Pharmacokinetic-pharmacodynamic and tissue distribution studies in physiological and cerebral ischemia-reperfusion injury rats after oral administration of anisodine hydrobromide tablets

ABSTRACT

Aim

We aim to establish a rapid and sensitive UPLC-MS/MS method to analyze the pharmacokinetics of oral anisodine hydrobromide (AH) tablets.

Methods

Comparison of pharmacokinetic differences between AH (present in vivo as anisodine) and its metabolite NOAT3 in two groups of rats after administration of AH at doses of 5, 10, and 20 mg/kg. In addition, plasma concentrations of AH were used as a pharmacokinetic parameter, and interleukin-1β (IL-1β) and lactic dehydrogenase (LDH) were selected as pharmacodynamic indicators to establish a pharmacokinetics-pharmacodynamics (PK-PD) combined model in physiological and CIRI model rats, to analyze the protective effect of AH against CIRI.

Results & conclusion

The study demonstrated that AH could be rapidly and extensively distributed to various tissues in rats. AH is a promising drug for the treatment of rat models of CIRI.

1. Introduction

The pathogenesis of cerebral ischemia-reperfusion injury (CIRI) is intricate and involves multifactorial and multi-pathway injury [1]. Among them, oxidative stress and inflammation are among the common pathological mechanisms of CIRI. It has been reported that during cerebral ischemia, cerebral vascular ischemia leads to cellular hypoxia, anaerobic metabolism, mitochondrial ATP reduction, failure of ion channel exchange, and electrolyte imbalance within and outside the cells, thereby damaging the blood–brain barrier [2,3]. After reperfusion, intravascular blood supply is restored. However, mitochondrial damage and electrolyte imbalance can cause an excessive increase in reactive oxygen species (ROS), leading to oxidative stress, endothelial dysfunction and a local inflammatory response. This, in turn, can cause cell death through various pathways and worsen brain damage [4]. Currently, numerous reports have demonstrated that the alkaloid components provide superior alleviation of oxidative stress and inflammation resulting from CIRI [5,6]. Anisodus tanguticus is a traditional Chinese medicine recorded in Shennong’s Classic of Materia Medica. Anisodine, a novel scopoletane alkaloid, is extracted from the roots of Anisodus tanguticus (Maxim.) Pascher, a member of the Solanaceae family. The structural formulae of anisodine and anisodine hydrobromide (AH) are shown in Figure 1(A,B) [7,8]. It has been reported that AH (existing as anisodine in vivo) can penetrate the blood–brain barrier, showing stronger central nervous system (CNS) effects than scopolamine of similar structure, and is significantly less toxic than other scopolamine alkaloids, such as atropine [9]. Pharmacological experiments demonstrated the significant therapeutic effects of AH against both acute and chronic cerebral ischemia-reperfusion injury models. In vivo, AH significantly reduced the infarcted area of brain tissue in CIRI rats and exerted a protective effect on CIRI rats by inhibiting the expression of anti-inflammatory and antioxidant-related factors as well as inhibiting neuronal apoptosis, which was manifested by decreasing the expression of P53 protein in rats with chronic ischemic injury, enhancing the expression of p-Akt protein in rats with acute ischemia/reperfusion, and activating the ERK1/2 signaling pathway and regulating the activity of ATPase activity to inhibit apoptosis. In vitro, AH promoted autophagy in hypoxic PC12 cells, protected neuronal cells, and alleviated oxygen–glucose deprivation/reoxygenation-induced injury in cerebral microvascular endothelial cells, demonstrating efficacy against acute cerebral ischemia/reperfusion injury [10–12]. More importantly, clinical studies have shown that AH injection has a good effect on acute massive cerebral infarction and improves cerebrovascular reserve function [13,14]. Although there are many studies on the pharmacokinetics of AH in normal animals [15,16], there is a lack of pharmacokinetic and tissue distribution studies analyzed in pathological models and pharmacokinetics-pharmacodynamics (PK-PD) combined model. Therefore, it is necessary to establish suitable methods to simultaneously monitor the in vivo metabolic profiles of AH and its major metabolites, N-oxide-anisodine (NOAT3) (Figure 1(C)), in rats.

Figure 1.

Structure of the three compounds to be measured. (A) Anisodine (B) Anisodine hydrobromide (C) N-oxide-anisodine.

The PK-PD model can comprehensively reflect the pharmacokinetic process of drugs in the body and changes in pharmacodynamic indicators, thus facilitating a more scientific and objective evaluation of the dynamic effects of drugs in vivo [17]. Hence, this study comparatively characterizes the pharmacokinetic and tissue distribution properties of AH tablets and their major metabolites in normal and CIRI rats using a validated ultra-high performance liquid chromatography/tandem mass spectrometry (UPLC-MS/MS) method. In addition, interleukin-1β (IL-1β) and lactic dehydrogenase (LDH) were selected as pharmacodynamic indicators to analyze the pharmacodynamic differences between physiological and model groups of rats after oral administration of AH. Finally, a combined PK-PD model was established, as well as the evaluation of its efficacy in relation to plasma concentration and time, which provided a certain basis for the further clinical application of AH, as well as the drug development and rational use of the drug for the treatment of CIRI.

2. Materials and methods

2.1. Materials and reagents

Anisodine hydrobromide (100% purity, as C₁₇H₂₁NO₅·HBr. Lot No. 100399–201802) and scopolamine hydrobromide (SCO, 91.7% purity, Lot No. 100049–201009) were obtained from the China Academy of Food and Drug Administration (NIFDC). N-oxide-anisodine (98.67% purity, Lot No. 210425-DZ) was provided by Chengdu First Pharmaceutical Co. The oral drug for experimental rats, anisodine hydrobromide tablets (Approval No. CNPD H51023615), was provided by Chengdu No.1 Pharmaceutical Co. Methanol, acetonitrile and formic acid were chromatographically pure, and all other reagents were analytically pure.

2.2. Animals

Healthy male and female Sprague-Dawley (SD) rats, 280–320 g, were purchased from Chengdu Dashuo Laboratory Animal Co., Ltd. (License No. SCXK (Sichuan) 2020–0030). All experimental rats were adaptively fed for 1 week under standard conditions (12 h light/dark cycle, 50% ± 5% humidity, 25°C ±2°C). Diets were fasted for 12 h prior to modeling and drug administration, and water was freely available. All animal experiments in this study followed the experimental protocols and guidelines approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (No. 20210705). All rats used in the experiment were euthanized by abdominal aortic blood collection following anesthesia with isoflurane inhalation.

2.3. CIRI model replication and neurologic scores

Middle cerebral artery occlusion (MCAO) surgery was performed using the method of Longa et al. [18]. Rats were anaesthetized after weighing. The rats were fixed on a thermostatic surgical plate and sterilized with iodophor. A wound of approximately 1 cm was opened with surgical scissors approximately 0.5 cm to the right of the midline of the neck. Then the right common carotid artery (CCA) and vagus nerve were bluntly dissected to the bifurcation using ophthalmic forceps. The proximal end of the CCA was ligated with 3–0 sutures and the distal end of the CCA was clamped with an arterial clip. The external carotid artery (ECA) was again ligated with a suture at the ECA and internal carotid artery (ICA) forks, and by following the ICA to find the bifurcation of the pterygopalatine artery (PPA) from the ICA, the PPA was temporarily clamped with a miniature arterial clip. A silicone-coated nylon monofilament with a diameter of 0.38 ± 0.02 mm was inserted through the CCA. At the same time, the nylon monofilament was angled to the left to deflect it into the ICA, then angled back to the right and the CCA was gently pulled so that the nylon monofilament entered the brain along the ICA to block middle cerebral artery (MCA) blood flow. The depth of the nylon monofilament insertion was 18–20 mm from the beginning of the ICA. The MCAO procedure was completed by tying the remaining loose knot and suturing the incision. After 1.5 h of ischemia, CIRI was completed by slowly pulling the nylon monofilament back to the bifurcation of the ECA and ICA. After the animals woke up (approximately 1.5 h of reperfusion) and 22.5 h of reperfusion, the Longa 5-point scale [18] was used to assess the replication of the CIRI model. Model rats with scores of 1–3 were selected and returned to the rearing environment to be housed separately for further experiments.

2.4. UPLC-MS/MS methodology validation

The levels of anisodine and NOAT3 in biological samples were detected using a Shimadzu UPLC-MS 8060NX chromatography-mass spectrometry coupled to a Waters Acquity UPLC BEH C₈ column (50 mm × 2.1 mm, 1.7 μm). The mobile phase for chromatographic separation consists of 5 mM ammonium acetate +0.05% formic acid (Phase A) and chromatographic-grade methanol (Phase B). Mass spectrometry conditions: +ESI ion source, multiple-reaction monitoring (MRM) mode. The full-scan protonated molecular ion pairs (m/z) of anisodine, NOAT3, and SCO (internal standard, IS) were 320.2→119.1, 336.0→172.1, and 304.0→138.1, respectively.

The stability, specificity, linearity, matrix effect, recovery, accuracy and precision of the analytical methods were validated using standard biological samples prepared according to the guidelines of the Chinese Pharmacopoeia for quantitative testing of biological samples.

2.5. Biological sample preparation

Take 50 μL of biological sample, add 50 μL of IS (scopolamine hydrobromide, 50% MeOH, 50 ng/mL), vortex for 10 s, then add 50 μL of 10% trichloroacetic acid (TCA) solution, mix well, then centrifuge for 10 min at 4°C, 14000 rpm, after which, the supernatant was taken and detected by UPLC-MS/MS method with an injection volume of 5 μL.

2.6. PK study

A total of 36 male SD rats (280–320 g) were randomly divided into 6 groups (n = 6): CIRI low-dose group, CIRI medium-dose group, CIRI high-dose group, physiological low-dose group, physiological medium-dose group, and physiological high-dose group. The doses administered were 5, 10, and 20 mg/kg. The animals were fasted for 12 h prior to dosing, and water was freely available. Blood samples of 0.3 mL were collected from the retro-orbital venous plexus in 1.5 mL sodium heparin centrifuge tubes before and 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2.5 h, 4 h, 6 h, 8 h, 12 h, and 24 h after drug administration. After centrifugation at 4°C and 4500 rpm for 10 min, the plasma was pipetted into a new centrifuge tube and immediately frozen in a −80°C refrigerator until use. The model group was administered 24 h after cerebral ischemia-reperfusion and operated in the same way.

2.7. PD study

2.7.1. Grouping, administration and sample collection of experimental animals used for TTC staining and Nissi staining of brain tissue

Male SD rats (280–320 g) from the same batch as the PK study were used and were randomly divided into five groups (n = 6): physiological (control) group, CIRI group, CIRI low-dose group, CIRI medium-dose group, and CIRI high-dose group. The doses administered were 5, 10, and 20 mg/kg. Fasting began 12 h before modeling and drug administration, and water was freely available. The model administration group was administered 24 h after cerebral ischemia-reperfusion. The rat brain tissue from the physiological and CIRI groups was harvested immediately after 24 h of cerebral ischemia-reperfusion. The obtained rat brain tissue was immediately frozen at −80°C for about 5 min, and then removed and cut into 5 pieces for 2,3,5-Triphenyltetrazolium chloride (TTC) staining and Nissl staining. For Nissl staining, the brain tissue was fixed in paraformaldehyde for 24 h, embedded in paraffin, and further stained to measure cerebral infarct volume and neuronal cell survival. The model administration group was administered 24 h after cerebral ischemia-reperfusion, and the rats in different dose groups were executed 24 h after administration, and intact brain tissues were taken for TTC staining and Nissi staining.

2.7.2. Grouping, administration and sample collection of experimental animals for IL-1β and LDH assays

Experiments were conducted using physiological high-dose and CIRI high-dose groups, with six animals in each group. All administered at a dose of 20 mg/kg. Blood samples of 0.5 mL were collected from the retro-orbital venous plexus in 1.5 mL sodium heparin centrifuge tubes before and 5 min, 10 min, 20 min, 30 min, 45 min, 1 h, 2.5 h, 4 h, 6 h, 12 h, and 24 h after administration in the high-dose physiological and high-dose CIRI groups. After centrifugation at 4°C for 10 min at 4500 rpm, the plasma was pipetted into a new centrifuge tube and immediately frozen at −80°C until use. The expression levels of interleukin-1β (IL-1β) and Lactic dehydrogenase (LDH) in plasma samples collected at different time points were detected using commercial enzyme linked immunosorbent assay (ELISA) kits and LDH biochemical kits, respectively.

2.8. Establishment of the PK-PD model

The detected IL-1β and LDH values were imported into Phoenix WinNonLin 8.3.5 software, respectively, and the categories of the best pharmacodynamic models were determined by PD fitting. Subsequently, non-compartmental analysis (NCA) derived preliminary PK parameters, which informed the initial estimates for compartmental modeling to determine the optimal PK structural model. This process culminated in an integrated PK-PD model. Model adequacy was evaluated using the Akaike Information Criterion (AIC) and coefficient of determination (R²), with lower AIC values and R² values approaching 1 indicating superior model fit.

2.9. Tissue distribution study

A total of 96 SD rats (280–320 g) were randomly divided into control and CIRI groups. There were 24 female and 24 male rats in each group. The doses administered was 20 mg/kg. Fasting began 12 h before modeling and drug administration, and water was freely available. The model administration group was administered 24 h after cerebral ischemia-reperfusion. Twelve rats (6 males and 6 females) were sacrificed at 20 min, 1 h, 4 h, and 24 h after administration in the physiological group and CIRI group, and the heart, liver, spleen, lung, kidney, brain, stomach, small intestine, large intestine, skeletal muscle, bladder, prostate, testicles, oophoron, and fatty tissue were collected. After washed with saline, the water was blotted out with filter paper. The same portions were weighed and a quantitative amount of saline was added at a ratio w: v = 1:4. Then homogenized at 4°C, 1800rpm for 80 s and cycled 4 times. After the tissue homogenate was centrifuged at 4°C for 10 min at 4500 rpm, the supernatant was pipetted into a new centrifuge tube and immediately frozen at −80°C until use.

2.10. Data analysis and statistics

Data from UPLC-MS/MS assays were analyzed using the Lab Solution workstation. Pharmacokinetic parameter matching and PK-PD combined modeling analysis were performed using Phoenix Winnonlin 8.3.5. Statistical analysis was performed using SPSS 26.0, and differences between groups were analyzed using one-way AONVA test or independent samples t-test, with p < 0.05 considered statistically different. Relevant data were plotted using Graph Pad Prism 10.0.

3. Results and discussion

3.1. Model characterization and validation of efficacy

TTC staining of brain tissues showed that, the CIRI group had obvious cerebral infarction compared to the control group after 24 h of cerebral ischemia-reperfusion, with an average infarction rate of 31.90% (p < 0.0001). However, in contrast to the CIRI group, the rates of cerebral infarction in the rats were all significantly reduced in a dose-dependent manner after 24 h of drug administration. The results of the staining and the determination of the cerebral infarction rate were shown in Figure 2(A, B). Nissl staining results confirmed the results of TTC staining. As shown in Figure 2(C, D), the number of cells in the cortex was significantly reduced after 24 h of modeling, and then improved noticeably after drug administration. Similarly, the number of cells in the CA1 region of the hippocampus recovered after drug administration, but it was not statistically significant, as shown in Figure 2(C, E). The above results indicated that AH was able to ameliorate CIRI-induced brain tissue damage in rats in a dose-dependent manner.

Figure 2.

CIRI model establishment and TTC staining and Nissl staining results after drug administration. (A) Results of TTC staining at different groups. (B) The cerebral infarction rate was obtained by image J, and the significance between different groups was compared by one-way ANOVA, ^####p < 0.0001 compared with control, **p < 0.01, ***p < 0.001, and ****p < 0.0001 compared with CIRI, n = 6. (C) Representative results of Nissl-stained histologic images assessing neuronal survival in the ipsilateral cerebral cortex area and hippocampal CA1 area. Scale bar = 50 μm, n = 3. (D) The survival counts of neuronal cells in the cerebral cortex area obtained from image J analysis were compared for significance between the different groups according to the one-way ANOVA method, ^##p < 0.01 compared with control, *p < 0.05, **p < 0.01, and ***p < 0.001 compared with CIRI, n = 3. (E) The count of hippocampal CA1 neuronal cell survival obtained from image J analysis was compared for significance between different groups according to one-way ANOVA method, ^#p < 0.05 compared with control, *p < 0.05, **p < 0.01, and ***p < 0.001 compared with CIRI, n = 3.

3.2. UPLC-MS/MS methodology validation

3.2.1. Specificity

The analysis results of blank biological samples, mixed standard solutions of biological samples, and biological samples after drug administration showed that the biological sample components did not interfere with the detection of AH and NOAT3. The measured components had good definition and peak profiles. This indicated that the assay showed good specificity. Representative chromatographic results were shown in Fig. S1(A, B, C).

3.2.2. Linearity and lower limit of quantitation

The AH standard solutions were mixed with rat blank plasma and liver tissue homogenate supernatant, respectively, and diluted to a series of mixed standard solutions of biological matrix with AH concentrations of 2.00, 5.00, 10.00, 50.00, 200.00, 400.00, 600.00, 800.00, 1000.00 ng/mL. NOAT3 standard solutions were mixed and diluted with rat blank plasma to a series of mixed standard solutions of plasma with NOAT3 concentrations of 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 1.00 ng/mL. The samples were examined by UPLC-MS/MS and the integrals were calculated using the internal standard method. Least squares linear regression was used to determine the slope, intercept and correlation coefficient of the linear regression equation. The regression equation was weighted as 1/X. X was the concentration of the analyte in biological matrix, and Y was the ratio of the analyte peak area to the internal standard peak area. The regression equations and correlation coefficients for AH in plasma and liver tissue homogenate supernatant were Y_plasma = 0.0225X + 0.0117, R_plasma = 0.9996; Y_liver = 0.0087X + 0.0041, R_liver = 0.9995, separately. The regression equation and correlation coefficient for NOAT3 were Y = 0.1661X + 0.0044, R = 0.9991. The lower limit of quantitation (LLOQ) were 2.00 ng/mL and 0.10 ng/mL, respectively.

3.2.3. Accuracy and precision

Based on the linear range, four concentrations of AH and NOAT3, LLOQ, low-concentration quality control (LQC), medium concentration quality control (MQC) and high-concentration quality control (HQC), were selected for investigating accuracy and precision in a single batch and over three different batches. The RSD (%) value was used to represent precision, and the relative deviation RE (%) value was used to represent accuracy. The RSD (%) values of the QC samples for both analytes were less than 6% and the accuracy RE (%) values ranged from 94% to 102%. Detailed results were summarized in Table S1.

3.2.4. Matrix effects and recovery

QC samples with two concentrations of AH and NOAT3 (LQC and HQC) were selected for evaluating the internal standard normalized matrix effect and pretreatment recovery of AH and NOAT3. The matrix effect results were expressed as RSD (%) values and the recovery rates were expressed as mean ± SD (%). The RSD (%) of matrix interferences were all less than 15% and recoveries in plasma could reach 90%–105%, which meant that the method can accurately detect and analyze biological samples. Detailed results were summarized in Table S2 and Table S3.

3.2.5. Stability

QC samples at four different concentrations (LLOQ, LQC, MQC, and HQC) were prepared and used to investigate the stability under specific conditions. The study conditions were as follows: Samples were repeatedly freeze-thawed 5 times at −80°C, stored at room temperature for 48 h, stored at 8°C for 24 h and stored at −80°C for 60 d. The results showed that the AH and NOAT3 were stable in all these conditions, with the RSD (%) less than 10%. Detailed results were summarized in Table S4 and Table S5.

All the results demonstrated that the method was reliable and met the requirements for the quantitative detection of drugs in biological samples that can be used for pharmacokinetic studies.

3.3. PK study

The drug concentration in the plasma of physiological rats and CIRI model rats after oral administration (5, 10, 20 mg/kg) was measured by the UPLC-MS/MS method to evaluate the pharmacokinetic differences. The plasma concentration versus time curve of AH was shown in Figure 3(A). Pharmacokinetic parameters were obtained using a non-compartmental model fit, and the main pharmacokinetic parameters were summarized in Table 1. Unfortunately, Pharmacogenetic parameters of NOAT3 were not smoothly obtained from some rats owing to the fact that the plasma concentration corresponding to some blood collection time points was lower than the LLOQ. Therefore, the pharmacokinetic parameters of NOAT3 were obtained by averaging the calculated mean values of the detected blood concentrations and then fitting them as shown in Table 2. The plasma concentration versus time curve of NOAT3 was shown in Figure 3(B). The mean plasma concentration of AH and NOAT3 after drug administration of different doses were summarized in Table S6 and table S7. The difference of the time for concentration maximum (T_max) of AH in plasma between the CIRI group and the physiological group was small. The concentration maximum (C_max) of AH in plasma of the CIRI group was slightly lower than that of the physiological group, but it was higher than that of the physiological group at the intermediate dose, which might be caused by excessive individual differences. The area under the curve (AUC_0-t and AUC_0-∞) in plasma of the CIRI group was higher than that of the physiological group, while it was significantly lower than that of the physiological group at the high dose (p < 0.05). The half-life (t_1/2) of the CIRI group was longer than that of the physiological group. In addition, the apparent distribution volume (V_z/F_−obs) of AH in the CIRI group was larger than that of the physiological group, and the difference was significant at the high dose (p < 0.05). The clearance rate (Cl/F_−obs) was lower than in the physiological group, yet the clearance rate was significantly higher in the high-dose group (p < 0.05). The comparison of NOAT3 pharmacokinetic parameters in the physiological and the model groups showed similar trends as AH, but there were no statistically significant differences (p > 0.05). From the C_max and AUC values, it was clear that NOAT3 had a low plasma concentration and very low exposure in vivo compared with AH. It indicated that only a very small amount of AH was metabolized via the NOAT3 form after oral administration and that the main process was still via the primary drug. This suggested that the main component producing the pharmacodynamic effect was also AH.

Figure 3.

(A) Mean plasma concentration–time curves in vivo after oral administration of different doses of AH in physiologic and CIRI rats, n = 6. (B) Mean plasma concentration–time curves of NOAT3 in vivo after oral administration of different doses of AH in physiologic and CIRI rats, n = 6.

Table 1.

Pharmacokinetic parameters of AH in rats after oral administration (mean ± SD), n = 6.

Dosage (mg/kg)	Group	Cmax (ng/mL)	Tmax (h)	AUC0-t (h*ng/mL)	AUC0-∞ (h*ng/mL)	t1/2 (h)	Cl/F−obs (L/h/kg)	Vz/F−obs (L/kg)
5	Control	214.39 ± 58.93	0.28 ± 0.09	538.04 ± 152.80	592.37 ± 147.68	6.31 ± 1.35	8.88 ± 2.19	79.86 ± 20.74
	CIRI	199.81 ± 71.81	0.24 ± 0.11	594.67 ± 94.65	697.74 ± 75.05	9.72 ± 4.77	7.24 ± 0.81	102.42 ± 53.88
10	Control	266.90 ± 90.13	0.36 ± 0.26	1048.98 ± 212.34	1097.39 ± 197.63	4.69 ± 1.51	9.32 ± 1.38	63.50 ± 24.19
	CIRI	381.75 ± 166.29	0.40 ± 0.31	1207.59 ± 244.63	1284.55 ± 239.25	7.10 ± 2.44	8.04 ± 1.65	85.28 ± 46.88
20	Control	544.57 ± 158.57	0.54 ± 0.30	2119.29 ± 341.91	2182.08 ± 354.85	4.92 ± 0.96	9.40 ± 1.75	65.99 ± 13.21
	CIRI	530.25 ± 250.96	0.36 ± 0.24	1613.42 ± 217.28*	1734.71 ± 184.70	6.59 ± 2.21	11.65 ± 0.22*	110.61 ± 38.74*

Comparison of control and CIRI groups: *p < 0.05.

Table 2.

Pharmacokinetic parameters of NOAT3 in rats after oral administration, n = 6.

Dosage (mg/kg)	Group	Cmax (ng/mL)	Tmax (h)	AUC0-t (h*ng/mL)	AUC0-∞ (h*ng/mL)	t1/2 (h)	Cl/F−obs (L/h/kg)	Vz/F−obs (L/kg)
5	Control	0.39	0.17	0.27	0.36	0.42	13944.02	8439.51
	CIRI	0.45	0.17	1.70	5.33	16.69	938.92	22609.24
10	Control	0.48	0.5	0.42	ND	ND	ND	ND
	CIRI	0.66	0.33	0.86	1.00	0.85	9937.93	12171.30
20	Control	0.9	0.33	1.73	2.46	3.53	8144.75	41526.12
	CIRI	0.84	0.33	1.43	1.73	1.84	11568.28	30735.44

The pharmacokinetic parameters of AH and NOAT3 in the plasma of the physiological and model groups were compared by independent samples T-test. Comparative analysis showed that the apparent volume of distribution of the drug in the CIRI model rats was higher than that in the physiological group at different doses, and the half-life was higher than that in the physiological group. In the pathological state, the blood–brain barrier of rats is damaged, increasing cell membrane permeability [19], and drugs that would otherwise have difficulty entering the CNS can more easily cross the blood–brain barrier to accumulate in the brain to form reservoirs, so that the apparent volume of distribution of CIRI rats is higher than that of the control group, and then, the drugs slowly diffuse from the brain tissue back into the blood circulation, resulting in a slower rate of clearance of the drugs from the corpuscular circulation. Cerebral ischemia reperfusion is not limited to the brain, but systemic inflammatory responses and oxidative stress also affect the expression of metabolic enzymes and transporter proteins in the organism, which in turn affects the process of drug absorption, distribution, and elimination. Secondly, the expression of metabolic enzymes and transporter proteins is altered in organisms in pathological states, thus affecting drug absorption, distribution, and elimination processes [20–22]. The delayed half-life of AH in the plasma of CIRI rats may also be due to the disruption of the hepatic CYP450 enzyme system due to the pathological state, resulting in a slower rate of metabolizing the drug. Third, the reason why the plasma drug concentration in the model group was slightly lower than that in the physiological group may be due to the dysfunction of microcirculation in rats caused by CIRI [23], which affected the drug absorption. It was noteworthy that the AUC in the model rats was higher than that in the physiological group after low and medium-dose administration, whereas the AUC in the model group was significantly lower than that in the physiological group after high-dose administration. There were several possible reasons for this: Firstly, it may be due to the high concentration of the drug, and the rate of absorption from the gastrointestinal tract slowed down as the blood concentration raised. At the same time, with the increased clearance, part of the drug entering the blood is rapidly metabolized directly by the kidneys, while the other part was distributed in to the tissues and further metabolized by the liver and kidneys, resulting in a reduction in the overall exposure in the body. Secondly, it may be due to the secondary absorption after drug absorption, which was excreted in the bile into the duodenum and forms the enterohepatic circulation (EHC) [24]. It can also be seen from the plasma concentration versus-time plot (Figure 3) that the curve in the physiological group showed a second absorption peak between 4 and 8 h after high-dose administration, whereas the model group showed a single peak curve. Thirdly, it was conceivable that the disease caused changes in transporter expression that affect the secondary absorption of the drug [25]. The increase in V_z/F_−obs of AH in the model group ensured more effective distribution of the drug to the target organs and tissues to produce effects, which was one of the key factors contributing to the differences in pharmacokinetic behavior and pharmacological effects between physiological and model rats.

In addition, drug transporters and hepatic CYP450 metabolizing enzymes play a very important role in the pharmacokinetic process of drugs in vivo. Previous studies have shown that the expression of metabolic enzymes and transporter proteins was altered in organisms under pathological conditions, thereby affecting the absorption, distribution, and metabolism of drugs [21,26,27]. In the present study, the prolongation of the plasma half-life time of AH in rats in the CIRI model group may also be attributed to the disruption of the hepatic CYP450 enzyme system due to pathological conditions, which led to a slowing of the rate of its metabolism of the drug.

3.4. PD study

During CIRI, reperfusion triggers excessive ROS production that damages endothelial cells and induces pro-inflammatory factors (IL-1β, IL-6, TNF-α), thereby amplifying the inflammatory cascade [1]. IL-1β exists as an initiator of the modulation of the inflammatory response during CIRI [28]. Lactate dehydrogenase (LDH) is an indicator of oxidative stress and the most sensitive enzyme to brain tissue damage during the development of cerebrovascular disease [29]. Therefore, IL-1β and LDH were chosen as pharmacodynamic indicators for the study. The levels of IL-1β and LDH in rat plasma after administration of AH to physiological and model groups were determined according to the steps of the corresponding kit instructions to evaluate the pharmacodynamic effects of AH on CIRI. The mean plasma efficacy concentration–time profiles in the physiological and model groups of rats are shown in Figure 4(A, B). The results measured in physiological rats after 24 h of CIRI establishment and 24 h of CIRI+AH (20 mg/kg) were graphed to obtain histograms of changes in efficacy as shown in Figure 4(C, D). The results showed that with the absorption, distribution, and metabolism of the drug in the body after administration, the levels of IL-1β and LDH in rats of both physiological and model groups showed a trend of increasing and then decreasing. Furthermore, IL-1β levels (p < 0.0001) and LDH levels (p < 0.001) were significantly higher in the rats 24 h after modeling compared to the physiological group. After 24 h of treatment, LDH levels decreased significantly (p < 0.05) and IL-1β levels also decreased, but there was no statistical difference (p > 0.05). This implied that AH had an ameliorative effect on oxidative stress and inflammatory response due to CIRI injury. This was consistent with the results of a pharmacological mechanism study of AH for the treatment of CIRI [10,11].

Figure 4.

Results of IL-1β and LDH levels in rat plasma in physiological (control) and model (CIRI) groups, n = 6. (A)Curves of mean IL-1β concentration over time after administration in physiologic and model groups. (B) Curves of mean LDH viability values over time after administration in physiological and model groups. (C) Comparative results of IL-1β levels in control group, after 24 h of CIRI modeling and after 24 h of CIRI+AH (20 mg/kg). ****P＜0.0001，^nsP＞0.05. (D) Comparative results of LDH levels in the control group, after 24 h of CIRI modeling and after 24 h of CIRI+AH (20 mg/kg). *P＜0.05，***P＜0.001.

3.5. PK-PD analysis

To further evaluate the relationship between plasma drug concentration and drug efficacy, IL-1β and LDH and LDH were first used as indicators of drug efficacy. It was found that the time points of AH blood concentration and drug efficacy after drug administration were not in one-to-one correspondence, and the drug efficacy diminished with decreasing blood concentration. Subsequent analysis of mean plasma concentration–effect curves in the high-dose CIRI group. As shown in Fig. S2, the mean plasma drug concentration–effect curves for both IL-1β and LDH showed a counterclockwise direction, indicating the presence of a pharmacodynamic lag effect. This lag effect phenomenon justified the selection of an effect compartment model for integrated PK-PD analysis. Subsequently, based on the comparison of Akaike Information Criterion (AIC) and correlation coefficient (R²) [30], a two-compartment model without lag time was selected for the PK model, with a weight of 1/Yhat. For the PD link model, the Sigmoid-Emax model in the presence of baseline was selected. The Sigmoid-Emax model (Emax, EC₅₀, γ) is used to flexibly characterize diverse concentration–effect relationships, including those with baseline effects, saturation phenomena, and sigmoidal curves [31]. The Hill’s equation was formulated as follows:

$$\mathit{E}=E_0+\frac{{\mathit{E}}_{\mathit{max}}\ast C^{\gamma }}{C^{\gamma }+\mathit{E}{C}_{50}^{\gamma }}$$ (1)

E was the predicted effect of the drug, E₀ was the initial effect described by the efficacy, E_max was the maximum effect described by the efficacy, C was the plasma concentration of AH, and EC₅₀ was the plasma concentration corresponding to 50% of the maximum effect. γ was the slope of the midpoint of the curve and describes the shape factor for the sensitivity of the concentration–effect relationship. The parameters of the goodness-of-fit index of the PK-PD model fit were shown in Table 3. The results of the pharmacodynamic parameters obtained from the ultimate fit were summarized in Table 4.

Table 3.

PK-PD model evaluation indexes (AIC and R²) in model group.

Indicators	PK model evaluation		PD model evaluation		PK-PD model evaluation (AIC)
	AIC	R2	AIC	R2
IL-1β	37.5	0.994	22.1	0.910	268.65
LDH			58.7	0.871	348.56

Table 4.

PK-PD fitting parameters for IL-1β and LDH pharmacodynamic parameters.

Index	Parameters	Unit	Physiological group	CIRI group
IL-1β	Emax	pg/mL	11.25	19.65
	EC50	ng/mL	211.65	368.27
	E0	pg/mL	7.19	16.41
	γ		16.17	2.35
	Ke0	1/h	9.0	5.0
LDH	Emax	pg/mL	138.76	274.56
	EC50	ng/mL	91.56	92.37
	E0	pg/mL	562.27	1017.19
	γ		1.699	9.97
	Ke0	1/h	1.0	10.0

3.6. Tissue distribution study

The obtained supernatants of 15 rat tissue homogenates after drug administration were diluted 20-fold with the corresponding blank matrix before biological sample preparation operation, and AH concentration in different tissues was detected. The results of AH mean concentrations in each tissue at different time points are summarized in Figure 5 and Table 5.

Figure 5.

Mean concentrations of AH in tissues at different time points after a single oral administration in rats (mean ± SD, n = 6). (A) and (B) mean concentrations of AH in tissues at different time points after a single oral administration in control group. (C) and (D) mean concentrations of AH in tissues at different time points after a single oral administration in CIRI group.

Table 5.

Concentrations of AH in tissues after oral administration in control and CIRI rats (mean ± SD), n = 6.

Tissues name	Unit	0.33 h		1 h		4 h		24 h
		Control	CIRI	Control	CIRI	Control	CIRI	Control	CIRI
Heart	ng/g	422.46 ± 171.05	286.29 ± 157.24	179.78 ± 91.20	215.27 ± 87.49	24.49 ± 24.49	35.72 ± 37.92	3.44 ± 5.86	9.60 ± 7.30
Liver	ng/g	2979.53 ± 1876.90	994.82 ± 232.14*	879.71 ± 514.05	1454.86 ± 1148.46	108.44 ± 51.64	112.49 ± 49.18	24.03 ± 4.96	48.95 ± 29.51
Spleen	ng/g	1323.88 ± 579.8	1018.01 ± 525.62	613.48 ± 414.32	600.14 ± 200.89	77.65 ± 48.69	96.43 ± 44.55	2.59 ± 1.34	29.05 ± 19.46*
Lung	ng/g	950.91 ± 350.91	766.58 ± 327.94**	477.07 ± 214.34	457.23 ± 166.12	52.37 ± 31.17	70.47 ± 38.39	2.33 ± 3.89	18.68 ± 17.94**
Kidney	ng/g	2564.24 ± 550.29	1630.62 ± 775.86*	1413.63 ± 748.99	1648.50 ± 493.44	632.80 ± 97.71	334.42 ± 89.50****	201.99 ± 40.60	243.94 ± 50.94
Brain	ng/g	182.74 ± 84.99	193.42 ± 63.25	145.33 ± 104.83	199.85 ± 97.65	34.44 ± 16.79	35.54 ± 15.96	—	7.93 ± 7.20**
Stomach	ng/g	8994.52 ± 1835.42	10564.49 ± 5321.83	6084.37 ± 3036.20	7988.76 ± 2957.28	480.40 ± 551.91	1673.70 ± 388.47***	3.55 ± 0.82	450.10 ± 586.63
Small intestine	ng/g	12804.92 ± 10146.28	4970.21 ± 4949.12	8002.81 ± 8868.95	5092.76 ± 6222.98	572.17 ± 461.29	361.87 ± 208.16	16.84 ± 16.79	58.60 ± 42.20
Large intestine	ng/g	3416.24 ± 3256.65	4041.12 ± 2816.88	1039.54 ± 967.66	1921.26 ± 2379.72	85.59 ± 96.46	466.70 ± 319.95**	11.93 ± 4.73	144.27 ± 113.99*
Skeletal muscle	ng/g	334.16 ± 105.64	308.53 ± 198.03	197.85 ± 95.92	229.31 ± 97.46	25.47 ± 13.94	26.93 ± 8.91	—	8.56 ± 8.60**
Bladder	ng/g	1487.12 ± 2346.31	3267.30 ± 4420.87	350.54 ± 258.38	4002.42 ± 3669.05	255.29 ± 217.94	294.86 ± 173.58	5.49 ± 5.20	101.91 ± 124.99**
Prostate	ng/g	539.49 ± 241.06	1172.92 ± 802.01	323.82 ± 198.33	1226.47 ± 1267.56	86.31 ± 58.42	127.14 ± 138.62	2.08 ± 1.73	31.11 ± 28.68
Testicles	ng/g	274.68 ± 300.92	196.92 ± 108.85	197.73 ± 54.91	344.83 ± 223.52	69.88 ± 26.64	74.66 ± 30.81	1.61 ± 2.91	11.74 ± 9.83**
Fat	ng/g	51.97 ± 19.93	77.25 ± 31.45	44.22 ± 35.15	62.67 ± 21.85	6.22 ± 2.38	15.73 ± 13.54	0.78 ± 1.90	3.10 ± 3.00
Oophoron	ng/g	1098.54 ± 173.54	1351.77 ± 362.84	323.40 ± 91.29	964.04 ± 440.57*	137.32 ± 54.25	120.70 ± 34.39	1.56 ± 1.22	2.54 ± 0.46

Comparison of control and CIRI groups: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

The results showed that AH was rapidly and extensively distributed in tissues after drug administration, which was consistent with the results of plasma pharmacokinetic studies. In the physiological group, within 1 h after drug administration, the AH concentrations in the stomach and small intestine were the highest, followed by the kidney, liver, large intestine, spleen, lung and bladder, and in the brain and adipose tissue were relatively low. As an orally administered formulation in this study, AH undergoes primary absorption through the gastrointestinal tract. Consequently, elevated drug concentrations were observed in both gastrointestinal tissues and the liver. Second, renal, bladder and large intestine drug concentrations were also relatively high, suggesting that the drug is eliminated at a faster rate and is rapidly transported to the excretory tissues. In addition, AH could cross the blood–brain barrier, but the brain concentration is lower than other tissues, probably due to its general lipid solubility. Compared with the control group, the pathological state of CIRI altered AH’s pharmacokinetic profile. The overall absorption of AH in the pathological state of rats was reduced, and the in vivo retention time was significantly prolonged. The drug concentration in brain tissue was significantly higher than that in the physiological group, probably because the disruption of blood–brain barrier permeability in the pathological state led to the abnormal distribution and reservoir formation of the drug in the central nervous system, which enabled the drug to better exert its therapeutic effect. After 4 hours’ drug administration, in the CIRI group compared to the control group, AH concentrations were highest in the kidney, and relatively high in the bladder and large intestine. It was suggested that the drug was mainly metabolized and excreted by the liver and kidney, as well as by the formation of feces in the large intestine. AH was almost undetectable in brain and skeletal muscle at 24 h. In the CIRI group, after a single oral administration, AH concentrations were consistently highest in the rat stomach and lowest in fat. Interestingly, the drug concentrations in the stomach, large intestine, brain, bladder, prostate, fat, and ovary in the CIRI group were significantly higher than those of the physiological group. In addition, the concentration of AH in each tissue of the physiological group began to show a large downward trend at 1 h, while in the CIRI group, it began to decrease significantly at 4 h (Fig. S3). There may be several reasons: 1. Due to the influence of the brain-gut axis, CIRI caused intestinal flora dysbiosis in rats [32,p.33], which affected the digestive and absorption function and prolonged the time of drug accumulation. 2. CIRI-caused inflammatory response increased the permeability of the cell membrane in tissues [26]. 3. The CYP450 enzyme system was affected by the pathological state of CIRI, which slowed the rate of drug metabolism [34]. 4. The concentration of the drug in the brain tissue was high and maintained for a long time, which might be caused by CIRI increasing the permeability of the blood–brain barrier in rats as well as the AH exerting its pharmacological effects to ameliorate the CIRI-induced brain injury [10,11,19].

4. Conclusions

In this study, we established a validated UPLC-MS/MS method for the simultaneous determination of plasma and tissue concentrations of AH and plasma concentrations of its metabolite NOAT3 in physiological and CIRI model rats. A combined PK-PD model was established in physiological and CIRI model rats using AH as a pharmacokinetic marker and IL-1β and LDH as pharmacodynamic indicators. The results of the PK study showed that the pharmacokinetic behavior of rats after CIRI differed in vivo, mainly in terms of wider tissue distribution, longer retention time in vivo, and slower metabolism. The results of the tissue distribution studies validated the plasma PK findings. Discrepancies in PK parameters occur for a number of reasons, including individual differences, pathological condition of the rat, cell membrane permeability, administered dose, drug-plasma protein binding, and metabolizing enzymes. PD studies had shown that AH had antioxidant and anti-inflammatory effects on CIRI and that there was a delayed effect in the onset of action. AH targets multiple pathological processes involved in cerebral ischemia-reperfusion injury, including microcirculation dysfunction, inflammation, oxidative stress, and apoptosis, which is in line with the complex pathophysiological characteristics of cerebral ischemia/reperfusion. Furthermore, as a selective muscarinic receptor antagonist with potent central and peripheral anticholinergic effects, AH exhibits greater blood–brain barrier permeability compared to anisodamine and a more favorable safety profile (lower toxicity) than atropine, representing significant advantages. In conclusion, this study demonstrates the promising therapeutic potential of AH for cerebral ischemia-reperfusion injury. Future research should focus on human-dose prediction based on PK characteristics from animal studies, with the objectives of defining the optimal effective dose, establishing the therapeutic window, and conducting comprehensive safety assessments.

Funding Statement

This work was supported by National Natural Science Foundation of China [81891012, 81630101, and U19A2010], Sichuan Province Science and Technology Support Program [2021JDRC0041, 2022ZYD0088], Innovation Team and Talents Cultivation Program of National Administration of Traditional Chinese Medicine [ZYYCXTD-D-202209], Sichuan TCM Science and Technology Industry Innovation Team [2022C001]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Article highlights

• We established a sensitive and rapid LC-MS/MS method for the first time to characterize the plasma kinetics and tissue distribution after oral administration of AH in physiological and CIRI model rats.

• Under physiological conditions, AH was rapidly absorbed into the bloodstream after oral administration, between 15 and 30 min, with a small difference in the peak time from that of the physiological group; under pathological conditions, the t_1/2 of AH in the plasma of CIRI rats was prolonged, which was more conducive to the exertion of the efficacy of the drug.

• There was a time lag in the efficacy of AH in CIRI model rats using PK-PD combined analysis with IL-1β and LDH as pharmacodynamic indicators.

Author contributions

Yujie Yu and Yanfang Liu: Conceptualization, Methodology, Formal analysis, Investigation, and Writing – Original draft. Jianlan Zhang: Validation and Writing – Original draft. Shu Dai: Writing – Original draft and Visualization. Rui Wu: Investigation. Feng Wan: Supervision. Chenhao Yao: Visualization. Yuxin Yao: Investigation. Feng Nan: Supervision, and Writing – Review and Editing. Yunxia Li: Conceptualization, Supervision, and Writing – Review and Editing.

Disclosure statement

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose

Ethical declaration

Our research adhered to the ARRIVE guidelines. The animal study protocol was approved by the Institutional Review Board (or Ethics Committee) of Chengdu University of Traditional Chinese Medicine (No. 20160705).

Data availability statement

The authors confirm that the data supporting the findings of this study are available within the article.

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/17576180.2025.2554567