Evaluation for pharmacokinetic exposure of cytotoxic anticancer drugs in elderly patients receiving (R-)CHOP therapy

(R-)miniCHOP therapy, which delivers approximately half-doses of the (R-)CHOP regimen, has shown efficacy and safety in patients who are more than 80 years old. This study aimed to compare the area under the plasma concentration–time curves (AUCs) of vincristine (VCR), doxorubicin (DXR), and cyclophosphamide (CPA) between (R-)CHOP and (R-)miniCHOP regimens. The AUCs were compared between patients aged 65–79 years receiving (R-)CHOP therapy and those aged 80 years and older receiving (R-)miniCHOP therapy. Age was not an independent variable for predicting the dose-adjusted AUCs (AUC/Ds) of cytotoxic anticancer drugs. The median AUCs of DXR and CPA were significantly smaller in the (R-)miniCHOP group than in the (R-)CHOP group (168.7 vs. 257.9 ng h/mL, P = 0.003, and 219.9 vs. 301.7 µg h/mL, P = 0.020, respectively). The median AUCs of VCR showed the same trend but the difference was not significant (24.83 vs. 34.85 ng h/mL, P = 0.135). It is possible that the AUCs of VCR, DXR, and CPA in patients aged 80 years and older receiving (R-)miniCHOP therapy may be lower than those in patients 65–79 years old receiving (R-)CHOP therapy.

In this study, we first investigated the factors that contribute to the effects of individual patient characteristics on the pharmacokinetics of VCR, DXR, and CPA in elderly patients aged 65 years or older. Next, we attempted to establish a simultaneous estimation method for area under the plasma concentration-time curves (AUCs) based on blood concentrations at the same sampling point to monitor the pharmacokinetic exposure of these cytotoxic anticancer drugs in the clinical setting. Finally, we compared the AUCs of VCR, DXR, and CPA between elderly patients aged 80 years and older receiving (R-)miniCHOP therapy and those aged 65-79 years old receiving (R-)CHOP therapy.

Results
Patient characteristics. Patient characteristics are listed in Table 1. A total of 20 patients (9 males and 11 females) were enrolled in this study. Eight patients were 80 years or older and receiving (R-)miniCHOP therapy. None of the patients had severe liver or renal dysfunction. There were no patients taking drugs that obviously affected the pharmacokinetics of VCR, DXR, or CPA, such as azole antifungal agents, cyclosporine, and drug-metabolizing enzyme-inducing antiepileptic drugs [14][15][16][17] . There were significant differences in median serum albumin (Alb) and estimated glomerular filtration rate (eGFR) between (R-)CHOP and (R-)miniCHOP groups (3.7 vs. 2.8 g/L, P = 0.047 and 70 vs. 47 mL/min, P = 0.020, respectively).

Pharmacokinetic analysis.
Variation of pharmacokinetic parameters. The pharmacokinetic profiles of each cytotoxic anticancer drug are shown in Fig. 1.
The variations of plasma concentration at each sampling point were larger for VCR and DXR than for CPA. The plasma concentrations of CPA and VCR after 9 and 9.5 h, respectively, from the start of administration generally decreased according to the first-order rate constant; the concentration of DXR showed a diphasic decrease.
Pharmacokinetic parameters of each cytotoxic anticancer drug are listed in Table 2. The coefficients of variation (%CV) of AUC and dose-adjusted AUCs (AUC/Ds) for all cytotoxic anticancer drugs were greater than 15% and those of VCR were the largest of the 3 agents (40.5% and 36.7%, respectively). The %CV of VCR t 1/2 was larger than that of CPA; the %CV of DXR t 1/2 could not be calculated because its concentration showed a diphasic decrease. Although the AUC of CPA was significantly correlated with the AUCs of DXR and VCR (rho = 0.549, P = 0.012, and rho = 0.550, P = 0.012, respectively), there was no significant correlation between the AUCs of DXR and VCR (rho = 0.096, P = 0.686).
Relationships between AUC/Ds and patient characteristics. The results of a univariate analysis of variance for AUC/D and t 1/2 are shown in Table 3. Although there was no significant correlation between any of the patient variables and AUC/D of VCR, body weight and body surface area (BSA) had significant correlations with AUC/D of CPA and DXR (P < 0.05). Stepwise multiple linear regression analysis showed that the BSA and Alb were independent factors for predicting the AUC/D of DXR (partial R 2 = 0.370, P = 0.001, and partial R 2 = 0.307, P = 0.001, respectively), and BSA was an independent factor for predicting the AUC/D of CPA (P = 0.002).

Discussion
This study is the first report to show differences in AUCs of VCR, DXR, and CPA between (R-)CHOP and (R-) miniCHOP regimens. In addition, a simple estimation method of AUCs for these cytotoxic anticancer drugs was established. Until now, very few clinical studies have focused on the pharmacokinetics of cytotoxic anticancer drugs in elderly patients receiving CHOP therapy. The AUCs of DXR and CPA were significantly lower in patients receiving (R-)miniCHOP therapy than in those receiving (R-)CHOP therapy. Although the AUC of VCR was not significantly different between the 2 groups, a similar trend was observed. Because the dose and dose range of VCR were lower and narrower than those of DXR and CPA, there may be no clear differences in these AUCs between groups. At this point, it remains unclear whether elderly patients aged 80 years and older should be treated with equal pharmacokinetic exposures of cytotoxic anticancer drugs as those for patients aged 65-79 years old. Still, it has been reported that the (R-)miniCHOP regimen provides a good balance between efficacy and safety in patients aged 80 years and older with DLBCL 9,11 . Therefore, the optimal pharmacokinetic exposure of cytotoxic anticancer drugs in these elderly patients may be lower than that of elderly patients aged 65 to 79 years old. Outcomes in elderly patients are worse than in younger patients, but numerous studies have shown that this difference cannot be explained by age alone 18,19 . Because it is important to complete the 6 cycles in CHOP therapy 20 , further investigations are required to determine whether the AUCs of cytotoxic anticancer drugs affect the relative DIs. Table 3. Correlations of dose-adjusted AUC and t 1/2 with patient variables. Sex (male:female), 9:11. Alb, serum albumin; ALT, alanine aminotransferase; AST, aspartate aminotransferase; AUC n1-n2 /D, dose-adjusted area under the concentration-time curve from n1 to n2 h; BSA, body surface area; eGFR, estimated glomerular filtration rate; T-Bil, serum total bilirubin; t 1/2 , elimination half-life.  www.nature.com/scientificreports/ In this study, age was not an independent variable for predicting the AUC/Ds of VCR, DXR, and CPA in elderly patients aged 65 years and older receiving (R-)CHOP or (R-)miniCHOP therapy. Baudry et al. reported that aging did not affect the pharmacokinetics of DXR and CPA in very old patients (≥ 75 years old) receiving R-miniCHOP 21 . Our results agree with the findings of this study. BSA, which was an independent variable for predicting the AUC/Ds of DXR and CPA in this study, has been reported to affect pharmacokinetics parameters in other studies [22][23][24] . In addition, in this study, a negative correlation between Alb and AUC/D of DXR was found in elderly patients. It has been reported that DXR clearance (CL) correlates with various liver function test values, including Alb 25,26 . DXR is eliminated primarily by hepatic metabolism and biliary excretion. Although decreased Alb may represent decreased metabolic capacity, other liver function test values did not affect DXR AUC/D in this study. Further studies are needed to evaluate the relationship between Alb and pharmacokinetic parameters of DXR. Unlike with DXR and CPA, patient factors were not found to impact VCR AUC/D in this study. To the best of our knowledge, the effects of patient factors, such as sex, BSA, and biochemistry covariates, on VCR CL in elderly patients have not yet been clarified. Recently, it has been reported that gene polymorphisms of drug-metabolizing enzymes or transporters affect the pharmacokinetics of these cytotoxic anticancer drugs 23,[27][28][29][30] . Thus, various factors have a potential impact on the pharmacokinetic exposure of elderly patients receiving CHOP therapy. However, these covariate factors alone could not fully explain individual variabilities in the pharmacokinetics of VCR, DXR, or CPA. Moreover, the associations between the AUCs of these cytotoxic anticancer drugs and the outcomes of patients with DLBCL are also unclear.
Plasma concentrations at 9-9.5 h after the start of administration were well correlated with AUCs within 24 h from the end of administration in patients receiving VCR, DXR, and CPA. Because accurate measurement of the AUC requires the collection and analysis of multiple blood samples, it is difficult to use this value as an indicator in the clinical setting. On the other hand, the simultaneous estimation method of AUCs of 3 cytotoxic anticancer drugs by a single blood collection at one sampling point is very convenient. The Bayesian estimation method provides a simple technique with good performance for estimating AUC of various drugs. Population pharmacokinetic parameters for DXR 21,31 and CPA 21,32 used in the Bayesian estimation method have already been reported. The plasma concentrations at 9-9.5 h after the start of administration are considered the best single sampling point for estimating AUCs of these cytotoxic anticancer drugs according to this Bayesian estimation method. Moreover, estimation of AUCs using a regression line is a simpler analytical method than Bayesian estimation, which requires the use of computer software for analysis. In the future, the AUC estimation formulas for VCR, DXR, and CPA constructed in this study may be useful for examining the relationships between pharmacokinetic exposure and relative DI in CHOP therapy in the clinical setting.
This study had a few limitations. First, the number of patients included in our study population was very small, so associations between AUCs and toxicities, such as severe myelosuppression and peripheral neuropathy, or efficacy, such as progression-free survival or overall survival, were not revealed. Therefore, pharmacokinetics-pharmacodynamics studies consisting of many elderly patients receiving (R-)CHOP or (R-)miniCHOP therapy are needed in the future. Second, the AUCs calculated in this study did not include area under the time during the infusion and the distribution phase, which has a rapid decrease of blood concentration immediately after the end of administration. However, because they cover most of the area of the elimination phase, they can be considered to be indicators of safety and effectiveness. Third, the AUC estimation formulas constructed in this study were derived from a very small number of patients receiving CHOP therapy; thus, validation studies in special populations, such as patients with liver dysfunction, low eGFR, or extreme BSA, are needed. However, our findings revealed that the AUCs of VCR, DXR, and CPA included in CHOP therapy can be simultaneously estimated by Dose intensity calculation. The DI of each cytotoxic anticancer drug was calculated as a percentage of the dose of the original CHOP regimen. For patients whose BSA exceeded 1.43 m 2 , the DI of VCR was calculated with 2 mg/body as 100%.
Assay of plasma concentrations of VCR, DXR, and CPA. Plasma concentrations of cytotoxic anticancer drugs were measured by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/ MS) using the ACQUITY UPLC System (Waters, MA, USA). Pretreatment of plasma samples and UPLC separation were performed based on the method we previously reported 33 . Plasma (100 µL) was mixed with 150 µL of acetonitrile and 10 µL of internal standards (50 ng/mL trofosphamide for DXR and 20 µg/mL cyclophosphamide-d for CPA). The mixture was vortexed for 30 s and centrifuged at 13,500 rpm for 5 min at room temperature. Next, 100 µL of supernatant was diluted with 100 µL of MiliQ for DXR measurement, and 20 µL of supernatant was diluted with 380 µL of MiliQ for CPA measurement. Samples for VCR measurement were prepared according to a different procedure than those for DXR and CPA measurement. Plasma (200 µL) was mixed with 300 µL of acetonitrile and 20 µL of internal standards (50 ng/mL vinblastine [VBL]). The mixture was vortexed for 30 s and centrifuged at 13,500 rpm for 5 min at room temperature. Next, 350 µL of supernatant was evaporated using the centrifuge evaporator and then redissolved with 100 µL of 30% acetonitrile. The mixture was vortexed for 30 s and centrifuged at 13,500 rpm for 1 min at room temperature. The sample was transferred to an autosampler vial and then 2 µL for CPA, 8 µL for DXR, and 10 µL for VCR were injected into an ACQUITY UPLC HSS C18 column (1.7 um, 100 mm × 2.1 mm) at 40 °C. The mobile phase consisted of (A) MiliQ with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid at a flow rate of 0.4 mL/min. Gradient conditions were as follows: 0-1.0 min, held in 5% B; 1.0-6.0 min, linear from 5 to 95% B; 6.0-7.0 min, held in 95% B; 7.0-7.1 min, linear from 95 to 5% B; 7.1-10.0 min, held in 5% B. The analyte and internal standard were ionized and detected using Xevo TQD (Waters, MA, USA). Positive electrospray ionization was performed in the multiple reaction monitoring mode. Ion transitions were as follows: m/z 413. 5   www.nature.com/scientificreports/ energies were 60 V and 40 eV for VCR and VBL, 30 V and 20 eV for CPA and cyclophosphamide-d, 30 V and 10 eV for DXR, and 30 V and 30 eV for trofosphamide, respectively. The lower limits of quantitation for VCR, DXR, and CPA were 0.5 ng/mL, 1 ng/mL, and 1 µg/mL, respectively. The calibration curve was linear in the following ranges: VCR, 0.5-10 ng/mL; DXR, 2-80 ng/mL; CPA, 2.5-40 µg/mL. If the plasma concentration of CPA exceeded the upper limit of the standard curve, the sample was diluted by one-third. The intra-and interday accuracy values (CV%) were all within ± 15% and precision values (CV%) were all less than 15% in each calibration curve range.
Pharmacokinetic analysis. Pharmacokinetic analyses with VCR, DXR, and CPA were carried out using a standard non-compartmental model with Phoenix WinNonlin (Pharsight Co, Mountain View, CA, version 8.1). Plasma samples of CPA, (VCR) and (DXR) were collected at 1 (1.5), 2 (2.5), 3 (3.5), 5 (5.5), 9 (9.5), 13 (13.5) and 25 (25.5) h after the start of each drug administration. The AUCs of each cytotoxic anticancer drug were calculated on the basis of plasma concentrations after initial blood sampling (Fig. 3). Each AUC was calculated using the linear trapezoidal rule. The elimination half-life (t 1/2 ) was obtained using the log-linear regression of the terminal phase of the concentration-time data for at least 3 sampling points (elimination half-life = ln2/k e ; k e = elimination rate constant).
Statistical procedures. The Shapiro-Wilk test was used to assess distribution. The characteristics of patients and pharmacokinetic parameters of VCR, DXR, and CPA are expressed as means ± standard deviation or medians and range. The Mann-Whitney U test was used to determine the difference in continuous values between groups. Spearman's rank correlation coefficient test was used to assess correlations in continuous values between groups, and all results are expressed as Spearman's rho values. Factors with P-values less than 0.05 in a univariate analysis were included in a stepwise multiple linear regression analysis to identify independent factors for predicting results. The estimation formulas were constructed using simple linear regression analysis. The percent variation that could be explained by the simple regression equation was expressed as a coefficient of determination (R 2 ). A P value less than 0.05 was considered statistically significant. Statistical analysis was performed with SPSS 26.0 for Windows (SPSS IBM Japan Inc., Tokyo, Japan).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.