Effects of external acupoint therapy using sinomenine powder against rheumatoid arthritis based on pharmacokinetic and pharmacodynamic fitting

To the Editor: Acupoint sticking therapy reportedly enhances the therapeutic efficacy of drugs,^[1] with corresponding acupoints for the treatment of diseases, such as rheumatoid arthritis (RA) and dysmenorrhea. However, reports on the differences in the concentration and efficiency of bioactive constituents for acupoint and nonacupoint applications are lacking. The pharmacokinetics/pharmacodynamics (PK/PD) model is suitable for explaining bioactive constituents and pharmaceutical effects in vivo, and the relationship between PK and PD has been applied in traditional Chinese medicine (TCM) studies. Accordingly, PK/PD analyses may improve our understanding of the scientific connotation of acupoint administration by observing changes in drug efficacy with dose and time after acupoint administration. However, it remains difficult to establish a PK/PD model with high accuracy after acupoint administration. The development of the microdialysis sampling technique has enabled continuous monitoring of reactions in vivo for pharmaceutical analysis.^[2] In the present study, we evaluated the external application of sinomenine powder, a classic TCM powder used to treat RA, as a marker of the efficacy of acupoint application. Additionally, we assessed differences in the concentrations and efficacies of bioactive constituents between acupoint and nonacupoint applications using our PK/PD model and the microdialysis sampling technique.

We used an egg albumin-induced RA model. The method for model generation is shown in Supplementary Table 1, https://links.lww.com/CM9/A970. The criteria for human RA published by the American College of Rheumatology were used to determine whether the model was successful. All animal experiments were performed in accordance with and were approved by the Animal Ethics Committee of Heilongjiang University of Traditional Chinese Medicine (No. 2016101601).

After successful modeling, 20 male New Zealand rabbits (2.5–3.0 kg, purchased from the Experimental Animal Center, Heilongjiang University of Traditional Chinese Medicine, certificate No. SCXK [Hei] 2013-004; Heilongjiang, China) were randomly divided into five groups [Supplementary Table 2, https://links.lww.com/CM9/A970]. The ST 36, GB 34, and nonacupoint groups were administered 400 mg/kg sinomenine topically onto 0.6 cm² skin on shaved legs. The oral administration group was administered 400 mg/kg sinomenine powder by gavage. Microdialysate samples were collected 30 minutes post-treatment and 1–14 hours posttreatment at a unit interval via microdialysis probes. All probe implantation and treatment methods of microdialysis samples are summarized in Supplementary Tables 3, https://links.lww.com/CM9/A970 and 4, https://links.lww.com/CM9/A970. The reference-substance solution was prepared with 2.08 mg sinomenine in 100 mL volumetric flasks.

Sinomenine concentration in the microdialysis samples was determined by ultra-high-performance liquid chromatography-tandem mass spectrometry. The liquid phase conditions and conditions for mass spectrometry are shown in Supplementary Table 5, https://links.lww.com/CM9/A970. PK parameters, such as the peak plasma drug concentration, were read visually from the concentration-time profiles. Other PK parameters, such as the area under the time-concentration curve, were obtained using the WinNonlin Examples Guide, version 5.2.1 (Certara, Princeton, NJ, USA). Treatment of blood samples is shown in Supplementary Table 6, https://links.lww.com/CM9/A970. Statistical analysis was conducted using SPSS 23.0 (IBM Corp, Armonk, NY, USA).

Changes in sinomenine concentration in the microdialysis solution in the knees of RA model rabbits are shown in Supplementary Figure 1, https://links.lww.com/CM9/A970. Analyses of sinomenine concentrations in the articular cavities of rabbits in the five groups based on the above parameters and chart analysis were reported previously. The three percutaneous application routes each had a slower absorption rate. However, the durations of the three percutaneous application routes, particularly for ST 36, were longer than those of oral administration. Further, ST 36 showed a higher area under the curve (AUC).^[3]

The inhibition rate (IR) or ratio was selected in accordance with the PD indices as the effect value E in the PD study. For IL-1, matrix metalloproteinase (MMP)-3, and rheumatoid factor (RF)-IgM, the IR was calculated as $E_{IR}\left(E_{IR}=\text{IR lrb}\%\right)=\frac{C_0-C_t}{C_0}\times 100\%$, where C₀ is the initial concentration and Ct is the measured value. The average E_IR values of the three PD indices in the blank control group within 14 hours were as follows: RF-IgM, −3.22 ± 4.04; IL-1, 2.34 ± 1.04; and MMP-3, −1.68 ± 2.1, indicating no changes in the E_IR values of the three PD indexes in the blank control group. Plots of the E_IR values for sinomenine concentrations in the joint cavity are shown in Figure 1A–E. The efficacy did not correspond to changes in plasma concentrations, confirming the existence of the effect chamber. The concentration ratio of OPG and RANKL in plasma was chosen as another effect value, E_ratio, calculated as follows: E_ratio = osteoprotegerin (OPG)/receptor activator of nuclear factor kappa-B ligand $\left(\text{RANKL}\right)=\frac{C_{OPG}}{C_{RANKL}}$. The average E_ratto of OPG/RANKL in the blank control group within 14 hours was 0.014 ± 0.008, indicating no changes in the E_ratio in the blank control group. However, significant changes were observed (P < 0.05). The plot of E_ratio values of sinome-nine concentrations in the joint cavity is shown in Figure 1F, showing that despite the gradual reduction in sinomenine concentration, the effect initially increased and then decreased.

Figure 1: (A-F) RF-IgM, IL-1, MMP-3, arginine, citrulline, and OPG/RANKL IR-concentration curve in plasma samples for each administration route. (G-I) F _max, EC _e50, and K _e0 value cumulative graph was obtained by adding the MMP-3, OPG/RANKL, IL-1, and RF-IgM parameters for every administration route. IL: Interleukin; IR: Inhibition rate; MMP-3: Matrix metalloproteinase-3; OPG: Osteoprotegerin; RANKL: Receptor activator of nuclear factor kappa-B ligand.

The PK/PD fitting parameters for four PK indicators are shown in Supplementary Table 7, https://links.lww.com/CM9/A970. As shown in Figure 1G, the cumulative value of E_max for the typical acupoint ST 36 was the highest. From the cumulative value of E_Ce50 shown in Figure 1H, the cumulated E_Ce50 value of the sham point group was the lowest, whereas that of the other three routes was the same. The cumulative K_e0 value for the ST 36 group was the lowest [Figure 1I].

PK results showed that the AUC of ST 36 was the highest, indicating that a higher bioactive component concentration was obtained with acupoint application compared to other drug delivery routes. This may be because drugs do not act exclusively on target organs but are otherwise transported or stored simultaneously. PD results verified the presence of effector sites. The concentration-effect curves of the four PD indicators showed a direct nonlinear correlation between the drug concentration and E_IR. The drug concentration in the articular cavity decreased gradually. However, the curative effect increased initially before decreasing, indicating that the PD effect lagged behind the change in blood drug concentration. A PK/PD fitting model was used to study the dynamic dose-effect relationship of sinomenine at 14 hours. Compared with other applications, ST 36 had the highest Emax, indicating that the prediction effect should be the best for ST 36. It also had the lowest K_e0, suggesting that drug elimination was the slowest and that efficacy would persist longer.

Our findings showed that sinomenine penetrated more deeply into the cuticle and then was slowly released to exert therapeutic effects after ST 36. Plasma concentrations and tissue distributions are generally positively correlated with pharmacological effects. Increases in sinomenine AUC, C_max, and E_max values after acupoint application can help strengthen the effects of sinomenine powder after ST 36 in RA animal models and aid the exploration of its mechanisms.

A real-time, dynamic, in vivo microdialysis sampling method was successfully applied before PK/PD analyses of sinomenine knee cavity solution after acupoint and nonacupoint applications were performed. The fitting parameters showed that acupoint application resulted in a longer drug release time and higher efficiency of sinomenine compared with other drug delivery routes. These results may provide a reasonable explanation for choosing acupoint application and clarify the mechanisms of the beneficial treatment effects obtained after acupoint application.

Acknowledgements

We would like to thank the staff and postgraduate students at Heilongjiang University of Traditional Chinese Medicine for their assistance in carrying out the pharmacy research.

Funding

This research work was supported by grants from the National Natural Science Foundation of China (Nos. 82074271 and 81473359), the Natural Science Foundation of Heilongjiang Province (No. H201472), the Central Government Supports Local College Reform Projects (No. 2020YQ05).

Conflicts of interest

None.