Effects of borneol on pharmacokinetics and tissue distribution of tetramethylpyrazine in transdermal patch

Abstract

Tetramethylpyrazine (TMP), a potent coronary vasodilator, enhances myocardial perfusion and shows promise for treating cardiovascular disorders. Its clinical utility is limited, however, by the short half-life of conventional tablets and injections, which necessitates frequent dosing. To overcome this drawback, we developed a transdermal patch that co-loadsTMP and borneol (BO-TMP). After optimization, patches containing 3% borneol as a permeation enhancer, exhibiting the highest cumulative penetration amount of TMP (6.80 mg·cm⁻² over 24 h) in vitro. Pharmacokinetic profiling revealed that borneol markedly improved TMP exposure. C_max rose from (1.96 ± 0.27) to (2.88 ± 0.72) mg/L, and AUC_0–t expanded from (47.73 ± 6.93) to (73.18 ± 13.86) mg/L*h in plasma; C_max rose from (12.99 ± 2.28) to (15.33 ± 4.24) mg/L, and AUC_0–t expanded from (220.86 ± 53.88) to (374.55 ± 111.84) mg/L*h in skin, C_max rose from (0.87 ± 0.12) to (6.20 ± 0.84) mg/L, and AUC_0–t expanded from (13.08 ± 3.35) to (79.80 ± 16.31) mg/L*h in heart when BO-TMP patches were administrated compared with TMP patches. Tissue distribution studies demonstrated that borneol redirected TMP distribution, shifting the order of tissue abundance from liver > kidney > brain > spleen > heart > lung to liver > kidney > heart > brain > spleen > lung, and thus enriched the drug at its therapeutic site. By selectively heightening cardiac exposure, the BO-TMP patch transform TMP into a cardiovascular-focused therapy, offering a convenient, long-acting dosage from with the potential to enhance efficacy and improve patient outcomes.

Graphical abstract

Introduction

Cardiovascular disease poses a serious threat to human life and health, especially among middle-aged and elderly individuals, and is characterized by high incidence, high disability and high mortality. As medical technology continues to advance, our understanding of the disease has improved qualitatively. However, despite the application of current advanced and comprehensive treatment methods, cardiovascular disease remains the leading cause of death worldwide. Over the past 30 years, the global prevalence of cardiovascular diseases has nearly doubled, and mortality has continued to rise [1]. According to the American Heart Association, cardiovascular disease causes more than 17.3 million deaths annually, and It is estimated that by 2030, over 23.6 million people will die from condition [2]. Drugs are essential in the treatment of cardiovascular diseases. However, the short half-life of many drugs necessitates frequent dosing, which not only inconveniences patients but also leads to poor compliance. This has become a significant factor limiting the affecticeness of pharmacotherapy in cardiovascular diseases. Therefore, developing an administration method that reduce dosing frequency while enabling continuous drug release is a top priority in cardiovascular treatment. Transdermal drug delivery system effectively address the limitations of injections and tablets and hold promising for the prevention and management of cardiovascular disease.

Transdermal Drug Delivery Systems (TDDS) are widely studied platforms that adhere to the skin or mucous surface, allowing drugs to permeate the skin layers or mucous membranes at a constant (or near- constant) rate, enter the systemic circulation, and produce local or systemic therapeutic effects. Compared with traditional administration methods (such as injection and oral administration), TDDS avoids the first-pass hepatic effect and gastrointestinal degradation; It enables drug to enter the body at a constant rate over an extended period, reducing dosing frequency and prolonging the interval between administrations; It maintain a constant and effective blood concentration by delivering the drug at the required rates; It is easy to use and can be removed at any time, making it especially suitable for infants, the elderly, or patients unsuitable for oral administration; It is painless, minimally invasive, and largely bypasses systemic metabolism. These features generally result in better patient compliance compared with injection or other invasive methods [3–5]. TDDS also overcomes some drawbacks of oral administration, such as extensive first-pass metabolism and low bioavailability [6].

At present, the application of percutaneous drug delivery in the field of cardiovascular diseases also shows significant advantage. It can improve bioavailability, prolong drug residence time, and maintain stable blood drug concentration in the study of hypertension [7, 8]. In the treatment of arrhythmia, it has been shown to reduce the incidence of postoperative atrial fibrillation, and the treatment effect of the patch group was more effective than that of the oral group [9–12]. It has also been demonstrated to improve cardiac function in rats with myocardial infarction, ameliorate adverse remodeling and myocardial perfusion, and prevented perioperative myocardial injury in high-risk patients [13, 14]. These findings suggest that the patch, as a new drug delivery route besides oral and intravenous injection, has been widely used in cardiovascular diseases both domestically and internationally.

Therefore, the research group selected TMP, which has anti-platelet aggregation, arteriolas dilation, microcirculation improvement, and blood circulation-promoting effects, as the model drug. TMP is mainly administered orally or by injection in clinical practice. It has a low molecular weight (136.19), a short biological half-life, and pronounced hepatic metabolism-characteristics that make it suitable for transdermal drug delivery system development [15]. However, the clinical effective dose of TMP is relatively high, and the amount of drug delivered across the skin via transdermal administration is insufficient. With technology advances, many methods wmerged to enhance percutaneous permeation, such as the addition of chemical permeation enhancers [16], microneedle technology [17], ethanol-based vesicles [18], soft vesicles, and ultrasound-mediated delivery [19]. These approaches enable drugs to reach effective doses and remain in the body for extended periods, reducing administration frequency and improving patient compliance.

Borneol was chosen as an adjunct drug. The combined application of borneol and TMP has been reported. TMP-loaded borneol liposomes significantly alleviate cerebral ischemia–reperfusion injury in rats [20], and two agents act synergistically in neuroprotection against global cerebral ischemia–reperfusion injury[21]. The combination also increase TMP permeability across the blood–brain barrier[22], enhance drug absorption [23], and exerts a synergistic protective effect on brain microvascular endothelial cells damaged by hypoxia [24]. However, reports on the impact of their combined use on pharmacokinetics and tissue distribution are lacking. Borneol is an effective permeation enhancer that elevates both the rate and extent of penetration of various drugs [25, 26]. Studys of its effect on TMP permeation-indicate two main mechanism: On the one hand, borneol disrupts the tight packing of the lipid bilayer, increasing its porosity and creating more favorable conditions for the TMP diffusion. On the other hand, borneol induces the formation of aqueous channels, markedly enhancing TMP permeability [27]. In traditional Chinese medicine theory, borneol also has the role of “Gui-xin Jing”.

Therefore, this study combined TMP with borneol to investigate whether borneol increases TMP distribution in heart tissue.

Material and methods

Compounds and agents

TMP (purity ≥ 98%) was purchased from Shanghai yuanye Bio-Technology Co., Ltd (Shanghai, China). Borneol (purity ≥ 98%) was purchased from Anhui Tongling Borneol Factory (Anhui, China). Carbamazepine and standard TMP (purity ≥ 99%) were purchased from National Institutes for Food and Drug Control (Beijing, China). Rohm Polyacrylic resin E100 was purchased from Rohm (Darmstadt, German). HPLC-grade methanol was purchased from Merck (Darmstadt, Germany). Triethyl citrate (TEC) (purity ≥ 98%), Succinic acid (SA) (purity ≥ 99%), glycerol (Gly) (purity ≥ 99%) were purchased from Shanghai Macklin Biochemical Co.,Ltd (Shanghai, China).

Experimental animals

Male Sprague–Dawley (SD) rats (body weight 200 ± 20 g, Specific-pathogen-free (SPF) grade) were purchased from SPF (Beijing) Biotechnology Co., Ltd. (Reg. No. SCXK (Jing) 2019–0010) and fed a normal diet. All animal experimental procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Research Ethics Committee of Yunnan University TCM (No. R-062021087).

Preparation of patches

The prescribed amounts of Gly, TEC, SA, and E100 were dissolved in 75% ethanol and stirred at 45 ℃ until completely dissolved. After adding precisely weighed TMP and borneol, the mixture was stirred to achieve homogeneity and then sonicated to degas. Evenly coated the film with 30 cm², heated for 2 h at 45 ℃, left at room temperature for 10 min, and covered with a protective film. The TMP patch was obtained for further study.

In vivo permeation studies

To evaluate the effect of borneol with different concentration (3%, 5%, 7%,10%, 14%) on the transdermal delivery of TMP, healthy SD rat skins (200 ± 20) were used. After anesthetized with pentobarbital, the abdominal areas of the rats were shaved and the full thickness abdominal skin of the rats was isolated; subcutaneous fat was removed and the rats were euthanized by inhalation of an overdose of carbon. The skin was mounted between the junction of the supplying cells and the receiving cells of the LOGAN automatic transdermal system (the diffusion area was 1.77 cm², the volume was 12 mL, the temperature of the transdermal diffuser was set at 32 ℃, and the speed of the magnetic stirring was 600 r/min), The patch prepared before the experiment was closely pasted on the cuticle of the skin. 12 mL of normal saline was added into the receiving chamber, and 5 mL of transdermal receiving solution was absorbed at 0.5, 1, 2, 4, 6, 8, 10, 12 and 24 h. The solution was filtered through a 0.22 µm microporous membrane, and an equal volume of blank receiving solution was added simultaneously. The concentration of TMP was determined.

For histological examination, skin samples were taken for HE staining at 3, 6 and 12 h after application of the BO-TMP patches and TMP patches.

Characteristic spectrum

For Characteristic spectrum, Raman imaging was performed with an inVia Pharmaceutical Analyser (Renishaw, Gloucestershire, UK) using 532 nm excitation; FTR-ATR spectra were recorded on a Thermo Scientific iN10 (FTIR, Zhongke Ruijie, China) over 400–4000 cm⁻¹; Thermal properties were assessed with a Setsys16 differential scanning calorimeter (DSC, Setaram, France) under 50 mL·min⁻¹ nitrogen, heating to 400 °C at 10 °C·min⁻¹; Approximately 10 mg of sample was weighed into tin cups and partially sealed.

Pharmacokinetic study of microdialysis in SD Rats

In vitro evaluation of probe recovery

In vitro the recovery rate of the probe was determined using the concentration difference method. The probe active dialysis window was completely immersed in a PBS solution containing TMP (1, 2, 4 μg·mL⁻¹). The PBS solution was used as perfusate at a flow rate of 1.0 μL·min⁻¹, stirred, and the positive recovery rate was obtained at 37℃. The probe active dialysis window was completely immersed in a PBS solution, and the PBS solution containing TMP (1, 2, 4 μg·mL⁻¹) was used as perfusate at a flow rate of 1.0 μL·min⁻¹, stirred, and the reverse recovery was obtained at 37℃.

In vitro the recovery rate of the probe was determined by velocity of flow difference method. The probe active dialysis window was completely immersed in PBS solution containing TMP 2 μg·mL⁻¹. The PBS solution was used as perfusate at a flow rate of 1.0, 1.5, 2, 2.5, 3 μL·min⁻¹, stirred, and the positive recovery rate was obtained at 37℃. The probe active dialysis window was completely immersed in PBS solution, and the PBS solution containing TMP 2 μg·mL⁻¹) was used as perfusate at a flow rate of 1.0, 1.5, 2, 2.5, 3 μL·min⁻¹, stirred, and the reverse recovery was obtained at 37℃.

In vivo evaluation of probe recovery

The recovery rate of probe was determined in vivo by anti-dialysis method. After the probe was inserted into the jugular vein, subcutaneous skin and heart, a PBS solution containing TMP (2 μg·mL⁻¹) was used as perfusate at a flow rate of 1.0 μL·min⁻¹. Following 1 h equilibrium, recovery rates in the three sites were measured overnight.

Microdialysis study

Twelve SD rats were randomly divided into 2 groups: the BO-TMP patches and the TMP patch group. Fasting for 12 h, drinking water freely; Neck and abdomen waxing. The rats were anesthetized with pentobarbital. probes were inserted into the jugular vein and the subcutaneous skin, the perfusion of PBS at the flow rate of 1.0 mL·min⁻¹ was balanced for 1 h. The BO-TMP patches and TMP patch were administered at a dose of 250 mg·kg⁻¹, respectively. Microdialysate was collected every 30 min for 48 h for LC–MS analysis.

A further twelve SD rats were randomly divided into two groups: the BO-TMP patches and the TMP patch group, chest waxing and a probe was inserted into the heart. Microdialysate was collected every 30 min for 24 h and analyzed by LC–MS analysis.

Tissue distribution study in vivo

For the tissue distribution study, 60 SD rats were taken and fasten for 12 h without drinking water, were randomly divided into two groups. One group received BO-TMP patches, and the other group received TMP patches. After intraperitoneal administration of the patch at a dose of 250 mg/kg, rats were euthanized by inhalation of an overdose of carbon dioxide at 1, 3, 6, 12, and 24 h, respectively. Subsequently, heart, liver, spleen, lung, and kidney were isolated. 0.9% normal saline was added according to 1:2 tissue weight, and the tissue was thoroughly homogenized by tissue crushing homogenizer. The homogenates of different tissues collected at different time points were taken 300 μL methanol and 20 μL internal standard solution (114 μg·mL⁻¹ carbamazepine solution) were added and vortexed for 5 min, respectively. After centrifugation at 14,000 r/min for 10 min, the supernatant was taken through a 0.22 μm filter membrane and analyzed.

Method validation

In accordance with bioanalytical validation guidelines, we have conducted a comprehensive validation of the method, covering specificity, linearity range, accuracy, precision, extraction recovery, matrix effects, and stability. The validation results are as follows: Linearity range: 0.03125–39.677 µg/mL; Accuracy: 87%–103%; Precision: less than 5%;

Extraction recovery ratio: 88%–108%, with RSD less than 8%; LOD: 13 ng/mL; LOQ: 35 ng/mL.

Detection of TMP concentration using LC–MS/MS method

TMP concentrations were determined by HPLC–MS/MS on an Agilent Eclipse Plus-C18 column (2.1 × 50 mm, 1.9 μm, Agilent, Santa Clara, CA, USA). The mobile phases were composed of component A (methanol, 30%) and component B (water, 70%). The column temperature was maintained at 30 ◦C, and the injection volume was 2 μL. The flow rate of the mobile phase mixture was set at 0.3 mL·min⁻¹. For quantitative analysis, the MS was run in the multiple reaction monitoring (MRM). The following optimized MS settings were used: Capillary voltage 3500 V; Dry gas flow rate 5 L·min⁻¹; Atomizing gas pressure 45 psi; Sheath temperature 250 °C; Transmission capillary temperature: 250 °C; Ion source temperature: 350 °C; Sheath gas flow rate 11 L· min⁻¹; Dry temperature 300 °C; Nozzle voltage:500 V; Quantitative ion pair: CHIm/z328/330.

Statistical analysis

DAS 2.0 pharmacokinetic program software (Chinese Pharmacology Society, Shanghai, China) was used to analyze the pharmacokinetic parameters, including peak concentration (Cmax), peak time (Tmax), AUC, cleaning half-life (T1/2), and the mean residence time (MRT). All parameters were expressed as the mean ± SD. Statistical analysis and comparisons between groups were carried out using SPSS 26, p < 0.05 was considered statistically significant.

Results and discussion

Ex vivo permeation studies

The maximum dose of promoting osmosis is usually 10% as a volatile component in the study of promoting osmosis; however, in this study, borneol serves not only as a promoting agent but also as one of the active ingredients in disease treatment, so its range was extended to 3%, 5%, 7%, 10% and 14%.

The permeation profile of TMP patches containing different concentrations of borneol through rat skin are shown in Fig. 1a. Statistical analysis (SPSS) indicated that 3% borneol concentration yielded the highest cumulative TMP permeation (6.80 mg·cm⁻² over 24 h, p < 0.05, n = 5), with an enhancement ratio of 1.35.

Fig. 1.

(a) The permeation profile of borneol with different concentrations on TMP patches through rat skin(n = 5). *p < 0.05, **p < 0.01, versus 3%BO. (b) Changes of stratum corneum over time after administration of different patches

At present, there is an increasing amout of research on the mechanism of osmotic enhants, which can enhance skin permeability by disturbing the orderly arrangement of stratum corneum lipids [28, 29], interfering with competitive hydrogen bonding between osmotic enhancer and cuticle ceramide [30, 31], and infiltrating the stratum corneum lipids. This leads to breakdown of the stratum corneum and the peeling flakes, resulting in C-H elongation and H-bond displacement between the osmotic enhancer and lipids [32]. Here, borneol facilitates TMP penetration by loosening the stratum corneum, an effect that more pronounced with the prolonged application, as shown in Fig. 1b.

Characteristic spectrum

In the process of preparation research, the influence of excipients on the preparation is crucial. Compared with drugs, excipients typically constitute a large proportion, and the active impurities in the excipients may interact with drugs, affecing the action of the APIs to form a characteristic peak. Various methods can be used to analyze these interactions. For instance, thermal analysis, spectral analysis, high-performance liquid chromatography, and scanning electron microscopy can effectively demonstrate the compatibility between raw materials [33, 34].

Raman spectra reveal that each excipient has its own characteristic peak, as shown in Fig. 2a, and that each excipient is evenly distributed within the patch, as shown in Fig. 2b. The results of DSC (Fig. 2c) and FTIR (Fig. 2d) indicate that BO-TMP patches exhibit their own characteristic peaks, which can be used for patch identification.

Fig. 2.

Characteristic spectrum: (a) Raman spectra: Characteristic peaks of raw materials and excipients. (b) Raman Distribution map of raw materials and excipients. (c) DSC thermogranms. (d) FTIR spectra

Microdialysis study

Microdialysis is used for in vivo sampling of molecules, providing a continuous, time-dependent profile of endogenous release and exogenous drug concentration [35]. A key feature is the semi-permeable membrane [36], roughly 80–90% of molecules cannot cross it, and only those of lower mass pass through[37]. when choosing a semi-permeable membrane, it is recommended to select one with a molecular weight cutoff value greater than that of measured compound to ensure that measurable compounds can be sampled in the experiment [38]. Saline solutions such as Ringer’s fluid or normal saline are routinely employed as perfusates because their ionic concentrations resemble those of extracellular fluids [39]. To date, no studies have examined whether these common perfusates affect the tissue surrounding the implanted probe or cause membrane fouling [40], so it is necessary to investigate the perfusion fluid during the experiment to ensure the stability of the recovery rate. In the course of this experiment, it was found that when normal saline was used as the perfusion solution, there was a large discrepancy between the forward and the reverse recovery rates, and a situation that has been reported in the literature. However, after changing the perfusion solution, the values of the forward and the reverse recovery rate were much close.

The concentration difference method was used to measure TMP concentration in vitro. This results, shown in Fig. 3a, indicated that the positive recovery rate was (84.98 ± 1.71) %, and the reverse recovery rate was (84.56 ± 0.98) %, which was close; The concentration of TMP in vitro was determined using velocity difference method. The results, shown in Fig. 3b, revealed that both forward and reverse recovery rates decreased with increasing perfusion flow rate. Microdialysis flow rates are usually in the range of 0.5–1 μL·min⁻¹ [41]. Evidence shows that ultra-low flow rates can cause mild tissue damage [42] and induce a local neuroinflammatory response [43]. Considering sampling time, safety, and recovery rate comprehensively, a flow rate of 1.0 μL·min⁻¹ was selected. At this flow rate, the positive recovery rate was (85.59 ± 0.81) %, and the reverse recovery rate was (84.56 ± 0.81) %, indicating that the anti-dialysis method was suitable for measuring the rate of the probe in vivo.

Fig. 3.

Recovery rate of the concentration difference method (a) and the velocity difference method (b) in vitro; Recovery rate in plasma and skin (c), and in heart (d) in vivo

In vivo recovery rates are shown in Fig. 3c and 3d. The results showed that the recovery rate in plasma was: (52.22 ± 2.24) %, the recovery rate in skin was (55.67 ± 2.11) %. the recovery rate in heart was (54.05 ± 1.10) %. As shown in our data, microdialysis recovery differs among tissues because of their inherent physiological and structural differences: dense organs such as the brain have narrow extracellular spaces and high diffusional resistance, leading to lower recoveries. In addition, probe length, pore size, and membrane material further determine extraction efficiency. Because each tissue requires a tailored probe, ignoring these factors allows any recovery bias to propagate directly into core PK metrics (Cmax, AUC), seriously compromising accuracy. Consequently, tissue-specific recovery must be determined in vivo and the resulting values used to calibrate the data before pharmacokinetic calculations, ensuring rigorous and reliable PK outcomes.

As observations had to be continued for 24–48 h, sporadic mid-experiment deaths were initially noted; once normothermia (37 ± 0.5 °C) was strictly maintained, mid-experiment mortality dropped to zero. Any other deaths occurred within the first post-operative hour, consistent with acute surgical stress. Hypothermia is known to increase peri-operative mortality to ~ 20% [44, 45]. Therefore, throughout both surgery and recovery, each animal was placed on a thermostatically controlled mat and given 5% glucose as needed to maintain normal physiological function.

Microdialysis probes implanted into other tissues and organs, such as the central nervous system, adipose tissue, and heart, had been reported to study drug distribution [46]., the technique is now widely applied to many tissues [47], and it measures the free concentration in peripheral tissue fluid, which has been shown to be useful for pharmacological studies [48], with advantages such as minimal tissue damage and no sample handling[49]. In this experiment probes were placed in the jugular vein, subcutaneous skin, and heart.

The concentrations of TMP in the jugular vein, subcutaneous skin, and heart, as measured by microdialysis, were lower than that of BO-TMP patches. The results, shown in Fig. 4, indicate that borneol promotes the penetration of TMP through the skin and increases systemic uptake. The pharmacokinetic behavior of the BO-TMP patches was superior than those of TMP patches (AUC_0–48 h and Cmax, P < 0.05), as presented in Table 1.

Fig. 4.

Concentration–time curves of TMP in the jugular vein (a), subcutaneous skin (b) and heart (c) after transdermal administration (mean ± SD, n = 6)

Table 1.

Pharmacokinetic parameters of TMP patch and BO-TMP patch in the jugular vein, subcutaneous skin and heart after transdermal administration (n = 6)

		AUC(0-t)(mg/h/L)	MRT(h)	Tmax(h)	Cmax (mg/L)	t1/2z(h)
plasma	BO-TMP	73.18 ± 13.86*	19.65 ± 0.61	7.38 ± 4.37	2.88 ± 0.72	16.43 ± 8.43
	TMP	47.43 ± 6.93	20.76 ± 0.55	11.25 ± 2.33	1.96 ± 0.27	10.45 ± 7.14
skin	BO-TMP	374.55 ± 110.84*	21.71 ± 3.37	15.88 ± 6.6	15.33 ± 4.24	4.85 ± 2.52
	TMP	220.86 ± 53.88	19.62 ± 3.45	16.50 ± 6.34	12.99 ± 2.28	6.88 ± 4.11
heart	BO-TMP	79.80 ± 16. 31***	13.83 ± 1.41	13.73 ± 6.82	6.20 ± 0. 84***	13.75 ± 6.83
	TMP	13. 08 ± 3. 35	13.09 ± 0.65	14.67 ± 0.09	0.87 ± 0.12	14.67 ± 0.09

Notes: Compared with TMP patches, * p < 0.05, ***p < 0.001, Values are expressed as mean ± SD

Tissue distribution of patches

Although drug distribution is a key determinant of pharmacological responses, it has historically received far less attention than absorption, metabolism, and excretion [50]. Recent studies now evaluate the targeting of drugs through tissue distribution studies. The process of drug distribution to the active site constituted a "link-bridge" of the pharmacokinetic/pharmacodynamic relationship. To improve selectivity, it is necessary to further study drug delivery system to ensure the specific delivery of drugs to specific organs or tissues [51]. This study focused on the effect of borneol on the distribution of TMP in different tissues.

The concentration of TMP in heart, liver, spleen, lung, kidney, and brain was determined at a series of time points after administration. As shown in Fig. 5, following percutaneous administration of BO-TMP patch, the TMP concentration in different tissues was in the order of liver, kidney, heart, brain, spleen, and lung. Following percutaneous administration of TMP patch, the TMP concentration in different tissues was in the order of liver, kidney, brain, spleen, hear,t and lung. The concentration in different tissues after administration of BO-TMP patches was higher than that after administration of TMP patches.

Fig. 5.

The concentration of BO-TMP patch (a) and TMP patch (b) in different tissues 1, 3, 6, 12, 24 h after transdermal administration (mean ± SD, n = 6)

TMP is primarily used to treat cardiovascular and cerebrovascular disorders, so the pharmacokinetics of TMP in the heart and brain tissue were analyzed. The results showed the pharmacokinetic behavior of the BO-TMP patch was significantly better than that of TMP patch (AUC_0–24 h and Cmax, P < 0.05), as shown in Fig. 6 and Table 2.

Fig. 6.

Drug time curves of BO-TMP and TMP in heart(a), liver(b), spleen(c), lung(d), kidney(e) and brain(f) tissues (n = 6)

Table 2.

Pharmacokinetic parameters of heart and brain following tissue distribution of BO-TMP patch and TMP patch (mean ± SD, n = 6)

Pharmacokinetic parameters	Heart (BO-TMP)	Heart (TMP)	Brain (BO-TMP)	Brain (TMP)
Cmax (mg/L)	2.083 ± 0.330**	1.448 ± 0.177	2.356 ± 0.450**	1.602 ± 0.200
Tmax (h)	2.667 ± 0.816**	1.333 ± 0.816	3.000 ± 0.100**	1.333 ± 0.816
T1/2 (h)	15.080 ± 7.330	1.935 ± 0.019	2.832 ± 2.456**	41.072 ± 23.111
MRT0–∞ (h)	22.330 ± 9.131	7.397 ± 0.120	7.893 ± 1.533**	60.311 ± 34.032
AUC (mg/h/L)	27.169 ± 2.009**	18.278 ± 0.682	23.672 ± 1.530**	28.103 ± 1.142

Notes: Compared with TMP patches, **p < 0.01, Values are expressed as mean ± SD

Given different patches, the amount of the drug in the liver and kidney was higher than in other tissues. The finding is precisely supported by the theory of Chinese medicine, which posits that blood is housed in the heart, hidden in the liver, rooted in the kidney, and flows through the vessels to nourish the body’s limbs.

In addition, Borneol increases the enrichment of TMP in the heart, a finding consistent with the traditional Chinese medicine (TCM) theory. In TCM, tropism (Gui Jing) denotes a drug’s selective therapeutic action for a particular organ [52]; for example, Platycodon grandiflorum has a tropism for the lung (Gui-fei Jing), and therefore has a selective action on the lung and respiratory system [53]; while borneol has a tendency towards the heart (Gui-xin Jing). Relevant data have confirmed this tendency. For example, borneol can significantly increase the enrichment of ferulic acid, a chemical component in Speedy Heart-Saving Pills, in the heart [54]. Borneol can also increase the content distribution of Notoginsenoside R1, Ginsenoside Rg1 and Re in the heart tissue [55]. These observationds confirm a link between tropism and tissue selectivity [56]. Recently, researchers both domestically and internationally have extensively investigated this tropism theory, attempting to elucidate the theory of channel tropism from several aspects, including receptors, pharmacodynamic parameters of active ingredients, changes in trace element content, and changes in cAMP/cGMP concentration. However, the theoretical basis and mechanism have not yet been clearly elucidated and still require further validation [52].

We preliminarily speculate that the cardiac tropism mechanism of borneol may be related to the following factors. First, borneol itself has a higher distribution in the heart, which may facilitate the co-delivery of TMP, resulting in a higher distribution of TMP in the heart as well. Second, borneol can loosen the tight junction structures between cells, thereby promoting the accelerated transport of substances and increasing vascular wall permeability of the brain [57]; whether borneol plays a similar role in the heart still requires in-depth research.

Conclusion

This study yielded two major findings. First, In the patch formulation, TMP was fully compatible with borneol, E100, triethyl citrate, succinic acid, and glycerol; 3% concentration of borneol gave the greatest permeation-enhancing effect on TMP patch, primarily by increasing cuticle shedding. As time progressed, the cuticle shedding became pronounced. Second, in subcutaneous microdialysis, the drug half-life (4.85 h for the BO-TMP patch, 6.88 h for the TMP patch) was significantly shorter than that observed in the plasma and heart (plasma: 16.43 h for the BO-TMP patch, 10.85 h for the TMP patch; heart: 13.75 h for the BO-TMP patch, 14.67 h for the TMP patch). This discrepancy aligns with the stepwise distribution sequence “subcutis → blood → heart”: the drug must first reach the systemic circulation via the capillary network and is then carried by blood flow to the myocardium, so the elimination phases in plasma and heart are inevitably prolonged, yielding longer apparent t₁/₂ values. Moreover, Tmax for the BO-TMP patch was shorter than that for the TMP patch in subcutaneous tissue, plasma, and heart, while the Cmax was higher for the BO-TMP patch than for the TMP patch. This suggests that borneol accelerates drug penetration through the stratum corneum and into the subcutaneous microvasculature, thereby shortening the onset time. Additionally, the AUC(0-t) in subcutaneous tissue, plasma, and heart was significantly higher with BO-TMP patch than with TMP group; the increase was most pronounced in the heart, where exposure rose approximately six-fold versus only 1.5-fold in plasma. This suggests that, beyond its permeation-enhancing effect, borneol may further amplify cardiac drug accumulation by increasing myocardial binding or expanding the apparent volume of distribution. Notably, cardiac AUC(0-t) (79.80 ± 16.31) and Cmax (6.20) exceeded the corresponding plasma values (73.18 ± 13.86 and 2.88), displaying the classic "tissue > blood" distribution profile and indicating additional affinity of the drug for myocardial proteins or lipid components, thereby raising the apparent volume of distribution above its anatomical value. Collectively, the incorporation of borneol markedly elevates total cardiac exposure.

Regarding tissue distribution, adding borneol to the patch increased the distribution of TMP in the plasma and skin, and improved the distribution of TMP in different tissues. The distribution order changed from liver, kidney, brain, spleen, heart and lung to liver, kidney, heart, brain, spleen and lung.

Based on the aforementioned findings, we believe that the topical formulation prepared with the combination of borneol and TMP holds significant potential and application value in the treatment of cardiovascular diseases. Easy to apply, non-invasive and simple to use, it is well suited for long-term treatment and can greatly improve patient compliance. This is especially crucial for patients with cardiovascular diseases who require prolonged therapy and whose quality of life stand to benefit. Moreover, borneol markedly amplifies total cardiac exposure to TMP, increasing concentration in the target tissue and providing a pharmacokinetic basis for enhanced efficacy. Whether this difference arises from an increased concentration of unbound drug, from greater non-specific binding, or from the traditional Chinese medicine concept of “Gui-xin Jing” that guides drugs into the heart will require clarification through future measurements of the unbound fraction and elucidation of transporter mechanisms. Identifying the precise molecular targets of the borneol–TMP interaction and defining its pharmacokinetic–pharmacodynamic relationships will provide a solid scientific foundation for more accurate and comprehensive transdermal therapies in patients with cardiovascular diseases.

Authors’ contributions

Conceptualization: Lili Cui, Yunshu Ma. Data curation: Lili Cui, Lichun Zha. Methodology: Jiahua Mei. Supervision: Rui Xue. Validation: Jie Wang, Ziwen Zhang. Writing-original draft: Lili Cui. Writing-review & editing: Lili Cui, Yunshu Ma.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No.82060723), Major science and technology Special Program of Science and Technology Department of Yunnan Province (Grant No. 202302AA310031), General Project of Basic Research Program of Science and Technology Department of Yunnan Province (Grant No. 202501AT070337), Education Department of Yunnan Province (Grant No.2024J0518).

Data Availability

The datasets used and/or analyzed are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

The animal study was reviewed and approved by Yunnan University TCM Committee on Animal Care and Use.

Consent for publication

All authors reviewed and approved to submit this manuscript.

Competing interests

The authors declare no conflicts of interest in this work.