Pharmaceutical composition for inhibiting vasodilation comprising lipid emulsion

Abstract

The present invention relates to a pharmaceutical composition that comprises a lipid emulsion and thus inhibits nitric oxide production, thereby inhibiting vasodilation.

Description

Pharmaceutical composition for inhibiting vasodilation comprising lipid emulsion

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

[0002]

[0003] Lipid emulsions treat systemic toxicity caused by local anesthetics. They also attenuate cardiovascular depression caused by toxic doses of lipid-soluble non-local anesthetics, such as antidepressants, antipsychotics, and cardiovascular drugs (such as verapamil and propranolol), in patients who do not respond to conservative treatment.

[0004] Adjunctive treatments currently used for cardiovascular depression induced by toxic doses of calcium channel blockers include gastrointestinal lavage, fluid administration, correction of metabolic acidosis and electrolyte imbalance, vasopressors, calcium, and high-dose insulin euglycemic therapy. Lipid emulsions are known to attenuate refractory cardiovascular depression caused by toxic administration of the antihypertensive agent amlodipine, a dihydropyridine L-type calcium channel blocker that acts on vascular smooth muscle. Furthermore, lipid-soluble and racemic amlodipine (Log P: 3), consisting of S-amlodipine and R-amlodipine, may additionally contribute to amlodipine-induced vasodilation by inducing endothelial nitric oxide synthase (eNOS) phosphorylation and nitric oxide production. Racemic and R-amlodipine induce nitric oxide production, whereas S-amlodipine does not. Therefore, amlodipine-induced vasodilation appears to be mediated primarily through L-type calcium channel inhibition and partially through endothelial nitric oxide mediated by S- and R-amlodipine. Furthermore, methylene blue, a nonspecific inhibitor of guanylate cyclase (GC), attenuated the amlodipine-induced blood pressure reduction in rats by inhibiting vasodilation via the nitric oxide-GC pathway in the cell signaling pathway involved in endothelial nitric oxide-induced vasodilation. Lipid emulsions also inhibit nitric oxide-induced vasodilation through inhibition of eNOS phosphorylation. Intralipid may also increase blood pressure and vascular resistance, but may decrease flow-mediated vasodilation and vascular compliance in humans, possibly due to decreased nitric oxide production. However, the effect of lipid emulsions on amlodipine-induced nitric oxide-mediated vasodilation is not yet known.

[0005]

[0006] The purpose of the present invention is to provide a pharmaceutical composition for inhibiting vasodilation.

[0007]

[0008] The present invention relates to a pharmaceutical composition for inhibiting vasodilation comprising a lipid emulsion.

[0009] In the pharmaceutical composition of the present invention, the vasodilation may be amlodipine-induced vasodilation.

[0010] In the pharmaceutical composition of the present invention, the lipid emulsion may inhibit vasodilation by inhibiting nitric oxide production.

[0011]

[0012] The pharmaceutical composition of the present invention can inhibit nitric oxide production by reversing stimulatory and inhibitory eNOS phosphorylation, thereby inhibiting vasodilation.

[0013]

[0014] Figure 1. Vasoconstriction and relaxation effects induced by phenylephrine and acetylcholine in isolated rat aortas with or without endothelium preservation.

[0015] Figure 2. Amlodipine (3 x 10 -7 Effect of endothelial removal on vasodilation by M). Data (N = 6) are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. N represents the number of rats. ***p<0.001 vs. endothelial preservation.

[0016] Figure 3. Effect of DMSO on phenylephrine-induced contraction in endothelium-preserved aorta.

[0017] Figure 4. N in endothelium-preserved rat aorta ω -Amlodipine (3 x 10) with nitro-L-arginine methyl ester (L-NAME, N = 7), methylene blue (N = 5), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, N = 5) and calmidazolium (N = 6) -7M) Effect on phenylephrine-induced vasodilation. Data are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. N represents the number of rats. **p<0.01 and ***p<0.001 vs. control.

[0018] Figure 5. (A) Amlodipine (3 x 10 -7 Effect of lipid emulsions on vasodilation by M). Data are presented as mean ± standard deviation and as a percentage of phenylephrine-induced contraction. N represents the number of rats. ***p<0.001 vs. control. (B) N ω -Amlodipine (3 x 10 -7 Effect of lipid emulsions on vasodilation by L-NAME (M). Data are presented as mean ± standard deviation and as a percentage of phenylephrine-induced contraction. N represents the number of rats. *p<0.05 and ***p<0.001 vs. L-NAME (10 4 M) alone. (C) Phenylephrine-induced contraction (%) time (30-80 min) of endothelial-stripped aortas treated with or without lipid emulsion. The difference in area under the curve was compared with L-NAME (10 -4 Effect of M). The difference in the area under the phenylephrine-induced contraction time curve in the endothelial-preserved aorta was defined as the area under the phenylephrine-induced contraction time curve (30-80 min) in the lipid emulsion-treated group minus the area under the phenylephrine-induced contraction time curve (30-80 min) in the lipid emulsion-free group. Data (N = 5) are presented as the mean ± standard deviation and are expressed as the difference in the area (min %) under the phenylephrine-induced contraction time curve (30-80 min). N represents the number of rats. *p<0.05 vs. L-NAME(10 4 Endothelialized aorta treated with M).

[0019] Figure 6. (A) Amlodipine (3 x 10-7 M) Linolenic acid on induced vasodilation (N = 8, 6 and 5 are control and 3 x 10 -6 and 10 -5 M linolenic acid). Data are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contractions. N represents the number of rats. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control. (B) Effect of amlodipine (3 × 10 -7 Effect of linolenic acid (N = 5) on phenylephrine-induced vasodilation. Data are presented as mean ± standard deviation and expressed as a percentage of phenylephrine-induced contraction. N represents the number of rats.

[0020] Figure 7. Lipid emulsion (LE, 1%, A, N = 3) in isolated aorta with preserved endothelium, N ω -Nitro-L-arginine methyl ester (L-NAME, 10 -4 M, B, N = 4) and linolenic acid (10 -5 M, C, N = 3) alone or in combination with amlodipine (3 x 10 -7 Effect of amlodipine on cyclic guanosine monophosphate (cGMP) production induced by amlodipine alone. Data are presented as mean ± standard deviation. N represents the number of rats. *p< 0.05, **p< 0.01, and ***p< 0.001 vs control. †p< 0.05, ††p< 0.01, and †††p< 0.001 vs amlodipine alone.

[0021] Figure 8. (A) Lipid emulsion (LE, 1%), PP2 (2 x 10 -5 M) and amlodipine (3 x 10 -7(B) Effect of 1% LE and amlodipine alone or in combination on phosphorylation of stimulatory endothelial nitric oxide synthase (eNOS, Ser1177) in human umbilical vein endothelial cells (HUVECs). Data (N = 5) are presented as mean ± standard deviation. N represents the number of independent experiments. *p < 0.05 vs. control. ††p < 0.01 and †††p < 0.001 vs. amlodipine. (C) Effect of 1% LE and amlodipine alone or in combination on phosphorylation of inhibitory eNOS (Thr495) in HUVECs. Data (N = 4) are presented as mean ± standard deviation. N represents the number of independent experiments. ***p < 0.001 vs. control. †††p < 0.001 vs. amlodipine.

[0022] Figure 9. Amlodipine (3 x 10 -7 M) and PP2 (2 x 10 -5 Effects of M) alone or in combination on caveolin-1 (Tyr14, (A)) and Src-kinase (Tyr416, (B)) phosphorylation in human umbilical vein endothelial cells. Data (N = 4) are expressed as mean ± standard deviation. N represents the number of independent experiments. *p<0.05, **p<0.01, and ***p<0.001 vs control. †††p<0.001 vs amlodipine.

[0023] Figure 10. Amlodipine (10) in human umbilical vein endothelial cells -6 Effect of lipid emulsion (LE, 1%) and N on intracellular calcium levels induced by M ω -Nitro-L-arginine methyl ester (L-NAME, 10 -4 Effect of M). Data (N = 5) are presented as median ± interquartile range (25–75%). N represents the number of independent experiments. *p<0.05 and ***p<0.001 vs control. †††p<0.001 vs amlodipine alone.

[0024]

[0025] The present invention is described in detail below.

[0026]

[0027] The present invention relates to a pharmaceutical composition for inhibiting vasodilation comprising a lipid emulsion.

[0028] Vasodilation can be drug-induced. The drug can be amlodipine, for example.

[0029] Lipid emulsions may inhibit vasodilation, for example by inhibiting nitric oxide production.

[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 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] 1. Materials and Methods

[0036] The Institutional Animal Care and Use Committee of Gyeongsang National University approved the experimental protocol (GNU-211217-R0106, December 22, 2021). The experiment was performed in accordance with the Animal Care and Use Guidelines.

[0037]

[0038] 1.1. Preparation of rat aorta and isometric tension measurements

[0039] Male Sprague-Dawley rats (Coatech, Pyeongtaek, South Korea) weighing 220–300 g were anesthetized with 100% carbon dioxide. The thoracic cavity was opened, and the descending thoracic aorta was extracted from the thorax. The extracted thoracic aorta was placed in Krebs solution containing 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 monopotassium phosphate (1.2 mM). Connective tissue and fat around the isolated rat aorta in Krebs solution were removed under a microscope. The isolated descending thoracic aorta was cut into 2.5-mm-long segments. Two 25-gauge needles were inserted into the aortic lumen, rolling the aorta back and forth to peel off the endothelium of a portion of the aorta. Isolated rat descending thoracic aortas were suspended on a Grass isometric transducer (FT-03, Grass Instrument, Quincy, MA, USA) in an organ bath maintained at 37°C. A baseline resting tension of 24.5 mN was maintained for 1.5 h to achieve steady state. During this period, the existing Krebs solution was replaced with fresh Krebs solution. The pH of the Krebs solution was maintained at 7.4 by supplying 95% oxygen and 5% carbon dioxide. The endothelial integrity of the endothelial-preserved rat aortas was examined as follows. Phenylephrine (10 -7 M) causes sustained and stable contraction followed by acetylcholine (10 -5 M) was added to the long-term bath. Aortas with acetylcholine-induced relaxation greater than 85% of phenylephrine-induced contraction were considered to have preserved endothelium (Fig. 1). Endothelial removal was confirmed as follows: Phenylephrine (10 -8 M) causes sustained and stable contraction of the aorta followed by acetylcholine (10 -5M) was added to the long-term bath. Aortas with less than 15% acetylcholine-induced relaxation were considered deendothelialized (Fig. 1). The aortas from endothelium-preserved and deendothelialized rats were washed multiple times to restore their baseline resting tension, and the following experimental protocol was performed.

[0040]

[0041] 1.2. Experimental Protocol

[0042] First, we investigated the effect of endothelial removal on toxic doses of amlodipine-induced vasodilation to determine whether amlodipine-induced vasodilation was endothelium-dependent. Phenylephrine (10 -6 After M) induced sustained and stable constriction of isolated rat aortas with or without endothelium, amlodipine (3 x 10) was added to the long-term bath to induce vasodilation in endothelium-preserved and endothelium-depleted rat aortas. -7 M) was added. The induced vasodilation was monitored for 80 minutes after the addition of amlodipine. In addition, amlodipine (3 x 10 -7 The effect of 0.1% DMSO, used to dissolve M), on phenylephrine-induced contractions was investigated. Phenylephrine (10 -6 After M) induced sustained and stable contractions, 0.1% DMSO was added to the long-term bath. Phenylephrine-induced contractions were monitored for 80 minutes after the addition of DMSO (Fig. 3).

[0043] Second, the cell signaling pathway involving NOS-nitric oxide-GC is involved in endothelial nitric oxide-induced vasodilation. Therefore, to determine whether vasodilation depends on the NOS-nitric oxide-GC pathway, we evaluated the effect of inhibitors on amlodipine-induced vasodilation in endothelium-preserved rat aortas. Endothelium-preserved rat aortas were treated with the NOS inhibitor L-NAME (10 -4 M), nonspecific GC inhibitor methylene blue (10 -6M), nitric oxide-sensitive GC inhibitor ODQ(10 -6 M) or calmodulin-regulating enzyme inhibitor calmidazolium (3 x 10 -6 After pretreatment with phenylephrine (10 M) for 20 minutes, -6 M) induced stable and sustained contraction. Amlodipine (3 x 10) was used to induce vasodilation in endothelium-preserved rat aortas with or without inhibitors (L-MAME, methylene blue, ODQ, and calmidazolium). -7 M) was added to the long-term bath. After that, amlodipine was added and vasodilation was monitored for 80 minutes.

[0044] Third, we investigated the effect of intralipid on amlodipine-induced vasodilation in endothelium-preserved rat aortas with or without L-NAME to determine whether lipid emulsion-mediated inhibition of amlodipine-induced vasodilation in endothelium-preserved rat aortas was dependent on endothelial nitric oxide. Some endothelium-preserved rat aortas were treated with the NOS inhibitor L-NAME (10 -4 After pretreatment with M) for 20 minutes, phenylephrine (10 -6 M) produced stable and sustained contractions. Subsequently, amlodipine (3 x 10 -7 M) was added to the long-acting bath, and some aortas were immediately posttreated with a fat emulsion (1%). Amlodipine-induced vasodilation was monitored for 80 minutes.

[0045] Fourth, we investigated the effect of linolenic acid, a long-chain fatty acid in intralipid, on amlodipine-induced vasodilation to determine whether linolenic acid-mediated inhibition of amlodipine-induced vasodilation was endothelium-dependent. Phenylephrine (10 -6 M) induced sustained and stable contractions in isolated rat aortas with or without endothelium, after which amlodipine (3 x 10 -7 M) was added to the long-term bath and the aorta of some rats was immediately treated with linolenic acid (3 x 10 6 and 10 5M) was added to the aortas of rats with preserved or removed endothelium, with or without linolenic acid, and amlodipine-induced vasodilation was monitored for 80 min.

[0046]

[0047] 1.3. Cyclic guanosine monophosphate (cGMP)

[0048] cGMP was measured using a cGMP complete kit (Abcam, Cambridge Science Park, Cambridge, England). The descending thoracic aorta with preserved endothelium was placed in Krebs solution in a 10 mL organ bath at 37°C for 60 minutes, including the drug treatment time. The aortic strips with preserved endothelium were incubated with amlodipine (3 x 10 -7 M) alone for 5 minutes, and lipid emulsion (Intralipid, 1%) alone for 25 minutes. Lipid emulsion (1%) or linolenic acid (10 -5 M) for 20 minutes, then amlodipine (3 x 10 -7 M) was treated for 5 minutes. L-NAME(10 -4 M) were treated for 30 min, followed by amlodipine (3 x 10 -7 M) was treated for 5 minutes. L-NAME(10 -4 M) for 15 minutes, then lipid emulsion (1%) for 15 minutes, then amlodipine (3 x 10 -7 After treatment with M) for 5 minutes, the descending thoracic aortic strips were frozen. They were treated with liquid nitrogen and stored for 10 -1 The aortic strips were homogenized with M hydrochloride. The acidic supernatant was acetylated, and cGMP was measured using ELISA with the cGMP Complete Kit. The cGMP concentration in each aortic strip was expressed as pmol / mL.

[0049]

[0050] 1.4. Human umbilical vein endothelial cell (HUVEC) culture

[0051] HUVECs (C-003-5C, American Type Culture Collection, Manassas, VA, USA) were cultured in endothelial cell medium (ECM) (ScienCell, Carlsbad, CA, USA) containing 15% fetal bovine serum (ScienCell), 1% endothelial cell growth supplement (ScienCell), 100 Units / mL penicillin, and 100 μg / mL streptomycin (ScienCell). Cells were grown in a humidified atmosphere at 37°C in a 5% CO2 incubator. Cells at passages 3–5 were additionally cultured in serum-free ECM for 4 h before drug treatment.

[0052]

[0053] 1.5. Western blot analysis

[0054] The expression of eNOS (Ser1177 and Thr495), Src-kinase (Tyr416), and caveolin-1 (Tyr14) phosphorylation in HUVECs was assessed using Western blotting. Cells were treated with amlodipine alone for 10 min, 1% Intralipid for 1 h, and then with amlodipine for 10 min, PP2 for 30 min, followed by amlodipine for 10 min, 1% lipid emulsion alone for 70 min, or PP2 alone for 40 min to determine the expression of stimulatory eNOS (Ser1177) phosphorylation. To determine the expression of inhibitory eNOS (Thr495) phosphorylation, cells were treated with amlodipine alone for 1 min, 1% lipid emulsion for 1 h, and then amlodipine for 1 min or 1% lipid emulsion alone for 61 min. Expression of Src kinase (Tyr416) ​​and caveolin-1 (Tyr14) phosphorylation was determined by treatment with amlodipine alone for 5 min and PP2 for 30 min, followed by either amlodipine alone for 5 min or PP2 alone for 35 min. After treatment, cells were harvested in radioimmunoprecipitation assay buffer (Cell Signaling Technology, Beverly, MA, USA) with a protease inhibitor cocktail (Thermo Fisher Scientific, Rockfield, IL, USA) and a phosphatase inhibitor cocktail (Thermo Fisher Scientific). Lysates were centrifuged at 20,000 × g for 15 min at 4°C, and the protein content of the supernatant was quantified using a bicinchoninic acid protein assay reagent kit (Thermo Fisher Scientific). After boiling for 10 minutes, protein samples were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). The membranes were incubated at 25°C for 60 minutes at 0.The cells were blocked in 5% skim milk in Tris-buffered saline containing 5% Tween-20 (TBST), and then treated with primary antibodies (anti-phospho-eNOS at Ser1177 [1:1000], anti-phospho-eNOS at Thr495 [1:1000], anti-eNOS [1:1000], anti-phospho-Src-kinase at Tyr416 [1:1000], anti-Src-kinase [1:1000], anti-phospho-caveolin-1 at Tyr14 [1:1000], anti-caveolin-1 [1:2000], and anti-β actin [1:10,000]) overnight at 4°C. After treatment, the membrane was washed three times with TBST for 10 min each and then treated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG diluted 1:5000 at 25°C for 60 min. Signal transmission of protein bands was analyzed by Western bright. TM The samples were stained with an ECL Western Blot Detection Kit (Advansta, Menlo Park, CA, USA) and imaged using a ChemiDocTM Touch Imaging System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Proteins were quantified using ImageJ software (version 1.45s, National Institutes of Health, Bethesda, MD, USA).

[0055]

[0056] 1.6. Measurement of intracellular calcium

[0057] Intracellular calcium [Ca] after amlodipine treatment was measured using a confocal laser microscope (IX70 Fluoview, Olympus, Tokyo, Japan). 2+ ] iLevels were measured. HUVECs were seeded and cultured on confocal cell culture dishes (SPL, Pocheon, Korea). Cells were incubated with Hanks Balanced Salt Solution medium containing Fluo-4 AM (2.5 μM, Invitrogen, Waltham, MA, USA) for 30 min, washed twice with phosphate-buffered saline solution, and then treated with amlodipine (10 -6 M) and intracellular calcium was measured. HUVECs were pretreated with 1% Intralipid or L-NAME and then treated with amlodipine (10 -6 M), 1% lipid emulsion, or L-NAME alone. Calcium levels were measured every 2.5 seconds at excitation and emission wavelengths of 485 and 520 nm, respectively. Intracellular calcium was analyzed using images expressed with Fluo-4 AM. Intracellular calcium was calculated by dividing the fluorescence intensity before drug treatment (F) by the baseline fluorescence intensity (F0). The net change in calcium ion was expressed as (Fmax-F0) / F0, where Fmax is the maximum calcium level of fluorescence intensity after treatment with amlodipine, lipid emulsion, and L-NAME alone or in combination. Intracellular calcium was measured for approximately 6 minutes.

[0058]

[0059] 1.7. Materials

[0060] All high-purity chemicals were commercially available. Intralipid (20%) was purchased from Fresenius Kabi (Uppsala, Sweden). Amlodipine, L-NAME, methylene blue, ODQ, phenylephrine, acetylcholine, linolenic acid, calmidazolium, and anti-β-actin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Amlodipine, calmidazolium, and ODQ were dissolved in DMSO (final DMSO concentration: 0.1%). Anti-Src kinase, anti-phospho-Src kinase, anti-phospho-eNOS (Ser 1177 and Thr 495), anti-caveolin-1, and anti-phospho-caveolin-1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-eNOS antibody was purchased from BD Bioscience (Franklin, NJ, USA).

[0061]

[0062] 1.8. Data Analysis

[0063] The primary outcome of the study was the effect of lipid emulsions, inhibitors, and linolenic acid (alone or in combination) on amlodipine-induced vasodilation in isolated rat aortas. This primary outcome was analyzed using a linear mixed-effects model (Stata version 14.2, StataCorp LLC, Lakeway Drive, College Station, TX, USA). The effects of lipid emulsions, inhibitors, and amlodipine (alone or in combination) on eNOS, Src-kinase, and caveolin-1 phosphorylation in HUVECs and on cGMP formation in isolated endothelial-exfoliated aortas were analyzed using a one-way ANOVA followed by Bonferroni's multiple comparison test (Prism 5.0, GraphPad Software, Inc., San Diego, CA, USA). The effects of lipid emulsions, L-NAME, and amlodipine (alone or in combination) on intracellular calcium levels in HUVECs were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison test.

[0064] The difference in area (min %) under the curve of phenylephrine-induced contraction (%) time (30-80 min) in endothelially damaged aortas with and without L-NAME treatment between the groups treated with 1% lipid emulsion post-treatment and those without was used to determine whether it was statistically significant, and for this purpose, an unpaired Student's t-test was used. p<0.05 was considered statistically significant.

[0065]

[0066] 2. Results

[0067] 2.1. Endothelial cell removal, N ω Effects of -nitro-L-arginine methyl ester, methylene blue, 1H-[1,2,4]oxadiazolo[4,3-a]quinosarin-1-one, calmidazolium, lipid emulsions, and linolenic acid or their combinations on amlodipine-induced vasodilation in isolated rat aorta.

[0068] Amlodipine (3 x 10 -7 M)-induced vasodilation was greater in endothelium-preserved aortas than in deendothelium-deprived aortas (Fig. 2, p<0.001 for 10-80 min). Dimethyl sulfoxide (DMSO, 0.1%), the same concentration used to dissolve amlodipine, did not affect phenylephrine-induced contraction in endothelium-preserved aortas (Fig. 3). In endothelium-preserved aortas, N, an inhibitor of nitric oxide synthase (NOS), was significantly increased. ω -Nitro-L-arginine methyl ester (L-NAME, 10 -4 M), nonspecific GC inhibitor methylene blue (10 -6 M), nitric oxide-sensitive GC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinosarin-1-one (ODQ, 10 -5 M), and the calmodulin-regulating enzyme inhibitor calmidazolium (3 x 10 -5 M) is amlodipine (3 x 10 -7M) inhibited vascular dilation induced by amlodipine (Fig. 4, p< 0.01 at 10 min and p< 0.001 at 20-80 min vs control). In endothelial-sparing aortas, lipid emulsion posttreatment inhibited amlodipine (3 x 10 -7 M) inhibited amlodipine-induced vasodilation (Fig. 5A, p < 0.001 vs control at 40-80 min). However, initially, it transiently increased amlodipine-induced vasodilation (Fig. 5A, p < 0.001 vs control at 10 min). Furthermore, the lipid emulsion inhibited amlodipine (3 × 10 -7 M) inhibited vasodilation by L-NAME (Fig. 5B, p < 0.05 at 10 min and p < 0.001 at 20-80 min vs. control). However, the difference in the area under the curve (minimum %) of phenylephrine-induced contraction (%) time (30-80 min) between the lipid emulsion-treated and -untreated groups in the endothelium-preserved aorta was greater in the absence of L-NAME treatment (Fig. 5C, p = 0.010). In the endothelium-preserved aorta, linolenic acid (3 x 10 -6 and 10 -5 M) is amlodipine (3 x 10 -7 Inhibited vasodilation by M) (Fig. 6A, 10 -5 M at 10-80 min p< 0.01; 3 x 10 -6 M at 30-80 min (p<0.05). However, in the deendothelialized aorta, linolenic acid (3 x 10 -6 and 10 -5 M) did not affect vasodilation (Fig. 6B).

[0069]

[0070] 2.2 Lipid emulsion on amlodipine-induced cyclic guanosine monophosphate formation in isolated endothelium-preserved rat aorta, N ω -Effects of nitro-L-arginine methyl ester and linolenic acid alone or in combination

[0071] Amlodipine (3 × 10 -7 M) increased cyclic guanosine monophosphate (cGMP) formation (Fig. 7A, p<0.01 vs control). However, lipid emulsion (1%) or L-NAME (10 -4 M) is amlodipine (3 x 10 -7 M) inhibited cGMP formation induced by amlodipine (Fig. 7A and B, p < 0.01 vs amlodipine alone). In addition, linolenic acid (10 -5 M) is amlodipine (3 x 10 -7 M) inhibited the formation of cGMP (Fig. 7C, p<0.05 vs amlodipine). However, lipid emulsion (1%) inhibited L-NAME (10 -4 M) pretreated with amlodipine (3 x 10 -7 M) did not affect the formation of induced cGMP (Fig. 7B).

[0072]

[0073] 2.3. Effects of lipid emulsions and PP2 on amlodipine-induced phosphorylation of endothelial nitric oxide synthase, caveolin-1, and Src-kinase in human umbilical vein endothelial cells.

[0074] Amlodipine (3 x 10 -7 M) increased stimulatory eNOS (Ser1177) phosphorylation in human umbilical vein endothelial cells (HUVECs) (Fig. 8A, p< 0.05 vs control) and decreased inhibitory eNOS (Thr 495) phosphorylation (Fig. 8B, p< 0.001 vs control). However, lipid emulsion (1%) had a significant effect on amlodipine (3 x 10 -7 M) reversed the increased stimulatory eNOS (Ser1177) phosphorylation and decreased inhibitory eNOS (Thr495) phosphorylation induced by amlodipine alone (Fig. 8A,B, p<0.001 vs amlodipine alone). In addition, the Src-kinase inhibitor PP2 (2 x 10 -5 M) is amlodipine (3 x 10 -7M) attenuated the stimulatory eNOS (Ser1177) phosphorylation induced by amlodipine alone (Fig. 8A, p<0.01 vs amlodipine alone). In addition, amlodipine (3 x 10 -7 M) induced caveolin-1 (Tyr14) phosphorylation in HUVECs (Fig. 9A, p<0.01 vs control). However, the Src-kinase inhibitor PP2 induced amlodipine (3 x 10 -7 M) inhibited caveolin-1 (Tyr14) phosphorylation induced by amlodipine (Fig. 9A, p<0.001 vs amlodipine alone). Amlodipine (3 x 10 -7 M) also induced Src-kinase (Tyr 416) phosphorylation in HUVECs (Fig. 9B, p<0.001 vs control). However, PP2 induced amlodipine (3 × 10 -7 Src-kinase (Tyr416) ​​phosphorylation induced by M) was inhibited (Fig. 9B, p< 0.001 vs. amlodipine alone).

[0075]

[0076] 2.4. Lipid emulsion and N on amlodipine-induced intracellular calcium levels in human umbilical vein endothelial cells W -Effects of nitro-L-arginine methyl ester

[0077] Amlodipine (10 6 M) increased intracellular calcium in HUVECs (Fig. 10, p<0.001 vs control). However, lipid emulsion (1%) and L-NAME (10 -4 M) is amlodipine (10 -6 M) inhibited the increase in calcium levels (Fig. 10, p< 0.001 vs amlodipine alone).

Claims

1. A pharmaceutical composition for inhibiting vasodilation containing a lipid emulsion.

2. A pharmaceutical composition for inhibiting vasodilation according to claim 1, wherein the vasodilation is amlodipine-induced vasodilation.

3. A pharmaceutical composition for inhibiting vasodilation according to claim 1, wherein the lipid emulsion inhibits vasodilation by inhibiting nitric oxide production.

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