has the biological effects on inhibiting a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, and a vascular obstruction, which are proved by the following experimental results.
Hereinafter are the detailed descriptions of the biological activity tests of Pulmodil.
1. Incubation of the Tracheal Smooth Muscle Cells (TSMCs):
The tracheal tissue of Wistar rat (200 g to 250 g) is aseptically obtained and the connective tissue around the tracheal tissues is removed. After clearance, tracheal tissue is aseptically sliced as fragments and spread on the T-25 flask. The T-25 flask is added with 6 ml DMEM medium (containing 20% (v/v) feotal bovine serum (FBS)) and incubated in a 37° C. incubator with 5% CO2. Later, the medium is refreshed by medium B (DMEM supplemented with 10% FBS) per three days. When 80% to 90% cell confluence was achieved, subcultures are performed.
The subculture process includes the following steps: decanting the medium at 80% to 90% cell confluence, rinsing cells with 2 ml phosphate buffered saline (PBS) once or twice, adding 1 ml solution including 0.25% trypsin and 0.02% EDTA (ethylenediaminetetraacetic acid) and incubated under 37° C., adding 10 ml medium B to cease the function of trypsin when cells come off the surface of the flask, collecting the medium containing the cells in the sterile centrifuge tube and discarding the supernatant after the centrifugation, resuspending cells with 10 ml fresh medium B, and culturing some suspended cells on 10 cm cell culture plate to proceed the subsequent experiments. Third to sixth generations of cells are used in the following experiments.
2. Identification of TSMCs:
First, morphology of the smooth muscle cells is observed under an optical microscope, and then the immunofluorescent assay described below is used to determine whether cells have a-actin.
The sterile cover glasses are disposed into the wells of 24-well culture plate and rat TSMCs are added into the wells by the concentration of 5×103 cells/ml per well. The cells are incubated at 37° C. in a humidified atmosphere of 5% CO2/95% O2 overnight to make cells attach on the surface of the cover glasses. The medium in the wells is removed and cells are washed with 0.5 ml ice-cold PBS solution for triple times. 1 ml formaldehyde is added into each well for 5 minutes at room temperature to fix cells. After removing the formaldehyde, PBS solution (1 ml) is added into each well and the culture plate is gently shaken on horizontal orbital shaker for 5 minutes. After removing the PBS solution, 0.5 ml prepermeabilized lysis buffer (BD. Pharmingen, San Diego, Calif.) is added into each well and reacted with cells for 15 minutes at room temperature. After removing the prepermeabilized lysis buffer, the cells are washed with PBS solution for triple times, 0.5 ml for each time. 0.5 ml FITC-conjugated monoclonal mouse anti-smooth muscle a-actin antibody (1:100 dilution) is added into the culture plate to darkly react for 2 hours at room temperature. After the reaction of the antibody, the wells are washed with washing buffer (containing 20 mM Tris base, 140 mM NaCl, 1% (v/v) Tween 20, pH 7.6) twice, and then the cover glasses are mounted on the glass slide with the fluorescent mounting medium for 30 minutes and visualized using a fluorescent microscope.
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3. MTT Assay:
Cell concentration is adjusted to 104 cells/ml by using a cell counter. Ten thousand cells (1 ml) are inoculated in 24-well culture plate for 24 hours, and cells are attached thereon. Medium B then is added into the culture plate and cells are cultured for another 24 hours. Subsequently, different concentrations of drugs are treated for 24 hours in low-oxygen incubator. 100 μL MTT (methylhiazolyldiphenyl-tetrazolium bromide, 5 mg/ml) is added darkly into the wells and the reaction is proceeded darkly at 37° C. for 3 hours. After MTT is decanted, 500 μL Isopropanol is added into the wells and the culture plate is shaken for 10 minutes and incubated for another 10 minutes. After the incubation, the supernatant (200 μL) is transferred to a new 96-well culture plate for determining the absorbance at 540 nm (OD540) and 630 nm (OD630). The effect of drugs on cell growth is evaluated by the value of “OD540-OD630”.
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Usage of Pulmodil is much safer than that of Pulmodul-1 or that of MSC in accordance with the results of MTT assay. Particularly, Pulmodil is a salt derivative of its major structural compound, and the structural difference therebetween is a hydrochloride molecule. However, Pulmodil represents a significant improvement in toxicity and gains the great benefits on medical treatment.
4. The Effect of Pulmodil on U46619 Induced Pulmonary Artery Hypertension:
Acute thromboxane A2 (TXA2)-mimetic U46619 can cause the hypertension and reduce oxygen content of the pulmonary artery by invoking the contraction of the pulmonary artery, and U46619 also results in the lack of NO by inactivating the function of eNOS and thus results in the decreased expression of cGMP/PKG. Therefore, the effects of Pulmodil on the mechanism of U46619 are studied by the pretreatment of U46619.
Hemodynamic measurement of mean arterial pressure (MAP) is carried out in male Wistar rats, weighting 300˜0350 g, anesthetized with pentobarbital sodium (40 mg kg−1) by intraperitoneal injection (hereinafter referred to as “i.p.”).A catheter is inserted into femoral artery, and the MAP and heart rate are recorded with a pressure transducer (Gould, Model P50, U.S.A.) connected with the catheter and a Pressure Processor Amplifier (Gould, Model 13-4615-52, U.S.A.). The catheter in the femoral vein could be used for the intravenous administration of Pulmodil and other drugs.
The mean pulmonary arterial pressure (MPAP) is recorded by catheterizing the pulmonary artery in closed-chest rats with a PE-50 catheter connected with a disposable diaphragm dome TA1019, a pressure transducer and an amplifier.
After hemodynamic measurement is balanced, Pulmodil (0.5-2.0 μg kg−1 min−1) and reference drugs, such as milrinone (1 μg kg−1 min−1), sildenafil (1 μg kg−1 min−1), zaprinast (10 μg kg−1 min−1), urapidil (100 μg kg−1 min−1), are administrated by intravenous infusion (hereinafter referred to as “i.v.”) for 20 minutes and subsequently followed by continuous intravenous infusion of U46619 (2.5 μg kg−1 min−1) for 30 minutes to achieve approximately a 2-fold elevation in MPAP of U46619 as control level. If Pulmodil is administrated by intraperitoneal injection, the dosage could be about 0.1-1.0 mg kg−1.
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5. The Effect of Pulmodil on the Monocrotaline (MCT) Induced Chronic Pulmonary Artery Lesions:
Eight-week-old adult Wistar rats are given with a single injection of monocrotaline (MCT, 60 mg kg−1, i.p.) on day 0 for inducing chronic PAH. Pulmodil (5 mg kg−1 day−1, p.o. and 1 mg kg−1 day−1, i.p. for 21 days) is administered to prevent from worsening of PAH. As shown in Table 4, after the treatment of Pulmodil for 21 days, MCT-induced chronic PAH is acutely decreased.
6. Pulmonary Artery Relaxation Tension Measurement:
The isolated rat pulmonary artery rings (2˜3 mm) are suspended under isometric conditions and connected to a force transducer (UGO BASLINE, Model 7004, Italy). Cumulative concentration-response curves are constructed in response to TXA2 receptor agonist U46619 (0.5 μM) and a-adrenoceptor agonist phenylephrine (PE, 10 μM). Amplitude of the contraction is expressed as a percentage of the maximal U46619-induced and PE-induced contraction. The amplitude of relaxation is expressed as percentage of the maximal amplitude of contraction induced by U46619 or PE application.
After equilibration, rings are contracted with U46619 or PE. When the contractile response to each agonist reaches a stable tension, cumulative concentration-response curves to Pulmodil (0.01˜100 μM) are carried out by cumulative addition of them after a steady-state response is reached after each increment. To examine the possible mechanisms of pulmonary artery relaxant effects of Pulmodil in some experiments, the relaxant effects of Pulmodil is tested in pulmonary arteries pretreated with sGC inhibitor ODQ (1 μM), NOS inhibitor L-NAME (100 μM) and adenylate cyclase inhibitor SQ22536 (1.0 μM), before a contraction was induced with U46619 (0.5 μM) or PE (1.0 μM). Contractile tension is recorded by a computer program. The preparations are stretched to a resting tension of 1 g, and allowed to equilibrate for 60±90 min.
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In Animal models of either acute or chronic PAH, Pulmodil causes a significant decrease of pulmonary artery pressure, without affecting systemic artery pressure. Further, based on the experiments regarding the pulmonary artery vasodilatation, it could be known that Pulmodil relieves the pulmonary artery hypertension by relaxing the contractions of the pulmonary artery rings.
In addition, it is known hypertension is also a risk factor for arteriosclerosis. When Pulmodil of the present invention is used to relieve the pulmonary artery hypertension, it is found that the risk of suffering a cardiovascular disease including a angina, a myocardial Infarction and a heart failure, a cerebral vascular accident, a diabetes, a diabetic retinopathy or a nephropathy is also decreased.
7. Blood Oxygenation Assay:
Blood samples are simultaneously obtained (at pretreatment and the end of each treatment) from femoral arteries, following intra-peritoneal injection and intravenous infusion of Pulmodil and reference agents. After final administration, blood is sampled under conscious conditions through the catheter and subjected to analysis using an automatic blood gas analyzer (AVL OMN™, U.S.A.). The analysis included arterial oxygen pressure (PaO2), arterial carbon dioxide pressure (PaCO2), pH value, O2 saturation (SO2%). U46619 (2.5 mg kg−1 min−1) alone decreases PaO2 to 89±3.1 mmHg in plasma of Wistar rats. Pretreatment with Pulmodil by intravenous infusion reverses this condition to 100.1±2.2 mmHg at the dose of 1 μg kg−1 min−1. U46619-induced decreases of plasma oxygenation is restored by sildenafil (1 μg kg−1 min−1, i.v.) to 100.6±3.0, and zaprinast (10 μg kg−1 min−1, i.v.) to 98±3.1 (as shown in Table 5).
8. The effects of Pulmodil on Ca2+-activated K+ (BKCa) currents:
Smooth muscle cells from rat pulmonary arteries are enzymatically isolated. After this equilibration step, arterial segments are incubated (37° C.) in 0.5 mg/ml collagenase IA, 0.6 mg/ml papain and 0.2 mg/ml dithioerythritol for 45 min. After enzyme treatment, the tissues are washed three times in ice cold isolation medium and triturated with a fire polished pipette to release the myocytes. Cells are stored in ice-cold isolation medium for use in the same day. Whole cell BKCa currents are measured using the conventional patch-clamp configuration.
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9. The Effects of Pulmodil on Ca2+ Mobilization and Concentrations:
The measurements of Ca2+-mobilization and concentrations of rat epithelial cell line are performed by a spectrofluorophotometer. Both cultured rat epithelial cell and primary pulmonary artery smooth muscle cells are loaded with Fura-2/AM to permit the measurements of Ca2+-concentration changes in single cells by a spectrofluorophotometer (Shimadzu, RF-5301PC, Japan).
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K+ and Ca2+-mobilization/sensitization regulate pulmonary vascular tension and remodeling and constitute potential therapeutic targets in the regression of PAH. Loss of K+-channel and opening activity may contribute to the pathogenesis of PAH by causing a sustained depolarization, which increases intracellular Ca2+ and K+, thereby stimulating cell proliferation. Therefore, it could be known a medial thickness and an endothelium dysfunction of pulmonary artery, and a vascular obstruction could be caused by the reduced K+-channel and the lost opening activity thereof.
10. The Effects of Pulmodil on the Expressions of eNOS/sGC/PKG/PDE5A/ROCKII Proteins:
Isolated pulmonary arterial rings are incubated in cell culture medium in the presence of U46619 (0.5 μM) for 60 min and then treated with Pulmodil (0.1˜100 μM) for 60 min. Expressions of the relevant proteins are analyzed using their mouse or rabbit monoclonal antibodies, respectively. Immunoreactive bands are visualized using horseradish peroxidase-conjugated secondary antibodies and subsequent ECL detection (Amersham Pharmacia, USA). Pretreatment with various inhibitors for 30 min before application of Pulmodil or the reference agents is followed by the same procedure.
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Based on the above protein assays, Pulmodil, a theophylline-based PDE5 inhibitor, obviously inhibits U46619-induced PDE5A and increase cGMP. Thus, a combination of the enhancing activities of cGMP, eNOS, sGC and PKG and the inhibiting activity of ROCK caused by inhibiting PDE5A is suggested to provide optimal achievement in inhibiting a medial thickness, an endothelium dysfunction or a vascular obstruction of the pulmonary artery. According to the recent studies regarding rho-kinase inhibitor, in addition to the effect on the vascular smooth muscle cells, it is also proved to be effective in relieving the cardiovascular disease. One example for such rho-kinase inhibitor is “Fasudil”, which is widely used in the therapy of cardiovascular disease. Therefore, one skilled in the art could reasonably anticipate that Pulmodil provided in the present invention could be applied to cardiovascular disease since Pulmodil is also a kind of rho-kinase inhibitors.
11. Histological Examination of Lung and Heart of MCT-Treated Rats:
For light microscopy studies, right and left pulmonary arteries, as well as intrapulmonary arteries, of six rats from each treatment group are isolated 3 weeks after MCT (60 mg/kg, i.p.) or vehicle administration. The samples are fixed in paraformaldehyde, soaked in formalin, dehydrated through graded alcohols, and embedded in paraffin wax for subsequent sectioning. The samples embedded in paraffin wax are cut into 5-μm-thick sections and subjected to hematoxylin-eosin (HE) staining before light microscopic examination.
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In in vivo hypoxic experiments, rats are divided into four groups:
Group 1. Normoxia, i.e. under normal growth condition;
Group 2: Hypoxia for 21 days;
Group 3: Hypoxia+Pulmodil for 21 days;
Group 4, Hypoxia+sidenafil for 21 days.
All rats are on a 12-h light/12-h dark cycle at 25±1° C. and are provided with food and water. Some rats are housed in standard normoxic conditions as control Group 1 and Groups 2-4 are continuously housed in a hypoxic chamber (10% O2) for 21 days, except for a 30-min interval each day when the chamber is cleaned. The hypoxic gas mixture was prepared from N2 (gas cylinders) and compressed air.
In in vitro experiments, isolated rat pulmonary artery is grown under normoxia (20% O2) and hypoxia (1% O2). To achieve hypoxia, a pre-analyzed gas mixture (95% N2-5% CO2) is infused into a CO2 incubator (Class II series, Thermo Form a, USA). The temperature is maintained at 37° C. during experiments for 24 hrs.
1. Pulmonary Arterial Pressure Measurements.
The measurements of heart rate and MPAP of male Wistar rats, 10 weeks-old, are described in detail in embodiment 3, wherein the rats are anesthetized with urethane (1.25 g/Kg). Please refer to Table 7, which shows the effects of Pulmodil and sidenafil on the artery pressure under the Hypoxia condition. MPAP of normoxic and hypoxia-treated rats are 12.9±0.9 mmHg and 26.5±0.6 mmHg (n=6), respectively. Long-term treatment with Pulmodil and sidenafil (5 mg kg−1 day−1, p.o. for 21 days) on hypoxia-treated rats, the MPAP is markedly attenuated to 16.9±1.1 mmHg and 19.8±0.7 mmHg (n=6) at 21 day. As to other measurements, Pulmodil and sidenafil do not significantly affect the heart rate, mean artery pressure (MAP) and heart weight/total weight of each group of rats. The values in Table 7 is presented by mean value±standard error, wherein * indicates P<0.05, compared to Normoxia group 1 and # indicates P<0.05, compared to Hypoxia group 2.
19.8 ± 0.7#
As shown in Table 7, it could be known that Pulmodil could specifically relax the pulmonary arteries and thereby attenuate the pulmonary arterial pressure and reduce the occurrence of the pulmonary arterial diseases.
2. Histological Examination of Lung and Heart.
Pulmonary artery wall thickness is detected on day 0 and day 21, following left lung resection. Please refer to
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3. Western Blotting Analysis.
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Based on the chronic hypoxia experiments, it is indicated that Pulmodil inhibits hypoxia-induced pulmonary artery hypertension (PAH) in rats through increase of eNOS/sGC/PKG and decrease of ROCK/VEGF expressions. Further, Pulmodil is more effective in eNOS than Sildenafil.
In addition to oral administration and intravenous infusion, it is proved in this invention that Pulmodil is effective by sublingual administration as well. The MCT-treated rats are used as samples for proving the effects of sublingual administration.
Adult male Wistar rats, weighted 200-250 g, are given a single subcutaneous injection of monocrotalin (MCT, 60 mg/kg) or vehicle and allowed 21 days to develop PAH. Sublinger preparation of Pulmodil (2.5 mg/kg/day/25 μL propylene glycol) is coated by a micropipette on sublingual cavity of rats. Rat mouth in non-analgesia condition is opened to allow application of Pulmodil with micropipette within 1 minute.
In the following relevant experiments, rats are divided into four groups:
Group 1: Control;
Group 2: MCT induction;
Group 3: sublingual administration of Pulmodil after the MCT induction; and
Group 4, sublingual administration of sidenafil after the MCT induction.
1. Pulmonary Arterial Pressure Measurements.
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In the four groups, medial thickness (μm) and medial wall area (calculated as the area between the internal elastic lamina and the adventitia) of the muscular layer of pulmonary arteries are determined using an Eclipse E600 microscope (Nikon, Champigny-sur-Marne, France) coupled to a color video camera (Sony, Paris, France). Measurements are obtained with Histolab software (Microvision instruments, Evry, France). For each animal, medial thickness and medial wall area determination are taken as the average of three measurements.
2. Histological Examination of Lung and Heart.
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3. Western Blotting Analysis.
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Comparing with sublingual Sildenafil, sublingual Pulmodil is more effective in reducing ROCKII expression and has similar effects on ET-1.
4. Plasma Concentration of Pulmodil and et-1.
Plasma concentration of Pulmodil (lower range: 0.1-20 ng/ml and higher range: 10-5000 ng/ml) in rats received 3.6 mg/kg Pulmodil, dissolved in propylene glycol and sublingually administered with a micropipet, is measured by an LCpMS/MS method. The plasma concentration of ET-1 is determined using an enzyme immunoassay kit, according to the commercial guidance of manufacturer (Biomedica Group, Wien, Austria). Cardiac puncture through needle (19G) on rats is performed to obtain blood and followed by centrifugation to have plasma sample. Plasma samples (0.8-1.0 ml) are acidified with 0.6% trifluoroacetic acid and centrifuged (2000 g, 48 degree C. for 15 mins).
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The merit of sublingual administration of Pulmodil is to increase the bioavailability, compared to oral administration. Further, sublingual administration of Pulmodil is certainly absorbed into plasma and could indicate the pharmacologic profile of long term administration of Pulmodil to prevent from MCT-induced PAR The long term effect for at least three weeks reflects the pharmacologic feature processed by the specific structure of Pulmodil.
Based on all of the above, it could be known that the inhibition of Pulmodil on drug-induced PAH is achieved by inhibiting PDE, enhancing NO/cGMP and increasing the K+-channel opening activity. As to the Hypoxia-induced PAH, the prevention effects on a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall, a vascular obstruction and a right ventricular hypertrophy is achieved by inhibiting ROCK/VEGF and activating eNOS/PKG. Such effect of increasing the activity of eNOS and thereby releasing NO is similar to that of statin drugs. Further, the various administration routs, e.g. an oral administration, an intravenous injection, a subcutaneous injection, an intraperitoneal injection, an intramuscular injection and a sublingual administration, increase the bioavailability. Therefore, regarding various drugs-induced PAH, the applications of Pulmodil in reducing a pulmonary artery endothelium dysfunction, a thickened pulmonary artery medial wall and a vascular obstruction are proved, and have wide-range effects than other drugs for treating PAH. Pulmodil provided in the present invention could be applied to acute or chronic PAH induced asthma, breathing disease, chronic obstructive pulmonary disease (COPD), anaphylaxis, pulmonary fibrosis, pulmonary emboli, or right ventricular hypertrophy or failure, for effectively reducing the death rate of the patients.
While the invention has been described in terms of what is presently considered to be the most practical and preferred embodiments, it is to be understood that the invention needs not be limited to the disclose embodiments. Therefore, it is intended to cover various modifications and similar arrangements included within the spirit and scope of the appended claims, which are to be accorded with the broadest interpretation so as to encompass all such modifications and similar structures.
| Number | Date | Country | Kind |
|---|---|---|---|
| 098114474 | Apr 2009 | TW | national |
