Abstract
A novel doxorubicin hydrochloride liposome injection was prepared to reduce toxicity and side effects, as well as extend plasma half-life in the treatment of breast cancer. In this study, a rapid and sensitive bioanalytical method was developed and validated to characterize the pharmacokinetic profile of total and free doxorubicin in plasma of 3 Chinese patients after intravenous infusion of this injection. Plasma samples were prepared by protein precipitation for the determination of total doxorubicin, while solid phase extraction was used to determine free doxorubicin. After plasma sample pre-treatment, total and free concentrations were quantified individually using a validated LC-MS/MS method. The calibration curves were found to be linear in the range of 0.20–500.0 ng mL−1 for total doxorubicin and in the range of 1.00–1,000 ng mL−1 for free doxorubicin. The free concentrations in plasma were only one sixth to one quarter of the total levels. Liposomal doxorubicin had a longer apparent half-life (>50 h) than the non-targeted drug (<10 h) reported in the reference. and a lower volume of distribution. This novel injectable formulation steadily released free doxorubicin from liposomes over a long period of time to reduce cardiac toxicity and side effects, while ensuring a clinical curative effect.
1 Introduction
Breast cancer is the most common cancer in women globally. In 2018, an estimated 4.3 million new cancer cases and 2.9 million new cancer deaths occurred in China [1, 2]. Treatment modalities include systemic therapy, of which chemotherapy is a major component. However, chemotherapy is associated with unavoidable side effects, which harm patients, and the development of drug resistance [3]. To solve these problems and to improve the anti-tumor effect of chemotherapeutics, a variety of tumor-targeting approaches have been explored to deliver drugs to tumor tissues and tumor cells [4, 5].
Nanoparticle (NP) drug delivery systems have recently become very popular for targeting. These systems can exploit small differences between the tumor tissue microenvironment and normal tissues to achieve targeted delivery of chemotherapeutic drugs, improving efficacy [6].
A liposome is an artificially prepared phospholipid bilayer that is very similar to a biological membrane structure. Liposomes have good biocompatibility because they are degraded into non-toxic end products in the body and exhibit no immunogenicity or other toxic side effects. Their clinical application has become increasingly extensive. The phospholipid molecule that constitutes the liposome is amphiphilic, consisting of a hydrophilic head and a hydrophobic tail. Consequently, liposome NPs can be used to load water-soluble drugs as well as lipid-soluble drugs. For some insoluble drugs, such as doxorubicin (DOX), paclitaxel, and amphotericin B, solubility and absorption can be greatly increased [7].
Doxorubicin hydrochloride liposome injection Doxil®, developed by Sequus Pharmaceuticals, was the first nanomedicine approved by the FDA in 1995 for clinical indications in breast cancer, ovarian cancer, myeloma, and AIDS-KS [7, 8]. The liposomal carrier is made of synthetic phospholipids that exhibit high stability and passive targeting of tumor tissue. In terms of pharmacokinetic features, the half-life of liposomal doxorubicin is around 80 h in patients with breast cancer [8, 9] and it has a lower volume of distribution than that of non-NP drugs [10]. These properties suggest the possibility of less frequent administration, sustained release, targeted therapy, and reduced toxicity. A novel doxorubicin hydrochloride liposome injection (DOX·HCl-PLI) formulation named “Lixing®” was developed and released in 2013 by Changzhou Kinyond Pharmaceutical Co., Ltd. (Chinese national medicine permission number 20123273 and its structure shown in Fig. 1). According to the patent document, its improved production techniques enable high encapsulation efficiency and stability of the nanoparticles. Considering that this DOX·HCl-PLI has been released on sale for about 10 years without any serious adverse drug reaction reports, it is believed that this DOX·HCl-PLI is safety and effective. In the absence of pharmacokinetic data, neither in Chinese breast cancer patients nor with this novel DOX·HCl-PLI, it is necessary to investigate the pharmacokinetic behavior of doxorubicin hydrochloride liposomes in Chinese breast cancer patients to lay the foundation for further research into clinical applications.
It is a remarkable fact that for anti-tumor NP formulations, the free drug concentration in blood or normal tissues is positively correlated with toxicity and side effects, while the free drug concentration in targeted organs such as tumor tissues is positively correlated with efficacy. Only the free drug released from liposomes is key to evaluating the efficacy and toxicity of anti-tumor NP formulations. However, most anti-tumor NP studies have focused on total concentration-based exposure and total concentration-based biodistribution in plasma and tissues, respectively, while ignoring the dynamic release of free drugs from anti-tumor NPs in vivo. Concentrations of anti-tumor NPs and the released free drug vary dynamically between the circulatory system, extracellular fluid, and intra-cellular fluid to reach a balance. After the anti-tumor NP enters the cell, there is a question of whether the free drug can be effectively released, and whether the released free drug can effectively combine with the target. Therefore, only demonstrating that the NP formulation can increase total drug concentration in the tumor does not mean that the free drug concentration in the tumor is increased, nor that the amount of drug bound to the target in the tumor is increased [11, 12]. Improvement of anti-tumor effect cannot be simply judged based on total drug concentration, but by the concentration of free drug compared to a drug that is entrapped in the NPs.
There were several analytical methods applied to the quantification of the DOX in the biological matrix, such as HPLC with fluorescence detector [13, 14]. However, the limit of quantification is too high to detect the trace amounts of the non-liposomal or called free doxorubicin (F-DOX) for encapsulated doxorubicin or total doxorubicin (T-DOX), which is not suitable for a comprehensive evaluation of the correlation between free drug and drug that is entrapped in the NPs (Liposome-entrapment of doxorubicin, L-DOX). Recently, more and more analytical researches about DOX have focused on the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method [11, 12, 15]. Compared with HPLC method, LC-MS/MS method is more sensitive and shows high resolution, and can significantly reduce analysis time and increase sample throughput. However, as for those reported LC-MS/MS methods, the sensitivities and rapidity were not enough to meet the increasing analytical requirements nowadays.
The objective of this study was to establish a sensitive and rapid LC-MS/MS method to determine the total and free concentrations of doxorubicin in the plasma of Chinese breast cancer patients after intravenous infusion of DOX·HCl-PLI to comprehensively evaluate the pharmacokinetics of liposomal doxorubicin. The study enables an understanding of the relationship between drug efficacy and toxicity of the NP formulation and provides a guide for efficient and accurate clinical application of anti-tumor NP.
Schematic diagram of DOX·HCl-PLI. mPEG-DSPE: methoxy polyethylene glycol-phosphatidylethanolamine
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01129
2 Experimental
2.1 Chemicals and reagents
Doxorubicin (DOX, purity >97%, Lot No. 2029075) and 7-deoxydoxorubicin aglycone (DDA, purity >75%, Lot No. 28-GHZ-48-1) were purchased from Shanghai Aladdin Bio-Chem Technology Co., Ltd. (Shanghai, China) and Toronto Research Chemicals Inc. (Toronto, ON, Canada), respectively. Buspirone hydrochloride (purity >99%), used as the internal standard (IS), was obtained from Sigma Co., Ltd. (St. Louis, MO, USA). DOX·HCl-PLI was prepared by Changzhou Kinyond Pharmaceutical Co., Ltd. (Changzhou, Jiangsu, China). HPLC-grade methanol and acetonitrile were obtained from Fisher Chemicals (NJ, USA). All other chemicals and solvents were of analytical grade. Pooled blank plasma from three female Chinese patients with invasive breast cancer before administration (analyte-free and collected in the heparinized tubes) was prepared in our laboratory. These three same patients enrolled in the present preliminary pharmacokinetic study.
2.2 Instrumentation and chromatographic conditions
The F-DOX (free doxorubicin) and T-DOX (total doxorubicin) were detected on an API 4000 Qtrap mass spectrometer (Applied Biosystems/MDS Sciex, Concord, ON, Canada) with an electrospray ionization source (ESI) operated in positive ion mode.
All plasma samples were separated on an Agilent Zorbax XDB C18 column (50×2.1 mm, 3.5 µm) using a Shimadzu HPLC system consisting of an LC-20AD pump, a DGU-20 A3 degasser, a SIL-20AC autosampler and a CTO-20A column oven (Shimadzu, Japan). Water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B) was used as mobile phase and delivered at a rate of 0.60 mL min−1 and the injection volume was 5 µL. The gradient program for measurement of F-DOX and T-DOX was: 0.00–0.50 min, 5% B, 0.50–1.00 min, 15%–95% B, 1.00–2.50 min, 95% B, 2.50–2.51 min, 95%–5% A, 2.51–3.5 min, 5% B.
The main MS working parameters were set as follows: ion spray voltage, 5.5 kV; ion source temperature, 500 °C; gas1, 55 psi; gas2, 55 psi; curtain gas, 15 psi. Analyte concentrations were determined using Analyst 1.6.2 software. The ion pairs chosen for F-DOX and T-DOX in multiple reaction monitoring (MRM) mode were m/z 544.2→397.1 for quantification and m/z 544.2→379.1 for identification, while m/z 386.3 →122.2 was used for the IS.
2.3 Preparation of DOX·HCl-PLI
DOX·HCl-PLI were prepared using the transmembrane gradient method. Briefly, the hydrogenated soybean phosphatidylcholine (HSPC) and methoxy polyethylene glycol-phosphatidylethanolamine (mPEG-DSPE) were dissolved in chloroform, which was removed by rotary evaporation. The dried lipid film, formed in a round-bottomed flask, was rehydrated with ammonium sulfate (NH4)2SO4 solution. The transmembrane gradient was formed by exchanging external ammonium sulfate with glucose solution using dynamic dialysis. DOX was added to the above-prepared liposomes followed by incubation for DOX·HCl-PLI. The injections were stored at 4 °C for further studies.
2.4 Preparation of solutions
Stock solutions (1.00 mg mL−1) of DOX and buspirone were prepared in DMSO. The buspirone stock solution was diluted with methanol to make an internal standard (IS) solution at a concentration of 500 ng mL−1 for the quantification of T-DOX. The IS solution for F-DOX was prepared similarly, replacing methanol with 5% glucose aqueous solution.
The stock solutions of both T-DOX and F-DOX were prepared in duplicate. One of the two stock sets was used to prepare calibration standard spiking solutions and the other set was used to prepare quality control standard spiking solutions.
The DOX stock solution was serially diluted with methanol to obtain working standard solutions with final concentrations ranging from 2.00 to 5,000 ng mL−1 for the preparation of T-DOX calibration curves. Similarly, the F-DOX calibration curves were prepared using stock solution diluted with 5% glucose to reach final concentrations ranging from 2.00 to 2000 ng mL−1. To prepare T-DOX calibration standards, working solutions (5 µL) were mixed with blank human plasma (50 µL) to final concentrations of 0.20–500 ng mL−1. For the F-DOX calibration standards, working solutions (50 µL) were mixed with blank human plasma (50 µL) to final concentrations of 1.00–1,000 ng mL−1. Four levels of quality control (QC) samples for DOX (0.20, 0.50, 20.0, 400 ng mL−1) were independently prepared using the same procedure as for the calibration standards. All samples were immediately stored at 4 °C.
2.5 Sample preparation
2.5.1 Solid phase extraction (SPE) procedure for the determination of F-DOX
F-DOX was separated from human plasma using Oasis® HLB SPE columns (1 cc/10 mg, Waters, MA, USA). After conditioning with methanol (1 mL) and water (1 mL) successively, columns were protein-coated with blank human plasma (0.10 mL) to diminish the retention of F-DOX [11, 16]. Following the addition of IS solution (100 µL, 500 ng mL−1 buspirone in 5% glucose solution) and 5% glucose solution (50 µL) to the plasma sample (50 µL), the sample was applied to the column and drawn through. The column was then washed with water (2 × 1 mL) and then the F-DOX was eluted with methanol containing 0.5% formic acid (500 μL). The eluate was collected into a 1.5 mL centrifuge tube and mixed completely by vortex for 1 min for subsequent analysis.
2.5.2 Extraction of T-DOX from plasma
Methanol (5 µL) was added to each plasma sample before spiking with IS solution (100 µL, 500 ng mL−1 buspirone in methanol-acetonitrile, 1:1, v/v) for protein precipitation. The mixture was vortexed for 1 min and then centrifuged at 14,000 g for 10 min. Aliquots (100 µL) of supernatant were transferred into HPLC vials for LC-MS/MS analysis.
2.6 Method validation
Method validation for the determination of DOX in human plasma was performed according to the FDA bioanalytical method validation and study sample analysis guideline (2022).
Selectivity was evaluated by analysing three replicates of blank human plasma samples from six different sources. The corresponding LLOQ plasma samples were used for comparison. There should be no significant response attributable to interfering components observed at the retention time of the analyte or the IS in the blank samples.
The calibration curve was plotted using the standard solution concentration as the abscissa and the ratio of the peak area of the analyte to IS as the ordinate. The least-squares linear regression method (1/x2 weighting) was used to determine the slope, intercept, and square regression coefficient (r2) of the linear regression equation.
Precision and accuracy were calculated by determining QC samples in 6 replicates at four concentration levels, including lower limit of quantitation (LLOQ), low, medium, and high concentrations in each analytical run (Intra-batch), and three analytical batches was used in total to evaluate Inter-batch accuracy and precision was evaluated by analysing each QC concentration level in 3 analytical batches. Precision was expressed as the relative standard deviation (RSD) and accuracy as the relative error (RE). The accuracy and the precision of each concentration level should be within ±15% RE and not exceed 15% (RSD%), respectively, except for LLOQ, where it should be within ±20% RE and RSD should not exceed 20%.
The extraction recoveries were evaluated through the ratio of mean peak areas between regularly prepared QC samples (LLOQ, low, medium, and high concentration) and spike-after-extraction plasma samples, which were prepared by adding the neat solutions of DOX and IS (prepared in methanol) to the post-extracted blank plasma matrix to obtain the final equivalent concentrations for the post-extracted samples at four levels. Similarly, the matrix effect was estimated by comparing the peak area of each analyte and IS in the plasma extracts-contained matrix with those in the water matrix at four QC levels.
The dilution integrity experiment was carried out at 20- and 100-times higher concentrations of the highest level of the calibration curve. This dilution integrity stock in plasma was then diluted 20 times and 100 times with blank human plasma to get dilution QC. Six replicates per dilution factor were prepared and processed along with a freshly prepared calibration curve. Their mean accuracy of the dilution QCs should be within ±15% of the nominal concentration and the precision (RSD%) should not exceed 15%.
The stability of the analyte in human plasma was evaluated by analyzing QC samples stored under the following conditions: at 20 °C for 24 h and at −80 °C for 30 days. The effect of three freeze/thaw cycles on the analyte was also examined. The mean concentration at each QC level should be within ±15% of the nominal concentration.
2.7 Subjects treatment and pharmacokinetic analysis
The clinical study was conducted between February 2020 and February 2021 at Beijing Friendship Hospital affiliated with Capital Medical University in accordance with the Declaration of Helsinki and Good Clinical Practice guidelines. All patients signed a written informed consent before inclusion in the study, and our institutional review board approved the study (No. 2019-P2-215-02).
Three female patients with invasive breast cancer, confirmed by preoperative puncture or intraoperative frozen section and postoperative paraffin section, were enrolled. The available plasma samples were entered into the study. The mean age of the patients was 49.7 years old.
The subjects were treated with 30 mg m−2 DOX·HCl-PLI combined with 600 mg m−2 cyclophosphamide infusion for 4 cycles, and each cycle of treatment was 21 days. Blood samples (0.50 mL) for were collected into heparinized tubes before the start of every infusion, at 3 h (at the end of every infusion) and at 8, 20, 28, 32, 44, 56, 68, 92, 116, 140, 240 and 480 h after the end of the fourth infusion treatment.
Plasma separation was achieved within 1 h of blood collection by centrifugation at 4,000 g for 5 min at 4 °C. To ensure the precision of T-DOX and F-DOX concentration determinations, the plasma was divided into two portions. One portion was diluted 100-fold with blank human plasma for T-DOX and the other was diluted 20-fold for F-DOX. Samples were quantified using the validated LC-MS/MS method.
The pharmacokinetic parameters of F-DOX and T-DOX, including apparent elimination half-life (t1/2) and area under the plasma concentration-time curve (AUC), were calculated using a non-compartmental method in WinNonlin 5.2 pharmacokinetic software (Pharsight Corporation, Cary, NC, USA). Data are reported as the mean ± SD.
Independent Student's t-test was used to evaluate statistical differences. Differences were considered statistically significant when P-values were less than 0.05. All statistical analyses were performed using SPSS 26.0 software (Chicago, IL, USA).
3 Results and discussion
3.1 Optimization of MS conditions
The operating parameters for MS detection of DOX and buspirone were optimized by flow injection using the standard solutions. The specific MS parameters of DOX and buspirone were shown in Table 1 and the MS2 spectra were recorded and are shown in Fig. 2. In the full-scan Q1 mass spectrum of DOX, the most abundant peak was [M + H]+ found at m/z = 544.2. The abundance of this ion peak ([M + H]+) was stable enough and sufficient for accurate quantification. The strongest DOX fragment was the ion at m/z 397.1. The transition m/z 544.2→397.1 was then used to optimize ion spray voltage, curtain gas pressure, nebulizer gas pressure, heater gas pressure, source temperature, and collision energy for DOX. In addition, the ion at m/z 379.1, the second strongest response of all fragments, was chosen for the identification of DOX. For buspirone, the most abundant peak was the protonated molecular ion [M + H]+ found at m/z = 386.3. Parameters for the buspirone transition m/z 386.3→122.2 were optimized in the same way as those for DOX.
MS parameters of doxorubicin (DOX) and buspirone determined by LC-MS/MS
| MS Parameters | DOX for quantification | DOX for identification | Buspirone (IS) |
| Q1 (m/z) | 544.2 | 544.2 | 386.3 |
| Q3 (m/z) | 397.1 | 379.1 | 122.2 |
| Ion Spray (V) | 5,500 | 5,500 | 5,500 |
| DP (V) | 101 | 101 | 100 |
| CUR (psi) | 15 | 15 | 15 |
| GS 1 (psi) | 55 | 55 | 55 |
| GS 2 (psi) | 55 | 55 | 55 |
| CAD | High | High | High |
| Capillary Temperature (°C) | 500 | 500 | 500 |
| CE (eV) | 15 | 26 | 40 |
| CXP (V) | 11 | 10 | 15 |
The MS2 product ion spectra and fragmentation patterns of DOX (a) and IS (b)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01129
An internal standard is usually required in LC-MS/MS analysis to eliminate the effects of matrix and extraction efficiency. Radio-labelled internal standard or daunorubicin [10], an analogy of DOX is the optimal choice but is too expensive to obtain in our study. Instead, buspirone, a readily available compound that has similar chromatographic retention behavior (tR = 1.51 min) to DOX, was selected as the IS [17].
3.2 Method validation
Typical chromatograms (Fig. 3) showed that, under the chromatographic conditions described, there was no obvious endogenous interference at the retention times of DOX or buspirone.
Typical MRM chromatograms for the determination of T-DOX in human plasma. (a) blank human plasma; (b) blank human plasma spiked with DOX (0.20 ng mL−1, LLOQ level of T-DOX) and IS; (c) an unknown human plasma sample collected 8 h after intravenous infusion of DOX·HCl-PLI to Chinese patients with breast cancer
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01129
The calibration curves for T-DOX and F-DOX exhibited good linearity over the concentration ranges of 0.20–500 ng mL−1 and 1.00–1,000 ng mL−1, respectively. The typical standard curve for T-DOX was described by the equation y = 6.08 × 10−3x + 2.51 × 10−7 and that of F-DOX by y = 4.13 × 10−2x − 3.62 × 10−5, where y is the peak area ratio of the component to buspirone, and x is the concentration of T-DOX or F-DOX. The square regression coefficients (r2) were both higher than 0.995.
The precision and accuracy of the method were assessed using QC samples (0.20, 0.50, 20, and 400 ng mL−1). The results, shown in Table 2, indicated high precision with intra-day precision ≤5.83% and inter-day precision ≤5.08% from the nominal values at each QC sample concentration, while the accuracy was in the range of 89.1–108%.
Intra-and inter-batch precisions and accuracies of doxorubicin (DOX) in human plasma determined by LC-MS/MS (n = 6 for Intra-batch, n = 3 for Inter-batch)
| Spiked Concentration (ng mL−1) | Precision (%, RSD) | Accuracy (%) | ||
| Intra-batch | Inter-batch | Intra-batch | Inter-batch | |
| 0.20 | 3.65 | 5.49 | 92.3 | 95.7 |
| 0.50 | 5.12 | 5.09 | 94.9 | 97.0 |
| 20.0 | 3.81 | 3.79 | 98.7 | 99.4 |
| 400 | 4.85 | 4.81 | 98.9 | 100.0 |
The recoveries of DOX varied from 86.2 to 94.3% and the matrix effects ranged from 89.3 to 95.7%. As for the dilution integrity experiment, the mean back-calculated concentrations of 1/20th and 1/100th diluted samples for DOX were 98.7 and 101.3% respectively to their nominal values. The coefficients of variance were 2.36 and 3.51%, which are both less than 15%.
DOX spiked into blank plasma was found to be stable for 24 h at 20 °C, for up to 30 days at −80 °C, and during three freeze-thaw cycles (Table 3). The stability was thus satisfactory for a routine pharmacokinetic study. Several pretreatment methods have been proposed to separate free drugs in plasma from liposome-entrapped drugs, including ultrafiltration [18], gel exclusion chromatography [13], and solid phase extraction (SPE)/online SPE [14, 19, 20]. In this study, a rapid and sensitive method for the determination of T-DOX and F-DOX in human plasma was established by optimizing the existing pretreatment methods. First, protein precipitation (PPT) was used to destroy liposomes and precipitate endogenous matrix from plasma for T-DOX measurement. To determine plasma levels of lipo-some-entrapped and free drugs, SPE was used to separate and enrich the free doxorubicin that was not encapsulated by liposomes, and simultaneously remove the endogenous matrix.
Stability results for the determination of doxorubicin (DOX) in human plasma determined by LC-MS/MS (n = 6)
| Stability | Spiked concentration (ng mL−1) | Determined concentration (mean ± SD, ng mL−1) | Accuracy (%) |
| 24 h at 20 °C | 0.20 | 0.210 ± 0.008 | 105 |
| 0.50 | 0.450 ± 0.021 | 90.0 | |
| 20.0 | 19.8 ± 1.91 | 98.8 | |
| 400 | 376 ± 9.29 | 93.7 | |
| 30 days at −80 °C | 0.20 | 0.198 ± 0.008 | 98.8 |
| 0.50 | 0.452 ± 0.011 | 90.4 | |
| 20.0 | 19.6 ± 0.814 | 98.0 | |
| 400 | 407 ± 21.0 | 102 | |
| Three freeze-thaw cycles | 0.20 | 0.191 ± 0.002 | 94.4 |
| 0.50 | 0.490 ± 0.019 | 93.1 | |
| 20.0 | 19.4 ± 1.27 | 97.1 | |
| 400 | 399 ± 9.00 | 99.8 |
Several pretreatment methods have been proposed to separate free drugs in plasma from liposome-entrapped drugs, including ultrafiltration [18], gel exclusion chromatography [13], and solid phase extraction (SPE)/online SPE [14, 19, 20]. In this study, a rapid and sensitive method for the determination of T-DOX and F-DOX in human plasma was established by optimizing the existing pretreatment methods [16]. First, protein precipitation (PPT) was used to destroy liposomes and precipitate endogenous matrix from plasma for T-DOX measurement. To determine plasma levels of lipo-some-entrapped and free drugs, SPE was used to separate and enrich the free doxorubicin that was not encapsulated by liposomes, and simultaneously remove the endogenous matrix.
Recent research has found that, for DOX, appropriate acidification of the final organic phase eluent could make the analyte present in an ionic state, increase polarity and improve elution efficiency [16]. Some conditions, such as eluent volume and formic acid content, were optimized in the pre-experimental phase. In this study, when the eluent was 0.5% formic acid/methanol and the elution volume was 500 µL, the elution efficiency was highest, and a better mass spectrometry response was obtained. This method got great recovery results (RSD < 2%), on behalf of no adsorption of F-DOX on the SPE column. The LLOQ (1.00 ng mL−1) of this F-DOX approach also met the requirements for F-DOX determination.
3.3 Pharmacokinetic data
The mean plasma concentration-time profiles of T-DOX and F-DOX are shown in Fig. 4. The main pharmacokinetic parameters, calculated with WinNonlin software using a non-compartmental model, are presented in Table 4.
The mean plasma concentration-time curves of T-DOX and F-DOX after intravenous infusion of DOX·HCl-PLI to Chinese patients with breast cancer (n = 3, 30 mg m−2)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01129
Pharmacokinetic parameters of total doxorubicin (T-DOX) and free doxorubicin (F-DOX) after intravenous infusion of liposomal doxorubicin to Chinese patients with breast cancer (n = 3)
| PK parameter | Drug form | |
| T-DOX | F-DOX | |
| t1/2 (h) | 70.1 ± 40.4 | 81.3 ± 45.6 |
| AUClast (h*mg L−1) | 5,245 ± 874 | 1,130 ± 398a |
| AUCInf (h*mg L−1) | 5,370 ± 1,002 | 1,175 ± 458a |
| AUCExtr (%) | 2.06 ± 2.43 | 2.88 ± 3.65 |
| Vz (mL m−2) | 535 ± 231 | 2,850 ± 576a |
| Vss (mL m−2) | 656 ± 89.3 | 3,533 ± 312a |
| CL (mL h−1 m−2) | 5.73 ± 1.17. | 28.3 ± 10.8a |
| MRT (h) | 109 ± 26.2 | 120 ± 22.4 |
P < 0.05 statistically significant difference against T-DOX
After intravenous infusion of DOX·HCl-PLI at a dose of 30 mg m−2, the apparent elimination half-life (t1/2) values of T-DOX and F-DOX were estimated to be 70.1 ± 40.4 h and 81.3 ± 45.6 h. The mean area under the plasma concentration-time curve from time zero to the last measurable plasma concentration point (AUClast) and the mean area under the plasma concentration-time curve from time zero to time infinity (AUCInf) values for T-DOX were 5,245 ± 874 and 5,370 ± 1,002 h*µg mL−1, respectively, and 1,130 ± 398 and 1,175 ± 458 h*µg mL−1 for F-DOX. Clearance (Cl), mean residence time (MRT), and volume of distribution (Vz) values for T-DOX were estimated to be 5.73 ± 1.17 mL h−1 m−2, 109 ± 26.2 h, and 535 ± 231 mL m−2, respectively. The corresponding parameters for F-DOX were 28.3 ± 10.8 mL h−1 m−2, 120 ± 22.4 h and 2,850 ± 576 mL m−2, respectively.
After intravenous administration of DOX·HCl-PLI to breast cancer patients, the T-DOX concentration in plasma was about 4–6-fold higher than that of F-DOX, indicating that DOX was mostly in the form of the liposome-entrapped drug in the human body. AUC, the volume of distribution and clearance were significantly different be-tween T-DOX and F-DOX. The free drug concentration in the body depended on the slow degradation of liposomes, which was consistent with the pharmacokinetic results in dogs reported in the literature [16].
A summary of pharmacokinetic studies of different types of liposome-entrapped doxorubicin is presented in Fig. 5. The pharmacokinetic features were completely different between traditional free doxorubicin and liposomal doxorubicin in humans [21]. Liposome-entrapment of doxorubicin leads to longer apparent half-lives, in the range of 50–80 h, while clearance and volume of distribution are lower compared with free DOX, meeting requirements for fewer side effects and long-acting efficacy [10]. Different brands with different production processes, such as replacing hydrogenated soybean phosphatidylcholine (HSPC) with distearoyl phosphatidylcholine (DSPC) in the formulation [8, 9, 22], have similar pharmacokinetic profiles, demonstrating that this novel DOX·HCl-PLI has equivalent properties.
Pharmacokinetics of different formulations of doxorubicin entrapped in pegylated liposomes administered to humans. a doxorubicin hydrochloride liposome injection (DOX·HCl-PLI) total doxorubicin (T-DOX, 30 mg m−2, n = 3), b DOX·HCl-PLI free doxorubicin (F-DOX, 30 mg m−2, n = 3), c F-DOX [21] (50 mg m−2, n = 4), d Doxil® Liposome-entrapment of doxorubicin [21] (L-DOX, 25 mg m−2, n = 8), e Doxil® L-DOX [9] (35 mg m−2, n = 6), f DSPC-PLD L-DOX [22] (30 mg m−2, n = 9), g Doxil® T-DOX [8] (60 mg m−2, n = 7)
Citation: Acta Chromatographica 36, 3; 10.1556/1326.2023.01129
It is known that DOX is a competitive inhibitor of arachidonic acid metabolism by CYP2J2 expressed in the heart, which may lead to cardiotoxicity [23, 24]. Cytochrome P450 reductase, the obligate redox partner of CYP2J2, reduces DOX to 7-deoxydoxorubicin aglycone (DDA), which probably contributes to anticancer activity and cardiotoxicity. We also determined DDA in our study, but neither the PPT method nor the SPE method found it in plasma. In female BALB/c nude mice, DDA was detected within 1 h of a single intravenous injection of DOX at a dose of 1.3 mg kg−1 [25], while that remained undetectable both in serum or ascites of patients with ovarian cancer [26]. Marked interindividual variation in the metabolism of DDA has been reported [26–28]. Since the biotransformation pathway could be subject to genetic polymorphism, it is speculated that DDA was not found in this study because of rapid metabolism in Chinese patients and inappropriate blood collecting times.
4 Conclusions
A rapid and sensitive LC-MS/MS method was developed and validated for the pharmacokinetic study of T-DOX and F-DOX following DOX·HCl-PLI dosing in Chinese breast cancer patients. In this study, T-DOX concentrations in plasma were 4–6-fold higher than those of F-DOX. The AUCInf of T-DOX was 5,370 ± 1,002 h*µg mL−1, which was about 5-fold greater than that of F-DOX (1,175 ± 458 h*µg mL−1). The results provide a scientific foundation for further clinical application research.
Declaration of interest statement
The authors report there are no competing interests to declare.
Acknowledgements
The authors are grateful to International Science Editing (http://www.internationalscienceediting.com) for editing this manuscript. This work was supported by the National Natural Science Foundation of China under Grant [No. 82174062 and 81573682].
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