Quantification of MDR-TB drug JBD0131 and its metabolite in plasma via UPLC-MS/MS: application in first-in-human study

Abstract

Aim: JBD0131, a novel anti-multidrug-resistant tuberculosis (MDR-TB) drug, can target and inhibit the synthesis of mycolic acids, which are crucial components of the cell wall of the Mycobacterium tuberculosis complex. To support the results of this clinical trial in healthy subjects, development of a specific and accurate quantification method for detecting JBD0131 and its metabolite DM131 in human plasma is needed.

Materials & methods: Samples with prior added stabilizer were pretreated by protein precipitation method and the extracts were subjected to ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The m/z transitions for the precursor/product ion pairs were 402.1/273 for JBD0131, 333.1/273 for DM131 and 386.1/257 for the internal standard (IS).

Results: This method showed good linearity from 1 to 2000 ng/ml for JBD0131 and 0.25 to 500 ng/ml for DM131 and was validated in terms of selectivity, linearity, accuracy, precision, matrix effect, recovery of pretreament and stability.

Conclusion: This method was sensitive and specific for measuring the plasma concentrations of JBD0131 and its metabolites. And it was applied for the investigation of the pharmacokinetics of JBD0131 and DM131 in a clinical trial.

Plain language summary

Article highlights.

• Necessity for the simultaneous determination of anti-TB drug JBD0131 and its major metabolite

  JBD0131 is a novel anti Multidrug-resistant tuberculosis (MDR-TB) drug, and it exhibited profound anti-M. tb activity in vitro and in preclinical study. DM131 is the major metabolite of it, although it didn't exhibited heart toxicity like its analog DM6705 (major metabolite of delamanid), it should be taken into concentration in this first-in-human study. So it is necessary to establish a quantitative method for the pharmacokinetic and dose-safety study.

• Establishment of a sensitive and specific LC–MS/MS method for JBD0131 and DM131.

  A sensitive and convenient LC–MS/MS method was established in Acquity™ Ultra Performance LC system and Xevo TQ-S triple quadrupole mass spectrometer (Waters).

• Development of a proper stabilizer formula for JBD0131 and DM131

  According to preclinical report, JBD0131 is unstable in plasma. For the accuracy and precision of quantification, we developed proper stabilizer for its storage before concentration detection.

• A linear response across a range of 1–2000 ng/ml for JBD0131 and 0.25–500 ng/ml for DM131 with only 0.05 ml plasma.

  The method easily detected JBD0131 at a level as low as 1 ng/ml and DM131 at a level of 0.25 ng/ml, and exhibit great selectivity, pretreatment recovery, accuracy and precision.

• Application in pharmacokinetic analysis in phase I clinical trial of JBD0131

  This method is successfully applied to the pharmacokinetic analysis in phase I clinical trial of JBD0131 on healthy subject. And result showed that when dose was climbed to 400 mg, the AUC of JBD0131 is no longer elevating.

1. Introduction

Tuberculosis (TB) is caused by infection with the bacillus Mycobacterium tuberculosis (M. tb). TB is a peril disease of the human population, and according to the WHO, tuberculous is a major cause of death in the human population after HIV/AIDS [1]. Multi and extensively drug-resistant (M/XDR) TB represents a real challenge for TB treatment. MDR-TB refers to patients who are resistant to at least one of the first-line anti-tuberculosis drugs rifampicin and isoniazid. It is generally caused by primary infection with drug-resistant M. tb or secondary drug-resistant bacteria caused by improper medication, and the cure rate is approximately 50–70%. In XDR-TB, M. tb-infected patients are not only resistant to isoniazid and rifampicin but also resistant to at least one of the fluoroquinolones and second-line injectable anti-TB drugs, and their cure rates are lower than those of MDR-TB [2–5].

Currently, drug-resistant pulmonary tuberculosis patients receive personalized treatment mainly based on the results of in vitro drug susceptibility testing. The course of treatment is long, and the cure rate is low [6–8]. Delamanid, as a part of the clinical treatment for MDR-TB, combined with optimized background therapy significantly increased the incidence of drug-resistant pulmonary tuberculosis in sputum culture conversion (SCC) (45.4 vs. 29.6%). Delamanid inhibits the synthesis of mycolic acids, a crucial component of the cell wall of the M. tb [9,10]. JBD0131 (Figure 1A) is a newly developed anti-MDR-TB compound that is also a nitroimidazole and inhibits M. tb through the same mechanism. According to preclinical studies, compared with delamanid, JBD0131 exhibited excellent activity against M. tuberculosis H37Rv in vitro. In mouse acute infection model, the pathogenic bacteria could be thoroughly suppressed with 100 mpk dose JBD0131 treatment and JBD0131’s relative distribution in lung (target organ) is more than delamanid. Besides, it exhibited reduced heart toxicity than delamanid [11] and unpublished data]. In summary, JBD0131 exhibited more superior efficacy and pharmacokinetic properties than delamanid in preclinical trial, making it a promising anti-MDR-TB drug.

Figure 1.

Chemical structure (A) JBD0131 (B) DM131 (C) Internal standard (IS).

A phase I clinical trial on the safety, tolerability, pharmacokinetic characteristics and food effects of JBD0131 tablets in healthy adult subjects was conducted in our center. The protocol was approved by the ethics committee of West China Hospital of Sichuan University (Chengdu, China) in September 2020. The dose used in this study was increased from 50 to 400 mg/kg when the AUC no longer increased with increasing dose. Considering that delamanid could be degradated by albumin in plasma [12], and which is worthy of note that the main metabolite (DM6705) of delamanid is the major factor that resulting to QTc prolongation [13,14]. The C_max of DM6705 under therapeutic dose is approximately four-times higher than hERG inhibition IC₅₀, that is why concentration of DM7605 was definitely investigated in research on delamanid. DM131 (Figure 1B) is the major active metabolite of JBD0131. Although there was no QT interval prolongation signal observed in the preclinical experiment of DM131, as an analog, it is worthy of attention in this first-in-human research. Thus, a suitable method for simultaneously detecting the plasma concentration of JBD0131 and DM131 is needed. There are some reports about determination of delamanid and its metabolite DM7605 by LC-MS/MS method [15–19]. Protein precipitation extraction or liquid-liquid extraction method were used to extract plasma samples, and extracts were analyzed using reversed phase UPLC-MS/MS and a electrospray ionization (ESI) source with a total run time of 4 to 12 min. While there is no quantification method for JBD0131 or its metabolites reported up to now. Here, we reported a method for determining the plasma concentration of JBD0131 and its metabolite DM131 by UPLC-MS/MS to evaluate its pharmacokinetic characteristics. In the method development procedure, the main problem we encountered was the instability of JBD0131 and DM131 in plasma and we developed a proper plasma stabilizer for this purpose.

2. Materials & methods

2.1. Chemicals & reagents

Reference JBD0131 (lot: 013, purity: 99.21%) was obtained from Changzhou Yinsheng Pharmaceutical Co., Ltd (Changzhou, China); DM131 (lot: EW22954–43-P1, purity: 95.65%) and Internal standard (IS, lot EW22954–5-P1, purity: 99.45%) (Figure 1C) was obtained from WuXi AppTec (Wuhan, China). Fat emulsion (20%, m/v) and physiological saline were supplied by Sichuan Kelun Pharmaceutical (Chengdu, China); MS-grade dimethyl sulfoxide (DMSO), methanol and acetonitrile were supplied by Fisher Scientific (Loughborough, UK); analytical grade ammonia, acetic acid and ammonium acetate were supplied by Chengdu Kelong Chemicals (Chengdu, China); and UPLC/MS-grade formic acid was supplied by Thermo Fisher Scientific (Waltham, MA, UK). Ultrapure water was produced by a Milli–Q purification system (Millipore, MA, USA).

2.2. Matrix

Blank human plasma and whole blood samples originating from healthy subjects were collected by additional blank blood collection protocol during the phase I clinical trial of JBD0131, and the protocol was approved by the Ethics Committee of West China Hospital [2020 (134)]. Lipemic matrix was obtained by adding blank plasma with 20% fatty milk (10%, v/v), while hemolyzed matrix was obtained by adding 2% (v/v) erythrocytes into blank plasma.

2.3. Stabilizers

The stabilizer A was prepared by mixing the supernatant of saturated ammonium acetate solution with acetic acid at a ratio of 10:3 (v:v). Mix stabilizer A and physiological saline in 1:2(v:v) to obtain stabilizer B.

2.4. Stock solutions, calibration standard, & quality control samples

JBD0131, DM131 and IS were dissolved in DMSO at concentrations of approximately 500 μg/ml to obtain stock solutions. Stock solutions were stored at −40°C.

Calibration samples of standards were prepared by diluting stock solutions with acetonitrile-water-stabilizer A (40:60:8 v:v:v) to concentrations ranging from 1 to 2 000 ng/ml (JBD0131) or 0.25 to 500 ng/ml (DM131) in plasma.

The quality control samples were prepared from another stock solution at concentrations of 1, 3, 120 and 1600 ng/ml for JBD0131 and 0.25, 0.75, 30 and 400 ng/ml for DM131.

2.5. Sample pretreatment

500 μl of internal standard solution (20 ng/ml IS in acetonitrile) was added to 50 μl of plasma sample to precipitate the protein. After vortexing for approximately 10 s, the mixtures were centrifuged at 13,000 rpm at 2 to 8°C for 5 min. 50 μl of the supernatant was transferred to a fresh vial and diluted with 150 μl of water. Finally, 4 μl of the mixture was injected into the UPLC-MS/MS system after vortex for approximately 30 s.

2.6. LC-MS/MS condition

The pretreated plasma samples were separated by an ACQUITY UPLC BEH C18 column (50 × 2.1 mm, 1.7 μm; Waters Corporation, MA, USA) equipped in an Acquity™ Ultra Performance LC system. The column temperature was maintained at 40°C. Ultrapure water with 0.05% (v:v) formic acid and 0.05% (v:v) ammonia was used as mobile phase A, and acetonitrile was used as mobile phase B. The gradient conditions was as follow: 35% A at 0 min, 50% A at 2.30 min, 100% A from 2.31 min until 2.90 min and 35% A from 2.91 min until 3.40 min, with a constant flow rate of 0.4 ml/min.

Quantification was performed in positive ion mode on a Xevo TQ-S triple quadrupole mass spectrometer (Waters, MA, USA) equipped with an ESI source. Multiple reaction monitoring mode was used to detect JBD0131, DM131 and IS at m/z values of 402.1/273, 333.1/273 and 386.1/257, respectively. Associated parameters were listed in Table 1. Data acquisition and processing were performed using UNIFI software version 1.7 (Waters, Milford, MA, USA.).

Table 1.

The mass spectrometer parameters of JBD0131, DM131 and IS.

Standard	Q1	Q3	Capillary voltage (kV)	Sample cone (V)	Source offset (V)	Source Temperature (°C)	MS collision energy
JBD0131	402.1	273	4	38	60	150	2
DM131	333.1	273	4	38	60	150	2
IS	386.1	257	4	38	60	150	2

2.7. Method validation

The method procedure was validated in accordance with the National Medical Products Administration (NMPA) (2020), Food and Drug Administration (FDA) (2018) and European Medicines Agency (EMA) (2012) guidelines for bioanalytical method validation [20–22].

2.7.1. Selectivity

Selectivity of the alalytical method was evaluated using the following matrices: lipemic plasma, hemolyzed plasma, plasma from six healthy subjects. A ratio was obtained by comparing the chromatograms of above blank matrix with the spiked matrices at the lower limit of quantification (LLOQ) level. For each matrix, the peak area of interfering peak in the blank matrix should not exceed 20% of the analyte peak area or more than 5% of the IS peak area in the LLOQ sample.

2.7.2. Crosstalk between JBD0131/DM131 & IS

To test if there is crosstalk between JBD0131/DM131 & IS, samples supplemented with IS but without JDB0131 or DM131 were used to assess IS's interference at the retention time of IS. Vice versa, upper limit of quantification (ULOQ) without IS samples were used to assess JDB0131 or DM131’s interference to IS. The acceptance criteria were the same as those used for selectivity.

2.7.3. Linearity & calibration curve

The calibration curve range, which included eight concentrations (ng/ml), was 1–2 000 for JBD0131 and 0.25–500 for DM131. The calibration curve was fitted by plotting the ratio of the analyte peak area to the IS peak area against the nominal concentration using weighted least-squares linear regression, with 1/x² as the weighting factor. The coefficient of determination (R²) should be ≥0.99. The accuracy of each calibration sample should be within 15%, except the LLOQ, where it should be within 20%.

2.7.4. Carryover

Carryover was assessed by injecting blank samples after the ULOQ samples. Generally, the carryover should not exceed 20% of the analyte response and 5% of the IS response in the LLOQ sample; otherwise, the carryover factor (CF) and impact factor (IF%) should be evaluated according to the following formula:

CF = the peak area of the highest carryover sample/peak area of the ULOQ sample before carryover sample

$$\text{IF}\%\mathit{ }=100\mathit{ }\%\times \frac{\text{CF}\times {\text{A}}_{\text{n}}}{({\text{A}}_{\text{n+1}}-\text{CF}\times {\text{A}}_{\text{n}})}$$

A_n represents the peak area of the n^th sample, and A_n+1 represents the peak area of the n+1^th sample.

If the IF% is greater than 5%, the n+1^th sample should be retested.

2.7.5. Accuracy & precision

The LLOQ, LQC, MQC and HQC concentrations (ng/ml) for the quality control plasma samples were 1, 3, 120 and 1 600 (JBD0131) and 0.25, 0.75, 30 and 400 (DM131), respectively. The accuracy and precision were elevated inter-run and intra-run by six quality control samples per concentration level. The accuracy was calculated as the relative error percentage (RE%) of the nominal concentrations, and the precision was calculated as the coefficient of variation percentage (CV%). The RE% and CV% should be within 15% within a run and between runs and within 20% in the case of LLOQ measurements.

2.7.6. Matrix effects & pretreatment recovery

To assess the matrix effects, QCs at low, median and high levels were prepared from six different human plasma samples, three hemolyzed plasma samples and three hyperlipidemic plasma samples. The matrix factor was expressed as the ratio of the peak area of the post extraction spiked QC sample to the peak area of the neat solution at the same concentration. The matrix factors of JBD0131 and DM131 were divided by the matrix factor of IS to obtain the normalized matrix factors.

The extraction recovery of the method was determined by comparing the peak area of analytes obtained from the blank plasma spiked before extraction to those obtained from the blank plasma spiked after extraction. The precision expressed by the CV% for both the matrix effect and recovery should not exceed 15%.

2.7.7. Stability

The stabilities of the analytes in the matrices were analyzed at the LQC, MQC and HQC levels under different conditions: long-term stability (storage at −80°C for 42 days, storage at -40°C for 8 days), short-term stability (storage at room temperature for 6 h), and three freeze–thaw cycles (-80°C to room temperature). The stability of the processed samples was assessed after 9 h of storage at 2−8°C and after 24 h of storage inside the autosampler (10°C). Whole blood stability was evaluated in six replicates after 3 h at room temperature. The tested samples were considered stable if the RE% and CV% of the mean concentration were less than 15%.

2.8. Application of the method

The fully verified method was successfully applied to simultaneously detect JBD0131 and its metabolite DM131 in the plasma of healthy subjects in a phase I, random and double-blind dose escalation trial. There were 94 subjects participating in this clinical trial and 2334 samples were collected for bioanalysis. Subjects were administered different doses of JBD0131 tablets after at least 10 hours of fasting. Whole blood samples were collected at 0 (pre-dose), 0.33, 0.67, 1, 1.5, 2, 2.5, 3, 3.5, 4, 5, 6, 8, 12, 24, 36, 48, 72, 96, 120 and 168 h to assess the pharmacokinetic profile. Venous blood (~3 ml) was sampled into an anticoagulant tube with lithium heparin and 90 μl of stabilizer B (3% (v/v)). The blood was centrifuged at 1 700 × g at 4°C for 10 min after mixing by inversion, and then the upper plasma layer (~1 ml) was transferred to cryogenic vials containing 20 μl of stabilizer A (2% (v/v)). The plasma samples were stored at -80°C.

3. Results & discussion

3.1. Method development

3.1.1. Optimization of MS/MS conditions

To develop the MS conditions, JBD0131, DM131 and IS were dissolved in a 25% acetonitrile solution at 100 ng/ml. The positive ion electrospray ionization mode used in the full mass scan permitted the detection of prominent peaks for JBD0131, DM131 and IS with m/z values of 402.1 and 355.31, 333.1 and 386.1, respectively. The most abundant and stable product fragments were observed at m/z 273 for both JBD0131 and DM131 and at m/z 257 for the IS (Figure 2). In addition, the mass spectrometry parameters were optimized mainly by the automatic optimization function of Waters-TQS. On the basis of automatic optimized parameter, we made some subtle adjustion to promote the response. After optimization, the ion spray voltage used was 4000 V and the desolvation temperature was 500°C. Other parameters for each analyte are listed in Table 1.

Figure 2.

Product ion scanning spectrum. (A) JBD0131. (B) DM131. (C) IS. m/z: Mass-to-charge ratio.

3.1.2. Optimization of LC conditions

The chromatography conditions were optimized by changing various parameters, including the proportions of the organic phase and aqueous phase in the flow and the pH of the aqueous phase. The best elution and tailing were obtained with 0.05% formic acid and 0.05% ammonia in pure water as phase A and acetonitrile as phase B. And the elution gradient is discussed above.

3.1.3. Optimization of the stabilizer

We screened different groups of buffer salts and acids in consideration of anti-oxidize or adjusting the pH value, including a saturated solution of Na₂S₂O₄, a saturated solution of Na₂S₂O₄ with 10% (v: v) formic acid, a saturated solution of ammonium acetate with 30% (v: v) acetic acid, a saturated solution of ammonium acetate with 30% (v: v) formic acid, a saturated solution of vitamin C and a saturated solution of KH₂PO₄ with 5% (v: v) phosphoric acid. To avoid hemolysis when added to whole blood, we chose physiological saline as the solvent for both groups. Finally, the combination of ammonium acetate and acetic acid was selected. We further explored the volume ratios (20:3, 10:3 and 2:1) of ammonium acetate and acetic acid and a volume ratio of 10:3 was selected; therefore, 10:3 (v: v) ammonium acetate and acetic acid were selected as stabilizer A. Finally, we screened the volume of stabilizer added to the plasma (0, 20, 40 and 60 μl in 1 ml of plasma, respectively) and 40 μl of stabilizer A in 1 ml of plasma (4%) was optimal. In whole blood, a heavy concentration of stabilizer will also lead to hemolysis, given that the whole blood was separated into plasma in a short period of time, so stabilizer A was diluted to stabilizer B and 30 μl of stabilizer B was added to 1 ml of whole blood. After centrifugation, the upper plasma layer was transferred to a fresh cryogenic vial supplemented with 20 μl of stabilizer A per milliliter {given that plasma account for approximately 55% of the volume of whole blood, so there is about 6% of stabilizer B (or equal to 2% of stabilizer A) – twice the original concentration in plasma after centrifugation, so additional 2% stabilizer A is needed for long-time storage}.

3.2. Method validation

3.2.1. Selectivity or specificity

As shown in Figure 3, there were no significant interfering peaks from the endogenous matrix that could be observed at the retention times of JBD0131, DM131 and IS. In addition, no interference was observed between JBD0131/DM131 and the IS (Figure 4). The retention times of JBD0131, DM131 and the IS were 2.4 ± 0.15, 0.9 ± 0.08, and 2.1 ± 0.18 min, respectively, with a total run time of only 3.4 min.

Figure 4.

Cross-talk between JBD0131/DM131 and internal standard in human plasma. Chromatograms of a blank plasma adding internal standard sample: (A) JBD0131, (C) DM131, (E) IS; chromatograms of a upper limit of quantification sample: (B) JBD0131, (D) DM131, (F) IS.

Figure 3.

Typical chromatograms of analyte and internal standard in human plasma. Chromatograms of (A) blank plasma, (B) lower limit of quantification, (C) upper quality control and (D) sample of a subject.

3.2.2. Linearity, LLOQ & carry-over

Figure 3B shows that the signal-to-noise ratio at the LLOQ was more than five fold, so the method could detect JBD0131 at a level as low as 1 ng/ml and DM131 at a level of 0.25 ng/ml. The calibration curve was calculated by plotting the peak area ratios (JBD0131 or DM131/IS) versus the concentration of JBD0131 or DM131 (ng/ml). The calibration curve showed good linearity in concentrations ranging from 1 to 2000 ng/ml for JBD0131 and 0.25 to 500 ng/ml for DM131. The fitted concentrations of all calibration curve samples by regression equation were within ±15% of the nominal value. The correlation coefficients were all greater than 0.99. No carry-over was detected in the blank samples analyzed after the ULOQ sample.

3.2.3. Accuracy & precision

The precision and accuracy of the method for JBD0131 and DM131 are summarized in Table 2. The intra-run CVs were less than 8.39% for JBD0131 and no more than 11.16% for DM131. The inter-run precisions were less than 6.88% for JBD0131 and less than 13.51% for DM131. The intra- and inter-run accuracies (RE%) ranged from -2.25 to 3.21% and from -6.90 to 8.23%, respectively, for JBD0131 and from -1.81 to 5.27% and from -3.37 to 9.93%, respectively, for DM131.

Table 2.

Summary of intra- and inter-batch precision and accuracy.

Analyte	Level	Inter-batch (n = 6)						Intra-batch (n = 18)
		Batch1		Batch2		Batch3
		CV%	RE%	CV%	RE%	CV%	RE%	CV%	RE%
JBD0131	LLOQ	3.79	-1.98	6.88	8.23	5.45	-6.90	8.39	-0.22
	LQC	2.55	5.24	2.48	5.12	2.94	-0.74	3.74	3.21
	MQC	3.00	1.90	1.88	2.84	3.21	-1.39	3.17	1.12
	HQC	2.75	-1.40	3.56	-1.14	2.54	-4.20	3.17	-2.25
DM131	LLOQ	13.51	9.93	9.70	6.20	8.83	-0.33	11.16	5.27
	LQC	4.57	7.36	1.96	3.20	3.50	3.22	3.84	4.59
	MQC	3.28	3.30	3.12	0.13	3.32	-0.99	3.57	0.81
	HQC	3.85	-0.34	2.08	-3.37	2.89	-1.70	3.15	-1.81

CV: Coefficient of variation; HQC: High quality control; LLOQ: Lower limit of quantitation, LQC: Low quality control, MQC: Middle quality control; RE: Relative error.

3.2.4. Matrix effects & recovery

The matrix effects of plasma JBD0131 and DM131 are displayed in Table 3. The CV of the normalized MEs was within 2.74% for JBD0131 and 3.22% for DM131 in plasma. For the two analytes and the IS, extraction recovery ranged from 89.18% to 97.92% in plasma.

Table 3.

Summary of extraction recovery and matrix effect.

Type	Statistic	JBD0131			DM131
		HQC	MQC	LQC	HQC	MQC	LQC
Recovery	CV%	4.04	7.46	2.72	4.42	6.20	2.85
	Measured%	115.54	99.43	87.66	96.61	88.08	87.93
Normal	CV%	2.74	1.38	2.33	3.22	0.70	2.19
	Measured%	96.76	101.84	92.59	98.72	99.77	90.47
Hemolyzed	Measured%	93.50	99.50	93.33	97.10	96.45	90.18
Hyperlipemic	Measured%	98.27	104.59	93.46	96.19	103.47	87.79

3.2.5. Stability

The results of the stability analysis are summarized in Table 4. When a stabilizer was added, the spiked plasma samples were stable for at least 8 days at -40°C and 42 days at -80°C. The extracted samples were stable for 9 h at 2–8°C and 24 h in an auto sampler (10°C). The whole blood sample was stable for 3 h at room temperature (25°C).

Table 4.

Summary of stability in different storage conditions. n = 6.

Stability	JBD0131			DM131
	LQC	MQC	HQC	LQC	MQC	HQC
Room temperature stability, 6 h
CV%	2.80	2.03	5.29	2.93	2.51	5.16
RE%	-4.43	-2.13	-5.45	2.53	0.16	-3.42
Freeze–thaw stability, -80°C, three cycles
CV%	2.01	3.10	5.00	2.31	2.27	1.88
RE%	-4.02	-5.77	-7.53	-1.47	-1.84	-5.69
Extracted sample stability, 2–8°C, 9 h
CV%	2.17	2.19	4.19	1.56	1.24	1.00
RE%	-5.98	-8.01	-9.68	-8.58	-5.13	-11.59
Autosampler stability, 10°C, 24 h
CV%	2.30	0.95	4.04	3.98	8.25	3.26
RE%	13.22	6.80	4.67	7.76	1.32	0.86
Whole blood stability, 25°C, 3 h
CV%	7.15	0.96	3.24	1.65	4.04	1.48
RE%	-6.94	-2.09	-6.80	-2.71	-1.17	-4.44
Long-term storage stability, -40°C, 8 days
CV%	3.75	1.03	3.21	5.60	1.80	2.03
RE%	-2.49	-2.04	-2.54	-7.38	-4.46	-2.02
Long-term storage stability, -80°C, 42 days
CV%	3.56	2.31	1.93	3.91	3.37	1.10
RE%	3.17	11.96	4.35	-0.16	7.25	3.83

CV: Coefficient of variation; HQC: High quality control; LQC: Low quality control; MQC: Median quality control; RE: Relative error.

3.3. Application of the method

This method was applied in a clinical pharmacokinetic study of JBD0131 in healthy subjects. The mean plasma concentration–time curves of JBD0131 (A) and DM131 (B) are depicted in Figure 5. For JBD0131, C_max values of 108.37 ± 44.97 ng/ml for 50 mg, 229.92 ± 95.37 ng/ml for 100 mg, 320.29 ± 138.17 ng/ml for 200 mg and 439.41 ± 193.61 ng/ml for 400 mg were achieved at approximately 2.47 h. The area under the concentration–time curve during the period of observation (AUC_0-t) was 1264.88 h·ng/ml for 50 mg, 2569.93 h·ng/ml for 100 mg, 5094.00 h·ng/ml for 200 mg and 6183.79 h·ng/ml for 400 mg. The increase in C_max and AUC_0-t was slightly lower than the increase in dose when the dosage was climbed to 400 mg. Subsequently, the plasma concentration decayed with mean elimination half-life (t_1/2) of 10.84 h. The mean apparent distribution volume (V_d) was 828.31 L, and the total clearance (CL) was 55.71 L/h. For DM131, C_max values of 11.83 ng/ml for 50 mg, 36.79 ng/ml for 100 mg, 36.12 ng/ml for 200 mg and 40.98 ng/ml for 400 mg were achieved at 3.18 h. AUC_0-t was 229.64 h·ng/ml for 50 mg, 583.30 h·ng/ml for 100 mg, 1016.93 h·ng/ml for 200 mg and 1334.88 h·ng/ml for 400 mg. The mean elimination half-life is 45.57 h, with a probability of metabolite accumulation.

Figure 5.

Mean plasma concentration-time curves of JBD0131 (A) and DM131 (B) in healthy subjects after administration of 50 mg (n = 8), 100 mg (n = 8), 200 mg (n = 8) or 400 mg (n = 8). Error bars represent standard deviations at individual time points.

4. Discussion

There is no doubt that MDR-TB is still a high-cost and low-cure rate problem, and novel drugs with higher effectiveness, shorter treatment duration and little side effect is urgent. Delamanid was approved to treat the MDR-TB by WHO in 2014, and was included in the European Union's Priority Medicines (PRIME) program by EMA. It is the third new anti-TB drug approved for marketing in nearly half a century. JBD0131 is an analog of delamanid that exhibited preferable anti-TB properties in preclinical studies. The phase I clinical trial was conducted in our center to evaluate the safety, tolerability and pharmacokinetics of JBD0131. In the method development of drug concentration determination, the LC-MS/MS method is the first choice, so a UPLC-MS/MS method was developed for the pharmacokinetic study in this clinical trial.

Of note, a preliminary experiment showed that JBD0131 is unstable in plasma and can be transferred to DM131 by albumin in plasma [unpublished Data], which is similar to that of Delamanid [14]. Thus, a suitable stabilizer is necessary for accurate sample concentration detection. According to the chemical structure of JBD0131 and DM131, we speculated that the pH of medium might be the major factor influencing their stability. After attempt of different groups of chemicals, ammonium acetate and acetic acid solution could meet the requirement. Besides, the hemolysis factor was also taken into consideration in determination of the ratio of stabilizer. Although subtle hemolysis happened in blood collection, it didn't affect the result according to the validation of matrix effect. According to the response result in MS, we thought the recovery rate of protein precipitation method for pretreatment could meet the requirement, and it can also satisfy the demand of large quantities of sample detection. The proportion of mobile phase favour the peak shape and the elution gradient in UPLC enable the method without interference and crosstalk. A sentinel experiment including 4 subjects administrated with 20 mg JBD0131 was conducted before the formal clinical trial. From that, the lowest concentration was confirmed and the highest concentration was accessed according to dose-concentration growth trend in preclinical trial of JBD0131 and the clinical trial of delamanid. One of the pities is that the internal standard in this method is not an isotope, despite that, its applicability was validated. From a generalized validation of this determination method, it is qualified to detect the JBD0131 and DM131 plasma concentration in a range of 1 to 2000 ng/ml and 0.25 to 500 ng/ml, separately.

According to the pharmacokinetic result of JBD0131 between 50 to 400 mg, the mean C_max in both dose groups exceed the minimum inhibitory concentration for drug-resistant tuberculosis in vitro, predicting the efficacy of the drug. Furthermore, JBD0131 and DM131 have shorter t_1/2 than delamanid and its metabolites, suggesting that JBD0131 may have higher security.

5. Conclusion

The novel nitro-dihydro-imidazooxazole anti-MDR-TB drug JBD0131 showed encouraging result in preclinical study that exceeding the analog delamanid. To support the clinical trial of it, the authors developed a fast, specific and robust UPLC-MS/MS method for the determination of the plasma concentrations of JBD0131 and its metabolite. The method was validated in terms of selectivity, calibration curve and range, precision, accuracy, carryover, matrix effect, recovery and stability according to the NMPA, FDA and EMA guidelines. This method can simultaneously determine the concentrations of JBD0131 and DM131 in wide ranges of 1–2000 ng/ml and 0.25–500 ng/ml, respectively, with a short run time of 3.4 min. The stabilizer we developed for JBD0131 and DM131 assist them to be stable in plasma under the research conditions. All in all, the method was successfully applied to investigate the pharmacokinetic properties of JBD0131 and DM131 in clinical trial. And from the result of quantification, we can see that the plasma concentration of JBD0131 and DM131 increase with the growth of dose until 400 mg. Meanwhile, the t_1/2 of them is short than that of delamanid, maybe indicating a higher security.

Financial disclosure

This work was sponsored by Chengdu Jiabaoyaoyin Medical technology Co., Ltd. (Chengdu, China). The authors have no other 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 apart from those disclosed.

Competing interests disclosure

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.

Writing disclosure

No writing assistance was utilized in the production of this manuscript

Clinical trial data disclosure

The authors certify that this manuscript reports original clinical trial data. Deidentified, individual data that underlie the results reported in this article (text, tables, figures and appendices), along with the study protocol will be available indefinitely for anyone who wants access to them. Trial Registration: www.chinadrugtrials.org. cn:CTR20202308.