1. Introduction
Hypertension is affecting one billion people and is the most common modifiable risk factor for cardiovascular disease (CVD) [
1,
2]. Hyperlipidemia, characterized by abnormal blood lipid levels, is also recognized as one of the important risk factors of CVD [
3,
4]. Hypertension and hyperlipidemia frequently coexist and are associated with elevated cardiovascular adverse outcomes [
5]. Globally, CVD is the primary cause of mortality [
6]. It is estimated that 7.8 million people will die prematurely from CVD by 2025 [
7]. Despite their increasing prevalence, the proportions of hypertension and hyperlipidemia awareness, treatment, and control are low [
8,
9]. The trial has demonstrated that lowering lipid levels and blood pressure decreases the incidence of major cardiovascular events more effectively than regulating hypertension or hypercholesterolemia separately [
10]. Therefore, significant joint control of blood pressure and blood lipids is needed for reducing the mortality rate of CVD.
Captopril (CAP), an orally active angiotensin-converting enzyme inhibitor (ACEI), is considered the preferred drug for antihypertensive treatment due to its effectiveness and low toxicity. Simvastatin (SIM) is a hydroxymethyl glutarate coenzyme A (HMG-CoA) reductase inhibitor that reduces total cholesterol and low-density lipoprotein cholesterol, thereby reducing the risk of atherosclerosis and CVD [
11]. CAP and SIM have profitable effects beyond blood pressure and lipid lowering, and when combined, they achieve greater improvements in outcomes than those associated with either monotherapies [
12]. However, when administered orally, CAP undergoes first-pass hepatic metabolism [
13,
14], its bioavailability is reduced to 55% in the presence of food, and its half-life is less than 2 h [
15]. SIM also has a significant liver first-pass effect, low bioavailability of only 5%, and a short half-life of 2 h [
16]. So, oral SIM and CAP both have common shortcomings, including significant liver first-pass effects, low bioavailability, and a short half-life.
The transdermal drug delivery system (TDDS) is one of the systems under the controlled drug delivery category that aims to deliver drugs through the skin at a predetermined and controlled rate [
17]. The TDDS has undergone the development of three generations [
18]. The first generation includes ointments, creams, sprays, gels, or patches [
18]. The second generation is involved in the use of different enhancements, including chemical enhancement, iontophoresis, and non-cavitational ultrasound [
19,
20]. The third generation correlates with novel chemical enhancers, electroporation, microdermabrasion, thermal ablation, cavitational ultrasound, and microneedle-based delivery [
20,
21]. Based on the above statements, the three generations of the TDDS all apply transdermal enhancer technology, known as physical enhancers or chemical enhancers. However, most physical enhancers rely on relatively costly, reusable devices [
20], and some physical enhancers, such as microneedles, have physical invasiveness, which raises additional safety and sterility concerns [
20]. Various chemical enhancers can be integrated into small, inexpensive patches that patients find convenient [
20]. Thus, chemical enhancers may have some advantage in the transdermal drug delivery system.
In 1979, the US Food and Drug Administration (FDA) approved the first transdermal patch (scopolamine transdermal patch) to prevent motion sickness [
22,
23]. Since then, many transdermal patches have been approved for the market, including Estradiol; Testosterone; Clonidine; Fentanyl; Nitroglycerin; Nicotine transdermal patches; and so on [
23]. The clonidine TDDS is used for the treatment of hypertension [
24]; it can improve compliance and patient acceptance in antihypertensive treatment, which may enhance the clinical treatment advantages [
25]. The nitroglycerin transdermal patch is used clinically to treat angina pectoris [
24,
26], and a 10 mg nitroglycerin transdermal patch has a significant therapeutic effect and can last for 24 h [
27]. Transdermal administration provides a leading advantage over injection and oral routes by improving patient compliance, avoiding first-pass metabolism, avoiding gastrointestinal irritation, and achieving sustained release of the drug [
17,
28]. Thus, preparing SIM and CAP TDDSs may solve the problems caused by the oral administration of SIM and CAP.
More than 64% of hypertensive patients also have dyslipidemia, and approximately 47% of patients with dyslipidemia suffer from hypertension [
29]. The frequent coexistence of hypertension and hyperlipidemia increases in cardiovascular disease events [
29]. Thus, simultaneous control of blood pressure and blood lipids is of great significance for reducing cardiovascular disease events. CAP and SIM are antihypertensive drugs and lipid-lowering drugs, respectively. However, when they are orally administered, they often show some drawbacks, whereas compared with oral administration, transdermal administration shows many advantages. So, the objective of our study is to develop an SIM and CAP combined transdermal delivery system. It has important significance for simultaneously controlling blood pressure and blood lipids, as well as reducing the risk of cardiovascular disease.
2. Materials and Methods
2.1. Materials
SIM (S129538) and EC (E809017, 18–22 mPa·s) were purchased from Shanghai Macklin Biochemical Technology Co., Ltd. (Shanghai, China). CAP (C837213) and carboxymethyl cellulose sodium (CMC-Na, S14016, 300–800 mPa·s), hydroxypropyl methylcellulose (HPMC, S14174, 4000–6500 mPa·s, HPMC K4m; 30,000 mPa·s, HPMC K30m; 100,000 mPa·s, HPMC K100m), polyacrylic acid resin II (S30569), and hydroxyethyl cellulose (HC, YY11927, 38,000–42,000 mPa·s) were purchased from Shanghai Yuan-ye Biotechnology Co., Ltd. (Shanghai, China). Azone, oleic acid (OA), polyvinylpyrrolidone (PVP K30, C20201, 3.4 mPa·s), and polyvinyl alcohol (PVA, D08019,44.0–54.0 mPa·s) were purchased from Tianjin Damao Chemical Reagent Co., Ltd. (Tianjin, China). Methanol and anhydrous ethanol were purchased from Tianjin Fengchuan Chemical Reagent Co., Ltd. (Tianjin, China). Acetonitrile was purchased from Tianjin Concord Technology Co., Ltd. (Tianjin, China). Phosphoric acid was purchased from Tianjin Huihang Chemical Technology Co., Ltd. (Tianjin, China). Acetonitrile and methanol were of high-performance liquid chromatography (HPLC) grade, while other reagents were of analytical grade.
2.2. Animals
Female Kunming mice (6 weeks old, weighing 16–18 g) and female Chinese rabbits (6 months old, weighing 2.0–2.5 kg) were purchased from Beijing SPF Biotechnology Co., Ltd. (Beijing, China). Kunming mice were kept in separate cages under standard laboratory conditions (12 h light/dark cycle, ~25 °C) and allowed to eat and drink freely. Chinese rabbits were kept in separate cages and fed a standard diet. Animal care and experimental protocols followed the institutional guidelines for the health and care of experimental animals. This research plan was approved by the Animal Experiment Ethics Committee of Nankai University in Tianjin, China.
2.3. Calibration Curve of SIM-CAP Compound
We used a CoM 6000 HPLC system (Albany, New York, NY, USA) with a Comatex C18-AB column (250 × 4.6 mm, 5 µm; 115 Corp, Plainfield, IL, USA) to measure the CAP and SIM concentrations in the samples. The wavelength was 230 nm. The mobile phase consisted of acetonitrile, water, and phosphoric acid (v/v/v, 300:100:0.1) at a 1.0 mL/min flow rate at 35 °C. All mobile phases were degassed; the sample volume was maintained at 20 µL. The SIM-CAP complex stock solution was prepared with five concentration gradients of 50, 100, 200, 300, and 500 µg/mL. The peak area values corresponding to different concentrations were determined according to the chromatographic conditions mentioned earlier. Linear analysis was performed with concentration as the x-axis (X) and peak area values as the y-axis (Y) to obtain a standard curve.
Figure 1 shows high-performance liquid chromatograms of simvastatin and captopril with injecting 200 µg/mL under the chromatographic conditions shown in
Section 2.3. The retention times of CAP and SIM are 2.6 min and 11.8 min.
Table 1 shows the peak areas corresponding to different concentrations of SIM-CAP compound. The standard curve was obtained with concentration as the x-axis (X) and peak area values as the y-axis (Y). The standard curve equation for SIM-CAP compound is Y = 45,307X − 1,695,273, with a correlation coefficient R
2 = 0.9956 (
Figure 2). This indicates a good linear relationship between the concentration of SIM-CAP compound and peak area within the concentration range of 50–500 µg/mL.
2.4. Preparation of Mice Abdominal Skin
After experimented Kunming mice were anesthetized, they were euthanized using the cervical dislocation method. First, we removed their abdominal hair with hair removal cream. Then, we cut off approximately 2 × 2 cm of abdominal skin with scissors and checked the integrity of the skin. After repeatedly rinsing the skin with physiological saline, we placed it flat in a culture dish moistened with physiological saline and stored it at −20 °C for later use. We thawed the material naturally at 25 °C before the formal experiments began.
2.5. Optimization of Transdermal Systems
Preliminary screening of the matrix composition of the SIM-CAP TDDS used the L
25 (5
5) orthogonal experimental table. The experiment used the primary skeleton material, secondary skeleton material, and enhancer as the three factors of the orthogonal experiment, with five different types of the three factors at the level of the orthogonal experiment (
Table 2). A Franz TP-6 transdermal diffusion instrument (Tianjin Jingtuo Instrument Technology Co., Ltd., Tianjin, China) was used to measure the drug release rate and determine the preliminary formula. Then, we used in vitro permeation experiments to screen different proportions of penetration enhancers and polyacrylic acid resin II (
Table 3) to determine the optimal formulation.
2.6. In Vitro Release Studies
We used the TP-6 transdermal diffusion meter to conduct drug release studies. Six TP-6 transdermal diffusion cells can be placed in the Franz transdermal diffusion meter. The effective diffusion area of TP-6 transdermal diffusion cells is 3.14 cm2. The excised skin of Kunming mice was firmly fixed between the donor and receptor compartments. Each receptor chamber was filled with 15 mL of saline solution, maintained at a temperature of 37 ± 0.5 °C, and stirred with a rotor to ensure uniform distribution. Sample aliquots (0.5 mL) were extracted from the receptor compartment at different time intervals (4, 8, 10, and 24 h) and replaced with an equal volume of saline solution. The samples were first filtered using a 0.22 µm pinhole filter (Scienhome Scientific Technology Development Co., Ltd., Tianjin, China) and then injected into HPLC instrument.
2.7. Preparation of TDDS
Figure 3 shows the preparation flowchart of the SIM-CAP combined transdermal drug delivery system. We used a solvent evaporation technique to prepare drug-containing transdermal systems according to the optimized formulation. PVA was dissolved in water, while the remaining components were dissolved in anhydrous ethanol. SIM and CAP are dissolved to form the drug-containing solution. OA and azone are dissolved to form the permeation-promoting solution. PVA and polyacrylic acid resin II are dissolved to form the pressure-sensitive liquid. They were mixed and magnetically stirred (60 °C, 30×
g) for 2 h to obtain the medicated gel solution. The evenly mixed solution was degassed via the use of ultrasound at room temperature for 30 min. Then, the mixed solution was dropped onto an anti-adhesion layer and dried at 60 °C for 120 min. After drying, release paper was attached to obtain transdermal patches. The prepared patches were placed in a sealed environment at 25 °C for the convenience of subsequent experimental research.
2.8. Scanning Electron Microscopy Analysis
A scanning electron microscope (QUANTA 200, FEI Company, Hillsboro, OR, USA) was used to analyze the external morphology of the TDDS.
2.9. Stability of the Prepared TDDS
For the high-temperature, cold resistance, and freeze–thaw experiment, the SIM-CAP TDDSs were placed at 50 ± 5 °C for seven days, 2–8 °C for seven days, and alternately in a freezer at −20 °C and in an oven at 37 ± 0.5 °C for eight days. The patches’ appearances and ductility were examined. Each process was repeated three times.
2.10. Stickiness of the Prepared TDDS
The methods for evaluating the adhesion of the SIM-CAP TDDS include peel strength testing; shear strength testing; and hand adhesion experiment. We referred to the previous experimental procedure in our laboratory for evaluating peel strength, shear strength, and hand adhesion [
30]. The time to completely peel off the SIM-CAP TDDS represented the peel strength. The time for the complete detachment of the TDDS was the shear strength. The displacement of stainless steel after contact with the patch indicated hand adhesion.
2.11. Drug Content in the Prepared TDDS
In total, 1 cm
2 of SIM-CAP transdermal patches was dissolved in 10 mL of ethanol, treated with ultrasound for 8 h, centrifuged at 12,000 rpm for 10 min, filtered, and analyzed by HPLC (the method is shown in
Section 2.3). This process was repeated six times.
2.12. In Vivo Pharmacokinetic Studies
The rabbits were divided into three groups: group A received blank patches, group B received oral gavage, and group C received the optimized SIM-CAP TDDS. The abdominal hair of the rabbits in groups A and C was shaved off using an electric hair removal device, and the skin was washed with physiological saline. The optimized TDDS was applied to the abdomen of group C rabbits, and the blank patch was applied to the abdomen of group A rabbits. The oral gavage (group B) process includes fixing the rabbits, placing a specially designed mouth expander into its mouth, and securing it to its mouth with a rope. We inserted an elastic rubber catheter through the small circular hole on the expander and entered the esophagus along the posterior pharyngeal wall. Then, a solution was added with the same concentration as the TDDS. After gastric perfusion, first the catheter was removed and then the mouth opener was removed. We collected 1 mL aliquots of blood from the ear vein of rabbits of different groups at different time intervals (1, 2, 4, 7, 12, and 24 h), placed them in heparinized tubes, and centrifuged them at 1500× g for 10 min. The blood serum was obtained and stored at −70 °C. We mixed 600 µL of blood serum with 900 µL of acetonitrile in a 1.5 mL polyethylene centrifuge tube, centrifuged it at 2500× g for 10 min, and then filtered it through a 0.22 µm membrane to obtain the sample for subsequent HPLC analysis.
2.13. Skin Irritation Studies
After 48 h, we removed the patches from the abdomen of rabbits in groups A and C and observed skin irritation condition, including erythema and edema. The scoring criteria for skin irritation referred to Literature 24 and are presented in
Table 4.
2.14. Data Analysis
Pharmacokinetic parameters include Cmax (observed maximum plasma drug concentration), Tmax (time required to reach maximum drug concentration), AUC(0–t) (area under the plasma concentration time curve from time 0 to t), and MRT (average time the drug molecule stays in the body).
We used the DAS 2.0 procedure based on statistical moment theory to analyze the pharmacokinetic parameters of group A and group C. AUC(0–t) was calculated using the trapezoidal rule. F-value (calculated relative bioavailability) referred to the ratio of AUC(0–t) of the TDDS to AUC(0–t) of oral administration. A bilateral t-test was performed for Cmax and AUC(0-t) analysis, and a non-parametric test was performed for Tmax analysis.
All results are represented as the mean ± standard deviation (Mean ± SD). One-way analysis of variance (ANOVA) and Student’s t-test were used to compare different groups. The statistical significance was set at p < 0.05.
4. Discussion
Transdermal drug delivery is a method for local or systemic treatment through the skin [
32]. Transdermal drug delivery systems (TDDSs) include the reservoir system, matrix system, and microreservoir systems [
17]. Matrix-type TDDSs are the most commonly used due to their simple design, ease of manufacturing, and good wear performance [
33]. Matrix-type TDDSs have a low risk of accidental overdose and a low likelihood of drug abuse, making it the preferred choice for patients [
34]. The SIM-CAP TDDS prepared in this study was designed as a matrix-type TDDS.
Matrix materials are important for TDDSs. Hydroxypropyl methyl cellulose (HPMC) is considered the most important hydrophilic carrier material for the preparation of controlled drug delivery systems [
35]. One of its most important features is high expansibility, which has a significant impact on the release kinetics of incorporated drugs [
36]. HPMC is widely used due to its advantages such as wide availability, ease of use, good biocompatibility, strong film-forming ability, and good biodegradability [
24]. Ethyl cellulose (EC) is a non-allergic and nontoxic polymer with excellent properties for the formation of tough films [
37]. Carboxyl methyl cellulose (CMC-Na) is an anionic derivative of carboxymethyl cellulose that exhibits good biocompatibility and stable water solubility [
38]. It has swelling properties and uniform viscosity and has been widely used in local drug formulations to achieve controlled and sustained targeted drug delivery [
39,
40]. Polyvinylpyrrolidone (PVP) is a non-ionic water-soluble linear polymer widely used in cosmetics and pharmaceuticals because of its good solubility, excellent affinity for resins and polymers, biodegradability, nontoxicity, and compatibility [
41]. Polyvinyl alcohol (PVA) has been widely used in biomedical and pharmaceutical applications due to its good biocompatibility, nontoxicity, hydrophilicity, and ability to form nanofibers and hydrogels [
42,
43]. In this study, we used an orthogonal experimental table to verify the cumulative release of the two-polymer combinations. The results showed that when both the main and secondary matrix skeleton materials were made of PVA, the cumulative drug release was higher. Therefore, PVA was selected as the matrix skeleton material for the SIM-CAP transdermal patches.
The skin, particularly the cuticle, forms an effective barrier to drug penetration, making it difficult to deliver drugs from the patches across the skin. The most widely used method for enhancing drug penetration through the cuticle employs chemical enhancers [
44]. We used two chemical penetration enhancers: OA and azone. The ability of OA to enhance skin penetration has been widely recognized in various in vitro studies [
45]. It disrupts highly stacked intercellular domain lipids in the cuticle [
46]. Azone was the first compound specifically designed as a skin penetration enhancer, and its penetration-enhancing properties reflected the molecule’s ability to reduce skin diffusion resistance [
47,
48]. Due to the limited enhancement effect of a single chemical enhancer on skin permeability, the combined use of chemical enhancers may achieve more satisfactory therapeutic effects and safety owing to their synergistic effects [
49]. This study evaluated the permeability of SIM-CAP transdermal patches using oleic acid and azone alone and in combination. The results showed that at the 16% concentration of OA/azone (1:1), the cumulative drug release was the highest and was selected as the enhancer for the SIM-CAP transdermal patches.
Pressure-sensitive adhesives (PSAs) are viscoelastic materials that can adhere to various solid surfaces under slight pressure quickly [
50]. They have the advantages of convenient use, good stability, simple manufacturing, and good appearance [
51]. Polyacrylic acid resin II is a type of polyacrylic acid resin pressure-sensitive adhesive that can adhere to biological tissues and shows excellent sustained release effects in the skin [
52]. We investigated the effect of different concentrations of polyacrylic acid resin II in SIM-CAP transdermal patches on drug release. The results showed that 2% polyacrylic acid resin II maintained the developed patch’s moderate adhesive qualities and optimized the cumulative release of SIM-CAP from the sustained-release TDDS.
The optimal formula for the SIM-CAP composite transdermal patch was determined by screening the skeletal material, penetration enhancer, and pressure-sensitive adhesive. The quality of the optimal formula was then evaluated, including quality indicators such as integrity, stability, and viscosity. SEM was used to inspect the integrity of the patch; the results showed that the surface of the prepared SIM-CAP composite transdermal patches was relatively smooth and uniform without solid particles or cracks. The SIM-CAP composite transdermal patches exhibited no significant changes in appearance and good stability under high-temperature, severe cold, and freeze–thaw conditions. The patches were also evaluated for their initial adhesion, adhesion retention, and peel strength. The results indicated that the SIM-CAP composite transdermal patch exhibited good adhesion.
We conducted pharmacokinetic analysis of oral administration and the SIM-CAP TDDS using rabbits. Compared with oral medication, the maximum concentration of the SIM-CAP TDDS was reduced, which avoids the side effects caused by peak oral administration. The Tmax of the SIM-CAP TDDS was prolonged to 4 h, showing that the speed at which drugs are absorbed into the body slows down. The extension of MRT from 5.34 h to 9.29 h indicated an increase in the duration of drug retention in the body. Compared with oral administration, the AUC(0–t) value of the SIM-CAP TDDS increased significantly, indicating that the patches had a longer duration of action and a more stable blood drug concentration. The AUC(0–t) ratio of the SIM-CAP TDDS and oral administration was compared. We found that the SIM-CAP TDDS gained a significant increase in relative bioavailability, reaching 235.16%. In summary, this pharmacokinetic parameter indicated that the SIM-CAP TDDS had a better absorption effect in vivo, longer action time, higher bioavailability, and more stable blood drug concentration and overcomes the first-pass effect of oral administration.
However, skin reactions may still occur with transdermal medications [
53]. The most common skin irritation of transdermal administration is redness erythema (redness) or edema (swelling) at the site of application [
54]. If the transdermal patch causes any skin irritation reaction, it will affect the patient’s use of the transdermal patch. Thus, evaluating skin irritation for transdermal patches is of great significance. So, we finally tested whether the transdermal patch would cause skin inflammation. The transdermal patch was applied to the abdominal skin of rabbits, and after removing the patch, redness and edema were observed. Then, we scored the redness and swelling on the skin. The results showed that the scores for erythema and edema were both less than 0.5, indicating that there were no obvious erythema and edema on the skin of the rabbits. This indicated that the SIM-CAP TDDS did not cause skin irritation.
Our study has established an SIM-CAP TDDS for simultaneously controlling blood pressure and blood lipids. Hypertension and hyperlipidemia are both recognized as important risk factors of CVD [
1,
2,
3,
4]. The coexistence of the two risk factors has more than an additive adverse impact on the vascular endothelium, which results in enhanced atherosclerosis, leading to CVD [
55]. CVD is currently the leading cause of death, accounting for one-third of deaths worldwide [
7,
56]. The SIM-CAP TDDS can slowly and continuously release SIM and CAP, increase the bioavailability of SIM and CAP, efficiently control blood pressure and blood lipids, and has great significance in reducing CVD incidence and improving human health.