Preparation Of Enalapril Buccal Mucosa Delivery System And Its Treatment On Spontaneously Hypertensive Rats

BackgroundIn recent years, common complications of hypertension and cardiovascular has increased steadily, causing serious adverse effects to the patients’quality of life. Hypertension is a long-term, chronic persistent disease, and the high blood pressure will be long-lasting damage to the endangium and smooth muscle reducing its systolic and diastolic function, as time passes, vasodilatation along with increased cardiac preload and afterload cause myocardial injury, vascular remodeling and arteriosclerosis. Hypertension bears the brunt of the heart. As mentioned earlier, left ventricular hypertrophy and myocardial pathological hypertrophy is mainly caused by hypertension. Therefore, how to effectively protect the cardiovascular system through antihypertensive therapy has important significance.Enalapril, as angiotensin-converting enzyme inhibitor (ACEI), has obvious antihypertensive effect, not only can diastole blood vessels effectively, but antagonize the action of aldosterone, namely conserving sodium and secreting potassium, reducing water and sodium retention; in addition, it also has a certain inhibitive effects on renin-angiotensin system, leading vasodilation of kidney, blood vessel wall and intracerebral vessels and collaboratively lowering blood pressure, thus, reducing the complications of heart, kidneys, brain and other vital target organs. However, it has low bioavailability for oral administration, so how to improve the bioavailability is one of the hotspots in clinical research, and will also have a positive role on improving drug effects. Therefore, this study aims to apply buccal mucosa administration in order to explore its feasibility through non-gastrointestinal mucosa administration. Theoretically, buccal mucosal blood vessels has intensive vascular, rich blood and fast drug absorption without the first-pass effect of the liver; moreover, buccal mucosal, with less enzyme content, has higher permeability rank only second in vivo to nasal mucosa and gastrointestinal mucous membrane, thus, as compared with other ways of mucosal administration, buccal mucosa delivery can be effectively up to a certain blood concentration in a short time with small fluctuation and stable,long-lasting efficacy; moreover, mucous membrane preparation medication has strong patient compliance for its conveniency.In order to investigate the effectiveness of mucous membrane preparation, this study selected first-line antihypertensive drugs (enalapril) to prepare buccal mucosa delivery system and to explore its bioavailability, clinical efficacy, and patients’ compliance. In vivo experimental, this study used spontaneously hypertensive rats as animal model, through treatment by common enalapril dosage preparation and enalapril oral buccal mucosa dosage preparation respectively, several indicators such as blood pressure, heart function, expression levels of reversing left ventricular remodeling, cardiac ACE, ACE2mRNA and its protein, serum aldosterone, angiotensin, nitric oxide, endothelin were observed and analyzed statistically to explore its mechanisms for the treatment of hypertension and provide new pharmaceutical dosage form, improve the therapeutic effect while reducing target organs’ damage, providing a new approach for clinical treatment.Objective1. In order to improve the therapeutic effects and patient compliance, enalapril buccal mucosa delivery system was prepared and characterized, and compared the biological effects with common enalapril to overcome the problem of low bioavailability;2. To observe the effects of enalapril and enalapril buccal mucosa delivery system on blood pressure, heart function, reversion of left ventricular remodeling in spontaneously hypertensive rats, and to explore the related mechanism.3. To observe the effects of enalapril buccal mucosa delivery system on the expression of ACE, ACE2 in heart of spontaneously hypertensive rats as well as the expression of serum aldosterone, angiotensin, nitric oxide and endothelin.Methods1. Preparation of enalapril buccal mucosa delivery systemThrough application of mucous membrane adhesive polymer and solvent coating technology, enalapril buccal mucosa delivery system was prepared into the form of buccal mucoadhesive films.2. Detection of SHR histological analysis,blood pressure and cardiac remodeling1. Groups of Experiment:30 SD rats were prepared into spontaneous hypertension model and numbered from 1-30.1) WKY control group:10 WKY rats were gavaged 5ml/kg dose of normal saline, qd.2) SHR control group:10 SHR rats were gavaged 5ml/kg dose of normal saline, qd.3) Common enalapril dosage group: 10 SHR rats were gavaged by 10mg/kg of enalapril dissolved in 5ml/kg distilled water, qd.4) Oral mucosal dosage group:10 SHR rats were given lOmg/kg dose of buccal mucoadhesive films, qd. The course of treatment for each group was 2 weeks.2. Blood pressure:The caudal artery in each group were detected, AMP, LVEDP, LVSP,+dp/dtmax and-dp/dtmax and other aspects of cardiac function data were recorded.3. Ventricular remodeling and histological analysis:Collagen content in left ventricular mass and cardiac tissue were detected, moreover, the change of pathological structure and myocardial ultrastructure in rat myocardial cells were observed by light microscopy and electron microscopy.3. The expressions of mRNA and protein of ACE and ACE21. Groups of Experiment:It is the same to experimental method with the second part.2. The expressions of mRNA of ACE and ACE2 in cardiac muscle tissue by agarose gel electrophoresis.3. The expressions of ACE and ACE2 protein:myocardial tissue samples were detected using immunohistochemistry and Western Blot4. Detection of ALD、AngⅡ, endothelin and nitric oxide1. Groups of Experiment:It is the same to experimental method with the second part.2. The radioimmunoassay was applied to detect the serum aldosterone, angiotensin and endothelin expression levels in each group.3. Nitrate reduction method was used to detect the expression levels of serum NO.Results1. The formula of buccal mucoadhesive films F2 and F4 showed good pharmacokinetic propertiesAll of the formulas showed a high drug percentage (96.45-98.49%). formula with good expansion characteristics displayed favorable dwell time. It was found in vitro that high viscosity carboxy methyl cellulose sodium (SCMC-HV) including mucoadhesive films (F2) showed the highest rate of drug release (fast drug release, 92.24% within 1.5 hours), followed by mucoadhesive films F4 (containing polyvinylpyrrolidone K-901% w/v, SCMC (HV) 1% w/v). The study found that by the end of 10 hours, drug penetration rate of F2 and F4 in vitro were 82.24% and 89.9% respectively.2. The effects of enalapril buccal mucosa delivery system on myocardial cells, blood pressure, collagen content and the effect of myocardial cell pathological damage under the light microscope and electron microscope:1) Observation of myocardial cells under the light microscope:WKY control group:The damage of myocardial cells in SHR control group was more serious than WKY control group. SHR control group:Under light microscope, normal muscle fibers of myocardial cell was losing arrangment with diameter thickening, visible of fracture phenomenon and interstitial edema; minority of myocardial interstitial saw collagen fibers deposition; there were large number of inflammatory cell infiltration, myocardial cells atrophy and partial dissolution or necrosis around small blood vessels. Common enalapril dosage group:The myocardial pathology changes in common enalapril group were significantly improved than those in the other two control groups. Under light microscope, the myocardial fibers in common enalapril group became slightly thicker with neat arrangement, and the bundle of muscle fiber slightly widen, the intact nuclei showed mostly single one at the center of the muscle fibers; there were some inflammatory cell infiltration with a small amount of imcomplete non-nuclear membrane. Oral mucosal dosage group:the oral mucosal dosage group changed slightly compared with common enalapril group, basically tend to normal.2) Myocardial ultrastructure observation:WKY group:myofibrils showed neat, compact, no fracture, large mitochondrial volume, huge quantity, tightly packed cristae in rich, large volume of sarcoplasmic reticulum, a small amount of interstitial collagen fibers attached to mesenchyme presenting majority of fine fibers.SHR control group:myocardial cell swelling significantly, myocardial fibers disarray, a large number of collagen fibers present in the stromal cells, dissolution of part myofibrillar with visible vacuole of varying sizes, especially at both sides of intercalated disc. Myofibril saw sarcomere misalignment and excessive contraction, slightly dilation of sarcoplasmic reticulum, or even expand into large cavities, swelling fracture with the number reducing, thick and thin collagen fibers in the mesenchyme interwoven net-like and interspersed. Mitochondrial showed swelling, cristae partially broken and dissolved in vague and formed vacuoles of varying sizes zones in mitochondria, and even dissolved. The light and dark zone was unclear with Z-wire twisted, distorted, broken filaments, dissolving to form a large number of muscle soluble focal. Common enalapril dosage groups:mild swelling of myocardial cells, myocardial fibers showed lattice-like arrangement in neat dense, the collagen fibers were more distributed in intercellular matrix. There were more myocardial mitochondrial swelling showing convergence with more chaotic arrangement and crest fracture. Oral mucosal dosage groups:myocardial fibers arranged in neat, little collagen fibers in stromal cells, most of the nucleus showed intact and clear while few nuclear membranes is not complete. Mitochondrial morphology showed normal and oval, the mitochondrial cristae were arranged regularly, there were some fracture and fragments formed in mitochondria. Mitochondrial volume decreased with near normal number.3) Myocardial cell pathological damage:Compared with myocardial biopsy of WKY control group, myocardial cell of SHR control group showed significant injury; compared with SHR control group, rat cardiac pathology in common enalapril group changed slightly, and cardiac pathology change in oral mucosal dosage group were even smaller compared with common enalapril dosage group; compared with WKY group, electron microscopy showed that the ultrastructure of myocardium in SHR control group were in serious disorder, ultrastructure of myocardium in oral mucosal dosage group and enalapril group were improved compared with that in SHR control group, and the improvement even more significant in in oral mucosal dosage group;4) In the aspects of blood pressure index:AMP, LVEDP, LVSP,+dp/dtmax,-dp /dtmax and other cardiac function parameters of SHR rats in the control group were significantly worse than those of WKY control group (P<0.05), the difference was not statistically significant,after drug treatment, the cardiac function parameters in common enalapril dosage group and oral mucosal dosage group were significantly improved (P<0.05), and the cardiac function parameters in oral mucosal dosage group were significantly better than that in common enalapril dosage group (P<0.05);5) Cardiac remodeling:In the aspects of myocardial tissue weight:there was no significant difference among each group in terms of LVM, LVMI (P>0.05). In the aspects of collagen content:in SHR control group were significantly higher than those in WKY control group (P<0.05), after drug treatment, collagen content in common enalapril dosage group and oral mucosal dosage group were significantly reduced (P<0.05), and the oral mucosal dosage group was significantly superior than common enalapril dosage group in terms of collagen content reductions (P<0.05).3. The effects of enalapril buccal mucosa delivery system on ACE and ACE21) The expressions of mRNA of ACE and ACE2 mRNA:The expression levels of cardiac ACE in SHR control group were higher than those in WKY control group (P<0.05), the difference was statistically significant,while cardiac ACE2 mRNA expression level was lower than WKY control group (P<0.05), the difference was statistically significant, cardiac ACE mRNA, ACE2 mRNA expression levels in common enalapril dosage group and oral mucosal dosage group were decreased and increased respectively after drug treatment (P<0.05), the difference was statistically significant,,and the degree of cardiac ACE mRNA, ACE2 mRNA expression levels in mucosal dosage group were more significant than those in common enalapril dosage group (P<0.05);2) The expressions of ACE and ACE2:The heart ACE protein expression levels in SHR control group was higher than that in WKY control group (P<0.05), the difference was statistically significant,while the cardiac ACE2 protein levels were lower than that in WKY control group (P<0.05), the difference was statistically significant,the cardiac ACE, ACE2 protein expression levels in common enalapril dosage group and oral mucosal dosage group after drug treatment were decreased and increased (P<0.05), the difference was statistically significant, and the degree of lower or higher levels of cardiac ACE, ACE2 protein expression in mucosal dosage group were more significant than those in common enalapril dosage group (P<0.05); cardiac ACE expression levels in SHR control group was higher than that in WKY control group (P< 0.05), the difference was statistically significant,while cardiac ACE2 expression levels were lower than that in WKY control group (P<0.05), the difference was statistically significant,the cardiac ACE, ACE2 expression levels in common enalapril dosage group and oral mucosal dosage group after drug treatment were reduced and increased (P<0.05), the difference was statistically significant,moreover, the degree of lower or higher levels of cardiac ACE, ACE2 expression in mucosal dosage group were more significant than those in common enalapril dosage group (P<0.05).4. The effects of enalapril buccal mucosa delivery system on ALD、AngⅡ、ET、 NO1) ALD、AngⅡ:In terms of Ang II, aldosterone expression levels, the degree of expression levels from high to low were SHR control group, WKY control group, common enalapril dosage group and oral mucosal dosage group in order.2) ET:In terms of endothelin expression level, the degree of expression levels from high to low were SHR control group, WKY control group, common enalapril dosage group and oral mucosal dosage group in order.3) NO:In terms of serum NO expression level, the degree of expression levels from high to low were SHR control group, WKY control group, common enalapril dosage group. Cell damage in both treatment groups than in the control group improved (P<0.05).Conclusions1. Preparation of enalapril buccal mucosa delivery system, formula F4 has a feature of quick release, rapid absorption and the longest residence time in vitro, which has provided potential bases for oral mucosa absorption formulations in future research and development.2. Enalapril buccal mucosa delivery system showed obvious reverse of left ventricular remodeling with significant antihypertensive effect compared with common enalapril dosage.3. The inhibitive effects of enalapril buccal mucosa delivery system on ACE2 expression and enhancing effects on ACE2 in heart of SHR were more significant compared with common enalapril dosage.4. The inhibitive effects of enalapril buccal mucosa delivery system on serum aldosterone, angiotensin, nitric oxide and endothelin expression in spontaneously hypertensive rats were more significant compared with common enalapril dosage.