Cell Medicine, Vol. 7, pp. 51–57, 2015 Printed in the USA. All rights reserved. Copyright Ó 2015 Cognizant Comm. Corp.
2155-1790/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/215517914X681802 www.cognizantcommunication.com
Synergistic Effects of Calcineurin Inhibitors and Steroids on Steroid Sensitivity of Peripheral Blood Mononuclear Cells Hironori Takeuchi,* Hitoshi Iwamoto,† Yuki Nakamura,† Toshihiko Hirano,‡ Osamu Konno,† Yu Kihara,† Naokazu Chiba,† Takayoshi Yokoyama,† Kiminori Takano,† Tatsunori Toraishi,§ Kiyoshi Okuyama,§ Chie Ikeda,† Sachiko Tanaka,‡ Kenji Onda,‡ Akiko Soga,* Yukiko Kikuchi,* Takashi Kawaguchi,* Shigeyuki Kawachi,† Sakae Unezaki,* and Motohide Shimazu† *Department of Practical Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan †The Fifth Department of Surgery, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan ‡Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan §Department of Pharmaceutics, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan
The steroid receptor (SR) complex contains FKBP51 and FKBP52, which bind to tacrolimus (TAC) and cyclophilin 40, which, in turn, bind to cyclosporine (CYA); these influence the intranuclear mobility of steroid–SR complexes. Pharmacodynamic interactions are thought to exist between steroids and calcineurin inhibitors (CNIs) on the SR complex. We examined the effect of CNIs on steroid sensitivity. Methylprednisolone (MPSL) sensitivity was estimated as the concentration inhibiting mitosis in 50% (IC50) of peripheral blood mononuclear cells and as the area under the MPSL concentration–proliferation suppressive rate curves (CPS-AUC) in 30 healthy subjects. MPSL sensitivity was compared between the additive group (AG) as the MPSL sensitivity that was a result of addition of the proliferation suppressive rate of CNIs to that of MPSL and the mixed culture group (MCG) as MPSL sensitivity of mixed culture with both MPSL and CNIs in identical patients. IC50 values of MPSL and cortisol sensitivity were examined before and 2 months after CNI administration in 23 renal transplant recipients. IC50 and CPS-AUC values of MPSL were lower in the MCG than in the AG with administration of TAC and CYA. The CPS-AUC ratio of MCG and AG was lower in the TAC group. IC50 values of MPSL and cortisol tended to be lower after administration of TAC and CYA, and a significant difference was observed in the IC50 of cortisol after TAC administration. Steroid sensitivity increased with both TAC and CYA. Furthermore, TAC had a greater effect on increasing sensitivity. Thus, concomitant administration of CNIs and steroids can increase steroid sensitivity. Key words: Steroid sensitivity; Pharmacodynamic interaction; Calcineurin inhibitors (CNIs); Tacrolimus (TAC); Cyclosporine (CYA)
introduction Current early stage immunosuppressant therapy in renal transplants generally consists of a four-drug combination therapy centered on calcineurin inhibitors (CNIs) and includes the antimetabolite mycophenolate mofetil (MMF), steroids, and basiliximab, an anticluster of differentiation 25 (CD25) antibody. CNIs and steroids are combined not only in renal transplants but also regularly combined for many types of organ transplants, although their clinical pharmacodynamic interaction has not yet been examined. The CNI-binding proteins FK506-binding protein (FKBP) 51 and FKBP52 (5), as well as cyclophilin 40 (Cyp40) (8) exist in steroid receptor (SR) complexes. Since these influence the affinity and intranuclear mobility of steroid–SR complexes, it is believed that steroids
and CNIs [tacrolimus (TAC) and cyclosporine (CYA)] interact at the SR complex level (3,4,7,10,12,14–16,19). That is, while it is thought that the steroid sensitivity of peripheral blood mononuclear cells (PBMCs) is influenced by TAC binding to FKBP51 and FKBP52 and CYA to Cyp40 (5,8), how this influences clinical immunosuppressant therapy in organ transplants remains unclear. The approval of the protein kinase inhibitor everolimus in Japan will likely lead to a variety of combination therapies. Thus, a full understanding of the pharmacodynamic interactions of these drugs would be valuable for evaluating comprehensive immunosuppressant effects. Therefore, we investigated 1) the influence of CNIs on the steroid sensitivity of PBMCs and 2) steroid sensitivity
Received May 21, 2014; final acceptance October 27, 2014. Online prepub date: December 12, 2014. Address correspondence to Hironori Takeuchi, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel/Fax: +81-42-676-5836; E-mail: [email protected]
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in renal transplant patients before and after coadministration (before and after transplant) of CNIs. MATERIALS AND METHODS Effects of CNIs on Steroid Sensitivity of PBMCs in Healthy Subjects Subjects. This study was approved by the Ethical Committee of Tokyo University of Pharmacy and Life Sciences, and informed consent was obtained from all healthy subjects. This study was performed from Sep tember 2010 to December 2010. Effects of CNIs on steroid sensitivity of PBMCs in 30 healthy subjects (14 males and 16 females) aged 22.9 ± 1.8 years (average ± SD) were assessed. Sensitivity Test of PBMCs (6,17). Venous blood was taken from each subject and heparinized (Terumo Corporation, Tokyo, Japan). The heparinized blood (10 ml) was layered on 3 ml of Ficoll-Hypaque solution (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) and centrifuged at 1,300 × g for 15 min, and the PBMCs, including lymphocytes, were separated. The cells were washed and resuspended in Roswell Park Memorial Institute 1640 medium (Gibco Laboratories, Rockville, MD, USA), containing 10% fetal bovine serum (Gibco Laboratories), 100,000 IU/L penicillin G (Invitrogen, Tokyo, Japan), and 100 mg/L streptomycin (Invitrogen), to a final density of 1 × 106 cells/L. The cell suspension (200 μl) prepared was placed in 96 flat-bottom wells of a microtiter plate (AGC Techno Glass CO., Shizuoka, Japan). Concanavalin A (Seikagaku Kogyo Co., Tokyo, Japan) was added to each well as the mitogen to activate mainly T-cells, to achieve a final concentration of 5.0 μg/ml. Subsequently, 4 μl of an ethanol solution (Wako Pure Chemical Industries, Osaka, Japan) containing methylprednisolone (MPSL) (Sigma-Aldrich, St. Louis, MO, USA) was added to the wells to give final concentrations of 0.1, 1, 10, 100, and 1,000 μg/L; to the control well, 4 μl of ethanol was added. The plate was incubated for 96 h at 37°C in a humidified chamber containing 5% CO2. The cells were pulsed with 18.5 kBq/well of [3H]thymidine (GE Healthcare Japan, Hino, Japan) for the last 16 h of incubation, collected on a glassfiber filter paper (Futabe Medical, Inc., Tokyo, Japan) by using a multiharvester (Futabe Medical, Inc.), and dried. The radioactivity on the filter paper was further processed for liquid scintillation counting (Hitachi Aloka Medical, Ltd., Tokyo, Japan). We determined the mean from triplicate counts obtained for each sample. The concentration–proliferation suppressive curves (CPS-AUC) of MPSL were generated. The concentration of MPSL that could inhibit 50% of PBMC mitosis (IC50) and the MPSL CPS-AUCs were determined from the concentration– response curve.
Takeuchi ET AL.
Estimation of Effects on Steroid Sensitivity of CNIs for PBMCs. After determining the CPS-AUC for MPSL alone in the PBMC sensitivity test described above, CPSAUCs were calculated for the CNIs [TAC (Astellas Co., Tokyo, Japan) and CYA (Novartis Co., Basel, Switzerland)] with the same method. Proliferation suppression rates were then determined from the curves for each CNI concentration (three concentrations each: TAC 0.1, 0.175, and 0.25 ng/ml; CYA 5, 10, and 25 ng/ml). The CNI concentrations were decided in preliminary testing as the concentrations at which the CPS-AUC was maintained during combination cultures. The CPS-AUC obtained by adding the proliferation suppression rate of these CNI concentrations to the steroid CPS-AUC served as the additive group (AG), while the CPS-AUC determined from a combined culture of steroids and the same CNI concentrations used in the AG served as the mixed culture group (MCG). MPSL sensitivity was assessed by comparing IC50 and CPS-AUC values between AG and MCG in identical subjects. If the MPSL IC50 and CPS-AUC values in MCG were found to be lower than in the AG, the CNI was determined to have a pharmacodynamic strengthening action on steroids. To compare the pharmacodynamic interaction of TAC and CYA on steroid sensitivity, AG to MCG ratios (AG/ MCG) for IC50 and CPS-AUC values were calculated and compared at three concentration ratios (titer ratios): 0.25 ng/ml:5 ng/ml (TAC:CYA = 1:20), 0.1 ng/ml:5 ng/ml (TAC:CYA = 1:50), and 0.25 ng/ml:25 ng/ml (TAC:CYA = 1:100). Statistical Analysis. Significant differences between IC50 and CPS-AUC values in the AG and MCG were determined using the statistical analysis software JMP version 8 (SAS, Cary, NC, USA). The Wilcoxon signed-rank test was used, with significance determined at p
2155-1790/15 $90.00 + .00 DOI: http://dx.doi.org/10.3727/215517914X681802 www.cognizantcommunication.com
Synergistic Effects of Calcineurin Inhibitors and Steroids on Steroid Sensitivity of Peripheral Blood Mononuclear Cells Hironori Takeuchi,* Hitoshi Iwamoto,† Yuki Nakamura,† Toshihiko Hirano,‡ Osamu Konno,† Yu Kihara,† Naokazu Chiba,† Takayoshi Yokoyama,† Kiminori Takano,† Tatsunori Toraishi,§ Kiyoshi Okuyama,§ Chie Ikeda,† Sachiko Tanaka,‡ Kenji Onda,‡ Akiko Soga,* Yukiko Kikuchi,* Takashi Kawaguchi,* Shigeyuki Kawachi,† Sakae Unezaki,* and Motohide Shimazu† *Department of Practical Pharmacy, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan †The Fifth Department of Surgery, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan ‡Department of Clinical Pharmacology, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan §Department of Pharmaceutics, Hachioji Medical Center, Tokyo Medical University, Hachioji, Tokyo, Japan
The steroid receptor (SR) complex contains FKBP51 and FKBP52, which bind to tacrolimus (TAC) and cyclophilin 40, which, in turn, bind to cyclosporine (CYA); these influence the intranuclear mobility of steroid–SR complexes. Pharmacodynamic interactions are thought to exist between steroids and calcineurin inhibitors (CNIs) on the SR complex. We examined the effect of CNIs on steroid sensitivity. Methylprednisolone (MPSL) sensitivity was estimated as the concentration inhibiting mitosis in 50% (IC50) of peripheral blood mononuclear cells and as the area under the MPSL concentration–proliferation suppressive rate curves (CPS-AUC) in 30 healthy subjects. MPSL sensitivity was compared between the additive group (AG) as the MPSL sensitivity that was a result of addition of the proliferation suppressive rate of CNIs to that of MPSL and the mixed culture group (MCG) as MPSL sensitivity of mixed culture with both MPSL and CNIs in identical patients. IC50 values of MPSL and cortisol sensitivity were examined before and 2 months after CNI administration in 23 renal transplant recipients. IC50 and CPS-AUC values of MPSL were lower in the MCG than in the AG with administration of TAC and CYA. The CPS-AUC ratio of MCG and AG was lower in the TAC group. IC50 values of MPSL and cortisol tended to be lower after administration of TAC and CYA, and a significant difference was observed in the IC50 of cortisol after TAC administration. Steroid sensitivity increased with both TAC and CYA. Furthermore, TAC had a greater effect on increasing sensitivity. Thus, concomitant administration of CNIs and steroids can increase steroid sensitivity. Key words: Steroid sensitivity; Pharmacodynamic interaction; Calcineurin inhibitors (CNIs); Tacrolimus (TAC); Cyclosporine (CYA)
introduction Current early stage immunosuppressant therapy in renal transplants generally consists of a four-drug combination therapy centered on calcineurin inhibitors (CNIs) and includes the antimetabolite mycophenolate mofetil (MMF), steroids, and basiliximab, an anticluster of differentiation 25 (CD25) antibody. CNIs and steroids are combined not only in renal transplants but also regularly combined for many types of organ transplants, although their clinical pharmacodynamic interaction has not yet been examined. The CNI-binding proteins FK506-binding protein (FKBP) 51 and FKBP52 (5), as well as cyclophilin 40 (Cyp40) (8) exist in steroid receptor (SR) complexes. Since these influence the affinity and intranuclear mobility of steroid–SR complexes, it is believed that steroids
and CNIs [tacrolimus (TAC) and cyclosporine (CYA)] interact at the SR complex level (3,4,7,10,12,14–16,19). That is, while it is thought that the steroid sensitivity of peripheral blood mononuclear cells (PBMCs) is influenced by TAC binding to FKBP51 and FKBP52 and CYA to Cyp40 (5,8), how this influences clinical immunosuppressant therapy in organ transplants remains unclear. The approval of the protein kinase inhibitor everolimus in Japan will likely lead to a variety of combination therapies. Thus, a full understanding of the pharmacodynamic interactions of these drugs would be valuable for evaluating comprehensive immunosuppressant effects. Therefore, we investigated 1) the influence of CNIs on the steroid sensitivity of PBMCs and 2) steroid sensitivity
Received May 21, 2014; final acceptance October 27, 2014. Online prepub date: December 12, 2014. Address correspondence to Hironori Takeuchi, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan. Tel/Fax: +81-42-676-5836; E-mail: [email protected]
51
52
in renal transplant patients before and after coadministration (before and after transplant) of CNIs. MATERIALS AND METHODS Effects of CNIs on Steroid Sensitivity of PBMCs in Healthy Subjects Subjects. This study was approved by the Ethical Committee of Tokyo University of Pharmacy and Life Sciences, and informed consent was obtained from all healthy subjects. This study was performed from Sep tember 2010 to December 2010. Effects of CNIs on steroid sensitivity of PBMCs in 30 healthy subjects (14 males and 16 females) aged 22.9 ± 1.8 years (average ± SD) were assessed. Sensitivity Test of PBMCs (6,17). Venous blood was taken from each subject and heparinized (Terumo Corporation, Tokyo, Japan). The heparinized blood (10 ml) was layered on 3 ml of Ficoll-Hypaque solution (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) and centrifuged at 1,300 × g for 15 min, and the PBMCs, including lymphocytes, were separated. The cells were washed and resuspended in Roswell Park Memorial Institute 1640 medium (Gibco Laboratories, Rockville, MD, USA), containing 10% fetal bovine serum (Gibco Laboratories), 100,000 IU/L penicillin G (Invitrogen, Tokyo, Japan), and 100 mg/L streptomycin (Invitrogen), to a final density of 1 × 106 cells/L. The cell suspension (200 μl) prepared was placed in 96 flat-bottom wells of a microtiter plate (AGC Techno Glass CO., Shizuoka, Japan). Concanavalin A (Seikagaku Kogyo Co., Tokyo, Japan) was added to each well as the mitogen to activate mainly T-cells, to achieve a final concentration of 5.0 μg/ml. Subsequently, 4 μl of an ethanol solution (Wako Pure Chemical Industries, Osaka, Japan) containing methylprednisolone (MPSL) (Sigma-Aldrich, St. Louis, MO, USA) was added to the wells to give final concentrations of 0.1, 1, 10, 100, and 1,000 μg/L; to the control well, 4 μl of ethanol was added. The plate was incubated for 96 h at 37°C in a humidified chamber containing 5% CO2. The cells were pulsed with 18.5 kBq/well of [3H]thymidine (GE Healthcare Japan, Hino, Japan) for the last 16 h of incubation, collected on a glassfiber filter paper (Futabe Medical, Inc., Tokyo, Japan) by using a multiharvester (Futabe Medical, Inc.), and dried. The radioactivity on the filter paper was further processed for liquid scintillation counting (Hitachi Aloka Medical, Ltd., Tokyo, Japan). We determined the mean from triplicate counts obtained for each sample. The concentration–proliferation suppressive curves (CPS-AUC) of MPSL were generated. The concentration of MPSL that could inhibit 50% of PBMC mitosis (IC50) and the MPSL CPS-AUCs were determined from the concentration– response curve.
Takeuchi ET AL.
Estimation of Effects on Steroid Sensitivity of CNIs for PBMCs. After determining the CPS-AUC for MPSL alone in the PBMC sensitivity test described above, CPSAUCs were calculated for the CNIs [TAC (Astellas Co., Tokyo, Japan) and CYA (Novartis Co., Basel, Switzerland)] with the same method. Proliferation suppression rates were then determined from the curves for each CNI concentration (three concentrations each: TAC 0.1, 0.175, and 0.25 ng/ml; CYA 5, 10, and 25 ng/ml). The CNI concentrations were decided in preliminary testing as the concentrations at which the CPS-AUC was maintained during combination cultures. The CPS-AUC obtained by adding the proliferation suppression rate of these CNI concentrations to the steroid CPS-AUC served as the additive group (AG), while the CPS-AUC determined from a combined culture of steroids and the same CNI concentrations used in the AG served as the mixed culture group (MCG). MPSL sensitivity was assessed by comparing IC50 and CPS-AUC values between AG and MCG in identical subjects. If the MPSL IC50 and CPS-AUC values in MCG were found to be lower than in the AG, the CNI was determined to have a pharmacodynamic strengthening action on steroids. To compare the pharmacodynamic interaction of TAC and CYA on steroid sensitivity, AG to MCG ratios (AG/ MCG) for IC50 and CPS-AUC values were calculated and compared at three concentration ratios (titer ratios): 0.25 ng/ml:5 ng/ml (TAC:CYA = 1:20), 0.1 ng/ml:5 ng/ml (TAC:CYA = 1:50), and 0.25 ng/ml:25 ng/ml (TAC:CYA = 1:100). Statistical Analysis. Significant differences between IC50 and CPS-AUC values in the AG and MCG were determined using the statistical analysis software JMP version 8 (SAS, Cary, NC, USA). The Wilcoxon signed-rank test was used, with significance determined at p
