Abstract
SZJ-1207 is an antidepressant natural product with a steroidal structure extracted from Stephanotis mucronata. It is a novel antidepressant candidate molecule and all its pharmacokinetic properties have not been reported. In this pharmacokinetic study in mice following oral administration, an accurate and sensitive UPLC-MS/MS method was established and evaluated to measure SZJ-1207 concentrations in mouse plasma and brain samples. The results provide information regarding the pharmacokinetics of SZJ-1207 as a potential antidepressant.
Introduction
Mental disorders such as Major depressive disorder (MDD) seriously threaten human health [1]. The Lifetime/12-month prevalence of MDD was estimated to be 6.1%/2.2% in Japan [2], 20.6%/10.4% in America [3] and 3.4%/2.1% in China [4]. Though there is a large amount of patients, most antidepressants work poorly, remission is only obtained in approximately 30% [5] of patients due to the complex mechanism of depression [6] while various side effects lead to poor compliance [7].
Chinese herbal medicine and its extracts have been proven to have favorable antidepressant activity with reliable safety and remarkable efficacy [8, 9]. Various components made it necessary to confirm the essential compounds in anti-depression in order to meet the needs of the standardized study of traditional Chinese medicine [10]. In fact, some natural products have been certificated to have obvious antidepressant effects [11–13].
Interestingly, many natural products with antidepressant activity have steroid structures, such as Lilium saponins [14], gypenosides [15], ginsenoside [16] and Panax notoginseng saponins [17], which suggest that natural products with steroidal structures may have potential antidepressant activity. Actually, the antidepressant drug allopregnenolone approved by FDA is also with a steroidal structure [18]. Steanthraniline A (STA) was a natural product extracted from Stephanotis mucronata (Blanco) Merr. with steroidal structure found to have anti-inflammatory effect, which was related to the inflammatory hypothesis of depression [19]. Based on this, natural products extracted from S. mucronata (Blanco) Merr. have been identified and two compounds with antidepressant activity have been reported [20].
(3S,8S,10R,12R,13R,14R,17S)-17-((S)-1-hydroxyethyl)-10,13-dimethyl-2,3,4,7,9,10,11,12, 13,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthrene-3,8,12,14,17-pentaol (SZJ-1207) is also an antidepressant natural product extracted from S. mucronata (Blanco) Merr. with a steroidal structure exhibited in Fig. 1. Preliminary studies [21] have shown that SZJ-1207 has significant antidepressant effects in a variety of depression mouse models, and no obvious acute toxicity and sedative-hypnotic side effects have been observed. However, the pharmacokinetic data of SZJ-1207 are still unclear, and the determination of SZJ-1207 in plasma and brain requires a quantitative analysis method.
To determine the mouse plasma and brain concentrations of SZJ-1207, a reliable analytical method development was conducted. Furthermore, pharmacokinetic parameters of SZJ-1207 in mouse were measured and pharmacokinetic properties of SZJ-1207 were preliminarily evaluated.
Experimental
Reagents and materials
SZJ-1207 (purity = 99.53%), Deacylmetaplexigenin (DMP, purity = 98.0%) were provided by the Natural Medicine Research Group of Hangzhou Medical College.
Methyl tert-butyl ether (MTBE), acetonitrile and pure water were purchased from TEDIA (USA), Merck (Germany) and Watsons (China), respectively. Heparin sodium was from Biosharp (China). Zoletil™ 50 was from Virbac (France). Blank mouse plasma and brain samples were collected in our laboratory.
Instruments and parameters for UPLC-MS/MS
AB SCIEX API 5500 UPLC-MS/MS System and Waters ACQUITY UPLC HSS C18 (2.1 × 100 mm, 1.8 μm) were prepared for analysis. The mobile phase consisted of water (A) and acetonitrile (B) while the flow rate was 0.3 mL min−1. Gradient elution was displayed as follows: 0–1.4 min, 60% A; 1.4–1.6 min, 60–10% A; 1.6–4.0 min, 10% A; 4.0–4.1 min, 10–60% A; 4.1–5.0 min, 60% A. The column oven temperature was 40 °C while the autosampler temperature was 4 °C. All samples were detected by mass spectrometry at 0–1.4 min, and then the elutions flowed into the waste liquid at the rest of time.
The negative mode was chosen for electrospray ionization source while multiple-reaction monitoring was selected as scan mode. Dominating mass spectrometer parameters were shown in Table 1. Ion source temperature was 550 °C. Ionspray voltage was −4500 V. As for curtain, nebulizing and auxiliary gas, the pressure was 35, 45 and 45 psi, respectively.
Ion pairs, declustering potential and collision energy of SZJ-1207 and DMP
| Compound | Q1→ Q3 (m/z) | Declustering potential (V) | Collision energy (eV) |
| SZJ-1207 | 381.4→245.1 | −206 | −39 |
| DMP | 379.3→343.3 | −180 | −27 |
Animals
ICR mice (SPF, 24 males, 24 females, body weight 20–25 g) were provided by the Experimental Animal Center of Hangzhou Medical College. Ethics was approved for all animal experiment schemes by Ethics Committee of Hangzhou Medical College (ZJCLA-IACUC-20060027).
Stock and working solutions preparation
A stock solution of SZJ-1207 (0.96 mg mL−1) for calibration standard was obtained in acetonitrile. Meanwhile, a stock solution of SZJ-1207 (1.06 mg mL−1) in acetonitrile was prepared for quality control (QC).
A stock solution of DMP was yielded in acetonitrile for internal standard (IS, 1.40 mg mL−1). A dilution with acetonitrile prepared working solutions of analyte and IS (25 ng mL−1). All solutions were stored away from light at 4 °C.
Sample preparation
Mixed 50 μL of plasma sample with 10 μL of DMP working solution for 1 min, then 400 μL of MTBE was used as an extraction reagent. Following this, samples were blended by a multi-tube vortexer and centrifuged for 5 min with the centrifugal force of 4000g. Organic phase was then separated and dried at ambient temperature. The residue was dissolved in acetonitrile-water (30:70, v/v) for 100 μL and 10 μL of the processed samples were injected for detection.
Took the cryopreserved mouse brain sample, smashed it with a clean pipette tip, put the pieces into a 2 mL homogenization tube, added normal saline (NS) at a mass-to-volume ratio of 1:4 (m Brain: V NS = 1: 4) and then homogenized for 30 s at 5,000 rpm. Following that, the same operations as for a plasma sample were carried out.
Method validation
By contrasting the chromatograms of blank plasma sample from six mice with the lowest limit of quantitation (LLOQ) sample, the selectivity was confirmed.
The sensitivity was measured by the signal to noise (S/N) ratio of LLOQ sample.
The carryover effect was evaluated by detecting two blank samples after ULOQ sample.
2.5 μL of working solutions of calibration standards was added into 1.5 mL centrifuge tube, dried the solution under nitrogen. Calibration standards at seven levels were prepared using 50 μL of blank matrix, the nominal concentration ratios of SZJ-1207 and DMP (X-axis) were plotted as a function of peak area ratios (Y-axis).
The precision and accuracy were evaluated by the QC samples at four levels in three independent batches.
The extraction recovery was obtained by comparing the peak areas of SZJ-1207 and IS of low, middle and high levels of QC samples (n = 6) with blank matrix extracts spiked with standard solutions at corresponding concentrations (n = 3).
The matrix effect was assessed by contrasting the peak areas of blank matrix extracts from six lots spiked with standard solutions at LQC (low quantitation control) and HQC (high quantitation control) with those of pure water extracts spiked with equivalent standard solutions (n = 3).
The matrix stability at LQC and HQC was studied after exposure to the following conditions: ambient temperature for 3 h, −70 °C for two freeze–thaw cycles.
The post-preparative stability was studied with low, middle and high levels of QC samples (n = 6). And the prepared samples were stored in autosampler for 24 h prior to injection.
Pharmacokinetic study
Before dosing, 48 mice (half male and half female) were fasted for solids but not for liquids overnight and administered orally with SZJ-1207 suspension at a single dose of 40 mg kg−1. Anesthetized mice using ZoletilTM 50, about 0.6 mL blood samples (3♀, 3♂) were collected from hearts quickly in 60 s and then hippocampi (3♀, 3♂) were isolated at pre-dose and 10, 20, 30, 60, 90, 120 and 240 min post-dose. Blood samples were stored on wet ice and followed by centrifuging at 8,000 g for 10 min within 0.5 h. Then plasma samples and hippocampi were kept at −70 °C until analysis.
Data analysis
Data were processed from Analyst 1.6.3 and analyzed by MultiQuant 3.0.2. Pharmacokinetic parameters were calculated by non-compartment model applying Phoenix WinNonlin software (version 8.1.0). The brain to plasma ratio (B/P) was calculated by the following formula: B/P = AUCbrain/AUCplasma
Results and discussion
Method optimization
Firstly, the MS parameters were optimized to achieve an excellent MS response. For SZJ-1207, poor MS response was observed in positive ionization mode, but it showed good response in negative ionization mode, so the negative mode was chosen for the method. And the product ion mass spectra in negative mode are shown in Fig. 2.
Product ion mass spectra of (A) SZJ-1207 and (B) DMP
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Product ion mass spectra of (A) SZJ-1207 and (B) DMP
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Product ion mass spectra of (A) SZJ-1207 and (B) DMP
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Sample extraction methods were evaluated before method validation. When samples at concentration of 0.5 ng mL−1 were prepared by protein precipitation with acetonitrile, the signal to noise (S/N) ratios of SZJ-1207 were lower than five, and interference peak made it difficult for precise quantification. When the same samples were prepared by liquid-liquid extraction with MTBE, SZJ-1207 showed higher response with lower interference.
The ratio of the reconstituted solution was optimized, water-acetonitrile (70:30, v/v) was proven to be benefit for good peak shape and high response. Isocratic elution was adopted to ensure good separation of peaks of SZJ-1207 and IS from impurity peaks. However, it was found that the mass spectral response of the latter sample decreased significantly when the sample was repeatedly injected, which was presumed to be caused by the matrix effect caused by the late distillate components (macromolecular non-polar substances) in the former sample. Therefore, a gradient elution procedure was added in which the proportion of organic phase was increased. As a result, the elution of non-polar substances was accelerated to eliminate the interference to the latter sample. The elution time of this method is slightly longer, and there is still room for optimization.
Method validation
Blank plasma samples were not found to have any significant interference peaks in six lots at the retention time of SZJ-1207 and DMP (Fig. 3).
Representative chromatograms of SZJ-1207 (A) and DMP (B) for blank matrix sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Representative chromatograms of SZJ-1207 (A) and DMP (B) for blank matrix sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Representative chromatograms of SZJ-1207 (A) and DMP (B) for blank matrix sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
The SZJ-1207 S/N ratio in the chromatogram of LLOQ sample was greater than five (Fig. 4).
Representative chromatograms of SZJ-1207 (A) and DMP (B) for LLOQ sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Representative chromatograms of SZJ-1207 (A) and DMP (B) for LLOQ sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Representative chromatograms of SZJ-1207 (A) and DMP (B) for LLOQ sample
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
The results indicated that the carryover effect of SZJ-1207 and DMP was acceptable.
Standard curves were validated at the concentration of 0.5–400 ng mL−1 for plasma and 0.5–100 ng mL−1 for brain. Correlation coefficients and back-calculated concentrations of each batch met the acceptable standard.
The data of SZJ-1207 at four QC levels were exhibited in Table 2, which demonstrated that the method was sufficiently precise and accurate.
Precision and accuracy of SZJ-1207 in mouse plasma and brain (n = 6)
| SZJ-1207 | Theoretical concentration (ng mL−1) | Intra-day (n = 6) | Inter-day (n = 18) | ||||
| Mean measured concentration (ng mL−1) | RSD (%) | RE (%) | Mean measured concentration (ng mL−1) | RSD (%) | RE (%) | ||
| Plasma | 0.50 | 0.479 | 6.5 | −4.3 | 0.486 | 10.7 | −2.9 |
| 1.50 | 1.39 | 3.5 | −7.6 | 1.44 | 7.0 | −3.9 | |
| 20.0 | 19.6 | 2.6 | −1.8 | 18.9 | 5.0 | −5.4 | |
| 320 | 360 | 2.3 | 12.6 | 354 | 3.8 | 10.7 | |
| Brain | 0.50 | 0.527 | 8.8 | 5.3 | 0.458 | 14.5 | −8.4 |
| 1.50 | 1.39 | 5.4 | −7.1 | 1.53 | 9.5 | 2.2 | |
| 10.0 | 10.1 | 3.8 | −5.4 | 10.2 | 4.4 | 1.9 | |
| 80.0 | 75.7 | 2.6 | −7.5 | 78.8 | 7.2 | −1.4 | |
As shown in Table 2, precision and accuracy were acceptable at four tested levels. SZJ-1207 concentrations in samples exceeding ULOQ could be diluted with blank matrix and then be detected.
The recovery of SZJ-1207 was reproducible, and the matrix effect on SZJ-1207 was ignorable (Table 3).
Recovery and matrix effect of SZJ-1207 in mouse plasma and brain (n = 6)
| SZJ-1207 | Theoretical concentration (ng mL−1) | Recovery | IS normalized matrix factor | ||
| Mean | RSD (%) | Mean | RSD (%) | ||
| Plasma | 1.50 | 90.9 | 8.7 | 110.5 | 8.4 |
| 20.0 | 97.6 | 3.7 | NA | NA | |
| 320 | 84.7 | 3.1 | 111.1 | 11.5 | |
| Brain | 1.50 | 74.4 | 3.9 | 104.6 | 4.2 |
| 10.0 | 76.5 | 4.2 | NA | NA | |
| 80.0 | 74.3 | 2.2 | 106.3 | 3.7 | |
In Table 4, the short-term and freeze–thaw stability of SZJ-1207 satisfied the acceptance criteria.
Matrix stability and processed sample stability of SZJ-1207 in mouse plasma and brain (n = 6)
| SZJ-1207 | Theoretical concentration (ng mL−1) | Storage at room temperature for 3 h | Two freeze–thaw cycles (−70 °C) | Processed sample stability stored at 4 °C for 24 h | |||
| RSD (%) | RE (%) | RSD (%) | RE (%) | RSD (%) | RE (%) | ||
| Plasma | 1.50 | 3.8 | 1.6 | 3.8 | −5.4 | 2.7 | 5.2 |
| 20.0 | NA | NA | NA | NA | 2.7 | −5.2 | |
| 320 | 7.5 | 10.1 | 0.9 | 7.7 | 3.0 | 8.8 | |
| Brain | 1.50 | 1.6 | 2.0 | 7.3 | −8.3 | 4.8 | 4.7 |
| 10.0 | NA | NA | NA | NA | 3.9 | 6.1 | |
| 80.0 | 2.6 | 2.1 | 4.0 | −10.9 | 2.4 | −2.3 | |
The results of processed sample stability demonstrated that SZJ-1207 in processed sample was stable at 4 °C for 24 h (Table 4).
Pharmacokinetic study
After intragastric administration of SZJ-1207 to mice, Fig. 5 shows the plasma and brain concentration-time curves. Table 5 shows the pharmacokinetic parameters of SZJ-1207. The high Cmax and AUC of SZJ-1207 in plasma samples reveals its fairish absorption while short terminal half-life time (T1/2) reveals its fast elimination. Moreover, the Tmax of SZJ-1207 in plasma and brain is within 1 h, demonstrating its rapid absorption and distribution.
Average plasma and brain concentration-time curves of SZJ-1207 in mice after intragastric administration of 40 mg kg−1 (n = 6)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Average plasma and brain concentration-time curves of SZJ-1207 in mice after intragastric administration of 40 mg kg−1 (n = 6)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Average plasma and brain concentration-time curves of SZJ-1207 in mice after intragastric administration of 40 mg kg−1 (n = 6)
Citation: Acta Chromatographica 2023; 10.1556/1326.2022.01102
Pharmacokinetic parameters of SZJ-1207 in mice after oral administration at doses of 40 mg kg−1 (n = 6)
| SZJ-1207 | Matrix | AUC(0-t) (mg L−1 min or mg kg−1 min) | Cmax (mg L−1 or mg kg−1) | Tmax (min) | MRT(0–t) (min) | T1/2 (min) | CL/F (L min−1 kg−1) | B/P |
| po | plasma | 1507.86 | 20.68 | 30 | 64.61 | 30.76 | 0.026 | 0.173 |
| brain | 260.35 | 3.81 | 30 | 67.81 | 54.26 | 0.15 |
Lower Cmax and AUC were observed in brain samples, the T1/2 was the same as the plasma samples, which indictes poor content, fast elimination in brain and poor B/P ratio of SZJ-1207. These pharmacokinetic properties might be related to its chemical structure. Poly-hydroxyl structure hinders compounds to cross the blood-brain barrier (BBB). Besides, potential transporters may also affect the progress of SZJ-1207 across the BBB and deserve further investigations.
In past studies, researchers generally tended to design lipophilic central nervous system (CNS) drugs because higher lipophilicity is beneficial for drugs to pass through BBB [22]. However, higher lipophilicity may increase non-specific binding to brain tissue, and highly lipophilic compounds are inclined to be efflux transport substrates, which leads to a reduction of free drugs in brain [23]. Besides, studies have found that with the increase in fat solubility, the risk of toxicity of the compound increased [24], which suggested that CNS drugs should not be too lipophilic. SZJ-1207 reduces its lipophilicity by increasing the number of hydrogen bonds, which may lead to low total concentration but relatively higher free drug concentration in brain, thus showing antidepressant activity. The reduction of total brain concentration also reduced the possible CNS accumulation toxicity.
In the early stage of development of CNS drugs, B/P > 0.3 is often used as the screening standard for CNS drugs [25]. However, some CNS drugs marketed may not meet this standard, such as paliperidone (B/P = 0.06), sulpiride (B/P = 0.078) and midazolam (B/P = 0.23) [26]. Therefore, this prompted researchers to consider a new strategy of drug design. The efficacy results of SZJ-1207 suggested that the idea of sacrificing the permeability of BBB to increase free concentration of drugs in brain has a real possibility of success. Some studies have also found that depression patients may be accompanied with changes in BBB permeability [27], and altered BBB permeability may allow some drugs with poor permeability to enter brain to produce therapeutic effects. Last but not the least, the low concentration in brain and no obvious toxic effects [21] have been proved to provide a certain feasibility for its continuous administration, which may be able to overcome the fast clearance rate of SZJ-1207 in vivo. For further development of SZJ-1207, pharmaceutical approaches, such as sustained-release preparation or controlled-release preparation, can provide means to improve phamacokinetic characteristics of this compound.
Conclusions
In this study, an accurate and sensitive UPLC-MS/MS method was established and evaluated to measure SZJ-1207 in plasma and brain samples in order to primarily understand its pharmacokinetic properties. The results provided information regarding the pharmacokinetics of SZJ-1207 as a potential antidepressant. The pharmacokinetic properties of SZJ-1207 proved that it is feasible to develop CNS drugs according to the idea of reducing lipophilicity, which may lead to poor penetration across BBB but high free drug concentration in brain.
Conflicts of interest
The authors declare no conflict of interest.
Abbreviations
| IS | internal standard |
| LLOQ | lower limit of quantification |
| ULOQ | upper limit of quantification |
| QC | quality control |
| CNS | central nervous system |
| BBB | blood-brain barrier |
| B/P | brain to plasma ratio |
| NA | not applicable |
| RE | relative error |
| RSD | relative standard deviation |
| DMP | deacylmetaplexigenin |
| MTBE | methyl tert-butyl ether |
Acknowledgments
This work was supported by Grant-in Aid from Zhejiang Provincial Science and Technology Council [2018C03071].
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