Pharmaceutical composition of theophylline for vasodilation

Abstract

The present invention relates to a pharmaceutical composition comprising theophylline or a pharmaceutically acceptable salt thereof and thus capable of endothelial cell-dependent vasodilation.

Description

Pharmaceutical composition for vasodilation of theophylline

[0001] The present invention relates to a pharmaceutical composition for vasodilation.

[0002]

[0003] Theophylline, a caffeate, is a nonselective phosphatase (PDE) and adenosine receptor inhibitor used to treat bronchial asthma and chronic obstructive pulmonary disease. However, theophylline's low safety margin leads to frequent side effects, such as electrocardiographic arrhythmias and hypotension. Theophylline intoxication increases endogenous catecholamine levels, resulting in increased contractility and heart rate, but also causes hypotension through β2-adrenergic receptor-mediated vasodilation. Nitric oxide (NO) produced in endothelial cells activates guanylate oxidase (GC) to produce cyclic guanosine monophosphate (cGMP), which induces vasodilation through cGMP-dependent protein kinase activation. PDE degrades cGMP to 5'-GMP, and PDE inhibitors, such as theophylline, can increase cGMP production, thereby increasing vasodilation. Therefore, theophylline-induced PDE inhibition may worsen the hypotension observed in patients with theophylline intoxication by inhibiting the degradation of cGMP and the cyclic adenosine monophosphate (cAMP) in the blood vessels.

[0004] Theophylline increases blood flow in rat ears, which is similar to the effect of N, an inhibitor of nitric oxide synthase (NOS). ω- is reversed by nitro-L-arginine methyl ester. In addition, the selective PDE inhibitor M&B 22948 induces endothelium-dependent vasodilation by increasing cGMP production by spontaneously released NO. In addition, vasodilation by the PDE inhibitor isobutylmethylxanine in cerebral vessels is endothelium-dependent, which relies on an NO-dependent increase in cGMP and cAMP levels. In summary, PDE inhibitors can induce NO-mediated vasodilation in endothelial cells and increase blood flow. In addition, a recent report of treatment with lipid emulsions in toxic doses of theophylline-induced shock that did not respond to conservative treatment suggested the possibility of inhibiting NO-mediated vasodilation by toxic doses of theophylline. However, theophylline-induced vasodilation in vessels pre-constricted with norepinephrine was inhibited by the NOS inhibitor N. ω -Theophylline-induced vasodilation is not inhibited by nitro-L-arginine methyl ester, and in human arteries, theophylline-induced vasodilation occurs independently of NO release. Considering these factors, the role of endothelial cell-generated NO in vasodilation induced by the PDE inhibitor theophylline remains unclear.

[0005]

[0006] The present invention aims to provide a pharmaceutical composition for vasodilation.

[0007]

[0008] The present invention relates to a pharmaceutical composition for vasodilation comprising theophylline or a pharmaceutically acceptable salt thereof.

[0009] In the present invention, the vasodilation may be endothelium-dependent arteriolar dilation.

[0010] In the present invention, the vasodilation may be mediated by endothelial NO release and phosphorylation inhibition.

[0011]

[0012] The pharmaceutical composition of the present invention can dilate arteries in an endothelium-dependent manner by inhibiting endothelial NO release and phosphorylation.

[0013]

[0014] Figure 1. A. Phenylephrine (10 -7 3×10 in -4 Original trace images of theophylline-induced arterial dilation in endothelium-sparing and endothelium-stripped rat aortas pretreated with M). B. Phenylephrine (10 -6 Effect of endothelial removal on theophylline-induced arterial dilation in isolated rat aortas pretreated with M). Data (n = 5) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. n indicates the number of rats used. **p < 0.01 and *p < 0.001 vs endothelial removal.

[0015] Figure 2. Phenylephrine (10 -6 N on theophylline-induced vasodilation in precontracted, endothelial-preserved isolated rat aortas ω Effects of -nitro-L-arginine methyl (L-NAME, A), methylene blue (B), and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, C). Data (n = 5) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contractions. n represents the number of rats used. *p<0.05, **p<0.01, ***p<0.001 vs. control.

[0016] Figure 3. Phenylephrine (10 -6 In isolated rat aortas pre-contracted with sodium nitroprusside (SNP, A), isoproterenol (10 -3Effect of theophylline on vasodilation induced by phenylephrine (M, B) and 8-bromoguanosine 3',5'-cyclic monophosphate sodium salt (bromo-cGMP, C). Data (n = 5) are presented as mean ± standard deviation and are expressed as percentage of phenylephrine-induced contraction (A, C) and percentage of phenylephrine-induced contraction to relaxation (B). n represents the number of rats. *p<0.05 and ***p<0.001 vs. control.

[0017] Figure 4. Phenylephrine (10 -6 Theophylline concentration-response curves in deendothelialized aortas preconstricted with phenylephrine or 60 mM potassium chloride (KCl). Data (n = 5) are presented as mean ± standard deviation and are expressed as the percentage of contractions induced by phenylephrine or KCl. n represents the number of rats used. ***p<0.001 vs 60 mM KCl.

[0018] Figure 5. A. Theophylline (3 × 10) on cyclic guanosine monophosphate (cGMP) formation in isolated rat aorta with preserved endothelium. -4 M). ***p<0.001 vs. control. B. Effect of sodium nitroprusside (SNP, 10 -8 Theophylline (3×10 M) induced cGMP formation -4 Effect of M). Data (n = 4) are expressed as mean ± SD. n represents the number of mice used. *p< 0.05 vs. control. †††p< 0.001 vs. SNP alone.

[0019]

[0020] The present invention is described in detail below.

[0021]

[0022] The present invention relates to a pharmaceutical composition for vasodilation comprising theophylline or a pharmaceutically acceptable salt thereof.

[0023] Theophylline is a compound having the structure of the following chemical formula 1.

[0024] [Chemical Formula 1]

[0025]

[0026] The term pharmaceutically acceptable salt means any organic or inorganic addition salt of acetylgentistin at a concentration that is relatively non-toxic and harmless to the patient and that does not diminish the beneficial effects of acetylgentistin due to side effects attributable to this salt.

[0027] The salt may be an acid addition salt or a metal salt obtained using a base. Any acid or base known in the art may be used without limitation.

[0028] Vasodilation can be arteriolar dilation. Vasodilation can be endothelium-dependent arteriolar dilation, which can be mediated by endothelial cell-produced NO and inhibition of kinases.

[0029] Since the pharmaceutical composition of the present invention can dilate blood vessels, it can also be used for pulmonary edema, pulmonary vascular disease, etc.

[0030] The pharmaceutical composition of the present invention can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injection solutions according to conventional methods. Carriers, excipients, and diluents that can be contained in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. When formulated, they are usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the above compound with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, and glycerogelatin.

[0031] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, and weight, but may be administered once or several times daily at a dosage of 0.001 to 100 mg / kg, preferably 0.01 to 10 mg / kg. Furthermore, the dosage may be increased or decreased depending on the route of administration, severity of the disease, sex, body weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any way.

[0032]

[0033] The present invention will be described in more detail with reference to the following examples.

[0034]

[0035] Materials and Methods

[0036] The Animal Care and Use Committee of Kyung Sang National University approved the experimental protocol (GNU-211217-R0106). All experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

[0037]

[0038] Preparation and isometric tension measurements of isolated rat aortas.

[0039] A total of 48 rats participated in the experiment. The rats were housed in cages at a temperature of 22–24°C, humidity of 30–70%, and a 12-h light / dark cycle. The rats had free access to sterile water and a standard chow diet. Male Sprague-Dawley rats (weight: 220–300 g; Koatech, Pyeongtaek, Gyeonggi-do, South Korea) were anesthetized with 100% carbon dioxide delivered through a small incision. The aorta was isolated, and isometric tension measurements were performed.

[0040] The thorax was opened, and the descending thoracic aorta was extracted from the rat thorax. The aorta was suspended in an organ bath (Grass Instrument, Quincy, MA, USA) maintained at 37°C and surrounded by Krebs solution containing the following components: sodium chloride (118 mM), sodium bicarbonate (25 mM), glucose (11 mM), potassium chloride (4.7 mM), calcium chloride (2.4 mM), magnesium sulfate (1.2 mM), and potassium monophosphate (1.2 mM). Fat and connective tissue around the aorta were removed using a microscope. The isolated rat thoracic aorta was sliced ​​into 2.5 mm-long segments. The endothelium of some thoracic aortas was removed by rolling them back and forth using two 25-gauge needles inside the aorta. The isolated descending thoracic aorta was suspended in a Grass isobaric transmitter (FT-03, Grass Instrument, Quincy, MA, USA) maintained at 37°C. A baseline stable tension of 2.5 g was maintained for 90 min, and the existing Krebs solution was replaced with fresh Krebs solution every 30 min. The Krebs solution was aerated with a gas containing 95% oxygen and 5% carbon dioxide to maintain the pH at 7.4.

[0041] Endothelial preservation in the endothelium-lined aorta was confirmed as follows:

[0042] 10 -7 After M-phenylephrine induced sustained and stable contraction in the endothelium-lined thoracic aorta, acetylcholine (10 -5 M) was added to the long-term bath and acetylcholine-induced relaxations greater than 80% from phenylephrine-induced contractions were considered to be endothelium-lined thoracic aorta.

[0043] Furthermore, the removal of the endothelium was verified as follows:

[0044] Phenylephrine (10 -8 After M) induced a stable and sustained contraction, acetylcholine (10 -5M) was added to the long-term bath and acetylcholine-induced relaxation of less than 15% from the phenylephrine-induced contraction was considered to be endothelialized thoracic aorta.

[0045] When acetylcholine-induced relaxation was confirmed in the deendothelialized thoracic aorta, it was repeatedly washed with fresh Krebs solution to reach baseline resting tension.

[0046]

[0047] Experimental protocol

[0048] Effect of endothelial removal on theophylline-induced vasodilation:

[0049] To determine whether theophylline-induced vasodilation is endothelium-dependent, the effect of endothelial removal was investigated. Phenylephrine (10 -6 After inducing sustained and stable contractions in endothelium-lined and endothelium-deprived femoral arteries using theophylline (10 -7 to 3Х10 -4 Theophylline-induced vasodilation was measured by adding M) little by little.

[0050] Effects of cell signaling pathway inhibitors:

[0051] To determine whether theophylline-induced vasodilation in endothelial femoral arteries was related to endothelial NO-mediated vasodilation, endothelial rat femoral arteries were treated with the NOS inhibitor N ω -nitro-L-arginine methyl ester (L-NAME, 10 -4 M), nonspecific GC inhibitor methylene blue (10 -6 M), and the NO-sensitive GC inhibitor 1H-[1,2,4] oxadiazolo[4,3-a] quinoxalin-1-one (ODQ, 10 -5 After 20 minutes of treatment with M), phenylephrine (10 -6 M) induced sustained and stable contractions. Theophylline (10 -7 to 3Х10 -4M) was added to measure vasodilation in the femoral artery with endothelium, and the effects of inhibitors (L-NAME, methylene blue, ODQ) were also confirmed in this process.

[0052] Effects of theophylline on vasodilation induced by a NO donor-induced cGMP-mediated vasodilator (sodium nitroprusside), a β-agonist-induced cAMP-mediated vasodilator (isoproterenol), and a cGMP analog (8-Bromoguanosine 3',5'-cyclic monophosphate sodium salt (bromo-cGMP)):

[0053] Theophylline (10) was injected into the deendothelialized femoral artery -4 After 20 minutes of treatment with M), phenylephrine (10 -6 After M) induced sustained and stable contraction, sodium nitroprusside (10 -10 to 10 -7 M) or bromo-cGMP (10 -10 to 3Х10 -5 M) was added little by little to induce vasodilation. At this time, the change in vasodilation was observed depending on the presence or absence of theophylline, and isoproterenol (10) was added in a similar manner. -3 The experiment was repeated using M).

[0054] Effects of potassium chloride (KCl) and phenylephrine-induced constriction on theophylline-induced vasodilation:

[0055] Phenylephrine (10 -6 M) or 60 mM KCl induced sustained and stable contractions in endothelialized rat femoral arteries, followed by theophylline (10 -7 to 3Х10 -4 M) was added little by little and the magnitude of theophylline-induced vasodilation was compared.

[0056]

[0057] Cyclic guanosine methyl phosphate (cGMP) measurement

[0058] Isolated rat thoracic aortas were used to determine cGMP concentrations. cGMP concentrations were measured using the cGMP Complete Kit (Abcam, Cambridge Science Park, Cambridge, England). The descending transcatheter aortas, treated with or without endothelium, were exposed to 10 ml of Krebs solution in an organ bath at 37°C for a total of 60 min, including the drug treatment time. The descending transcatheter aortas with endothelium were exposed to 3X10 -4 Theophylline was treated alone for 5 minutes at a concentration of M. The deendothelialized thoracic aorta was treated with 10 -8 M concentration of sodium nitroprusside alone for 5 minutes or 3Х10 -4 After 10 minutes of treatment with theophylline at a concentration of M, sodium nitroprusside (10 -8 M) was treated for 5 minutes. The treated aortas were then frozen in liquid nitrogen and solidified in 0.1 M hydrochloric acid. The acidic supernatant was acetylated and used to measure cGMP concentration using the cGMP Complete kit. The cGMP concentration obtained from each thoracic aorta was expressed as pmol / ml.

[0059]

[0060] cell culture

[0061] Human umbilical vein endothelial cells (HUVECs, C-0003-5C, American Type Culture Collection, Manassas, VA, USA) were maintained in endothelial cell medium (ECM) (ScienCell, Carlsbad, CA, USA) containing 15% fetal bovine serum (ScienCell), 100 units / μg / ml penicillin, 1% endothelial cell growth supplement (ScienCell), and 100 μg / ml streptomycin. Cells were cultured in serum-free ECM for 4 h prior to drug pretreatment at passages 3–5.

[0062]

[0063] Western blot analysis

[0064] Endothelial NOS (eNOS; Ser1177 and Thr495) in HUVECs was examined using Western blotting. Cells were treated with theophylline (3Х10) to determine eNOS (Ser1177 and Thr495) phosphorylation -4M) for 5, 10, 30, or 60 min. HUVECs were harvested in radioimmunoprecipitation assay buffer containing phosphatase and protease inhibitors (Cell Signaling Technology, Danvers, MA, USA). The lysate was centrifuged at 20,000 Х g for 15 min at 4°C, and the protein content of the supernatant was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific). After boiling for 10 min, samples containing 30 μg of protein were separated using SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.5% Tween-20 (TBST) for 60 min at 25°C and incubated with primary antibodies (anti-phospho-eNOS at Ser1177 (1:1,000), anti-phospho-eNOS at Thr495 (1:1,000), anti-eNOS (1:1,000), and anti-β actin (1:10,000)) overnight at 4°C. The membranes were washed three times for 10 min with TBST and then processed for reaction with mouse or rabbit immunoglobulin G diluted 1:5,000 for 60 min at 25°C. WesternbrightTM ECL Western blotting detection kit (Advansta, Menlo Park, CA, USA) was used to capture signals of protein bands, and ChemiDocTM Touch Imaging System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to capture signals of protein bands. ImageJ software (version 1.Proteins were quantified using a 45s antibody (National Institutes of Health, Bethesda, MD, USA).

[0065]

[0066] Measurement of intracellular calcium

[0067] Intracellular calcium was measured using a fluorescence diffraction microscope (IX70 Fluoview, Olympus, Tokyo, Japan). HUVECs were cultured in confocal cell culture dishes (SPL; Pocheon, Republic of Korea) and supplemented with Fluo-4 AM (2.5Х10 -6 M, Invitrogen, Waltham, MA, USA) for 30 min. Afterwards, HUVECs were washed twice with phosphate-buffered saline solution. HUVECs were treated with theophylline (10 -4 M) were treated, and intracellular calcium levels were measured every 2.5 seconds at emission and emission wavelengths of 520 and 485 nm. Intracellular calcium levels were analyzed using images rendered with Fluo-4 AM, which was calculated as follows: fluorescence intensity (F) divided by the baseline fluorescence intensity (F0) before drug treatment. The net change in calcium levels was expressed as (Fmax - F0) / F0, where Fmax is the maximum calcium level based on fluorescence intensity after theophylline treatment. Intracellular calcium levels were measured for approximately 6 minutes.

[0068]

[0069] substance

[0070] All chemicals used in the study were commercially purchased with high purity. Theophylline, L-NAME, methylene blue, ODQ, sodium nitroprusside, isoproterenol, bromo-cGMP, phenylephrine, and acetylcholine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-phospho-eNOS (Ser1177 and Thr495) and anti-eNOS antibodies were purchased from Cell Signaling Technology and BD Biosciences (Franklin, NJ, USA), respectively. ODQ was dissolved in dimethyl sulfoxide (DMSO), and the final DMSO concentration in the long-acting bath was 0.1%.

[0071]

[0072] Statistical analysis

[0073] The primary outcomes of this study were the effects of endothelial detachment and various drugs on theophylline-induced vasodilation, and the effects of theophylline on sodium nitroprusside, isoproterenol, and bromo-cGMP-induced vasodilation. The effects of endothelial detachment, various inhibitors, and vasoconstrictors (phenylephrine and KCl) on theophylline-induced vasodilation, and the effects of theophylline on sodium nitroprusside- and bromo-cGMP-induced vasodilation, were analyzed using linear mixed-effects models (Stata version 14.2, StataCorp LLC, Lakeway Drive, College Station, TX, USA). The effects of theophylline on isoproterenol-induced vasodilation were analyzed using an unpaired Student's t-test (Prism 5.0, GraphPad Software, Inc., San Diego, CA, USA). The effects of theophylline on eNOS phosphorylation were analyzed using one-way analysis of variance (ANOVA) followed by Bonferroni's test. The effects of sodium nitroprusside and theophylline, individually or in combination, on cGMP formation were analyzed using one-way ANOVA followed by Bonferroni's test or unpaired Student's t-test. The effects of theophylline on intracellular calcium levels were analyzed using unpaired Student's t-test.

[0074]

[0075] result

[0076] Theophylline-induced vasodilation was more pronounced in aortas with preserved endothelium than in deendothelialized vessels (Fig. 1A,B; p<0.01 vs. 3Х10 -6 to 3Х10 -4 M theophylline control). NOS inhibitor L-NAME (10 -4M) inhibited theophylline-induced vasodilation (Fig. 2A; p<0.01 vs. 10 -5 to 3Х10 -4 M theophylline control). In addition, the non-specific GC inhibitor methylene blue (10 -6 M) and NO-sensitive GC inhibitor ODQ (10 -5 M) reduced theophylline-induced vasodilation in aortas with preserved endothelium (Fig. 2B,C; methylene blue: p<0.05 vs. 3Х10 -6 M theophylline control; methylene blue and ODQ: p < 0.01 vs. 10 -5 to 3Х10 -4 M theophylline control). Theophylline (10 -4 M) is NO donor sodium nitroprusside and β-agonist isoproterenol (10 -3 M) increased induced vasodilation (Fig. 3A; p<0.05 vs. 10 -9 to 3Х10 -8 M sodium nitroprusside control) (Fig. 3B; p< 0.001 vs. control). In deendothelialized vessels, theophylline also increased bromo-cGMP-induced vasodilation, a cGMP analog (Fig. 3C; p< 0.05 vs. control at 3Х10 -6 to 3Х10 -5 M bromo-cGMP). Theophylline-induced vasodilation in deendothelialized vessels was induced by phenylephrine (10 -6 M) was greater in KCl (60 mM)-induced contraction than in induced contraction (Fig. 4; p< 0.001 at 10 -4 and 3Х10 -4 M theophylline). Theophylline (3Х10) in aortas with preserved endothelium -4 M) increased cGMP formation (Fig. 5A; p<0.001 vs. control). Sodium nitroprusside (10 -8M) increased cGMP formation (Fig. 5B; p<0.05 vs. control). In addition, theophylline (3Х10 -4 M) is sodium nitroprusside (10 -8 M) further increased the formation of cGMP-induced cGMP (Fig. 5B; p<0.001 vs. sodium nitroprusside alone). Theophylline (3Х10 -4 M) increased stimulatory eNOS (Ser1177) phosphorylation in HUVECs (Fig. 6A; p<0.01 vs. control at 10, 30, and 60 min). However, it decreased inhibitory eNOS (Thr495) phosphorylation (Fig. 6A; p<0.001 vs. control at 30 and 60 min). In addition, theophylline (10 -4 M) increased intracellular calcium levels in HUVECs (Fig. 6B; p<0.001 vs. control).

Claims

1. A pharmaceutical composition for vasodilation containing theophylline or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition for vasodilation according to claim 1, wherein the vasodilation is endothelium-dependent arteriolar dilation.

3. A pharmaceutical composition for vasodilation according to claim 1, wherein the vasodilation is mediated by endothelial NO release and inhibition of phosphorylation enzyme.

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