Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p = .0197, 2 mos: p = .0107, 3 mos: p = .0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.

Our findings suggest that choosing an optimal dosing time associated with the 24-h cycling of TNF-alpha could lead to effective treatment of rheumatoid arthritis by methotrexate.

We conclude that pre-administration of DOC may protect against ADR-induced toxic death and cardiotoxicity.

Abstract. Stiffness and cytokine in blood levels show 24-h rhythms in rheumatoid arthritis (RA) patients. We previously revealed that higher therapeutic effects were obtained in RA patients and RA model animals when the dosing time of methotrexate was chosen according to the 24-h rhythms to cytokine. In this study, we examined whether a dosing time-dependency of the therapeutic effect of tacrolimus (TAC) could be detected in collagen-induced arthritis (CIA) and MRL/lpr mice. To measure the levels of cytokines and serum amyloid A (SAA), blood was collected from CIA mice at different times. TAC was administered at two different dosing times based on these findings and its effects on arthritis and toxicity were examined. Plasma tumor necrosis factor (TNF)-α, interleukin-6 (IL-6), and SAA concentrations showed obvious 24-h rhythms with higher levels during the light phase and lower levels during the dark phase after RA crisis. The arthritis score and leukocyte counts were significantly lower in the group treated at 2 h after the light was turned on (HALO) than in the control and 14 HALO-treated groups. Our findings suggest that choosing an optimal dosing time could lead to the effective treatment of RA by TAC.

Our findings suggest that choosing an optimal dosing time associated with the 24-h cycling of TNF-alpha could lead to effective treatment of rheumatoid arthritis by methotrexate.

Studies s uggest t hat pr e-administration o f d ocetaxel ( DOC) in a driamycin ( ADR)-analysis o f h eart t issue from control, intermittent-dosing (DOC-ADR), and s imultaneous -dosing (ADR&DOC) groups. In DOC-ADR group, ADR was administered 12 h after DOC injection; i n A DR&DOC g roup, bot h dr ugs w ere a dministered s imultaneously; i n control group, saline was administered at the same timing as ADR injection of other groups. Heart samples were isolated from all mice 1 week after the treatment. The highly reproducible and sensitive method (FD-LC-MS/MS) identified nine proteins that were differentially expressed in heart tissue of control and the two treatment groups; seven of these nine proteins participate in cellular energy production pathways, including glycolysis, the tricarboxylic acid cycle, and the mit ochondrial electron t ransport c hain.Significantly higher e xpression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was observed in the DOC-ADR group, the g roup with the fewer cardiotoxic de aths, t han in the ADR&DOC group. Therefore, GAPDH may have potential as a drug target for protective intervention and a biomarker for 3 evaluation of the cardioprotective effects in pre-clinical studies.

Abstract. We previously reported that higher therapeutic effects were obtained in rheumatoid arthritis (RA) patients and RA model animals when the dosing-times of methotrexate and tacrolimus were chosen according to the 24-h rhythms of the inflammatory response. Mizoribine (MZR) is an immunosuppressive agent and is used against RA in the same manner as methotrexate and tacrolimus. In this study, we examined whether a dosing-time dependency of the therapeutic effect of MZR could be detected in collagen-induced arthritis (CIA) rats. To measure C-reactive protein (CRP) and tumor necrosis factor (TNF)-α levels, blood was collected from CIA rats at different times. MZR was administered at two different dosing-times based on these findings and its effects and toxicity were examined. CRP and TNF-α concentrations in blood showed significant 24-h rhythms. The exacerbation of arthritis and excessive increase in leukocytes in CIA rats were markedly lower in the group treated with MZR at the dark phase than those of the group treated with MZR at the light phase. These findings suggest that the therapeutic index of RA therapy may be improved by administering MZR at a time in the day when the inflammatory reaction begins to activate.

Abstract. We revealed that pre-treatment with docetaxel (DOC) 12 h before adriamycin (ADR) administration significantly reduced ADR-induced toxic death compared with the simultaneous dosing schedule that was commonly used in previous studies. We considered that pre-treatment with DOC relieves ADR-induced cardiotoxicity. In this study, we investigated the influence of DOC on the pharmacokinetics and pharmacodynamics of ADR in order to clarify the mechanism by which DOC pre-treatment relieves ADR-induced cardiotoxicity. When ADR and/or DOC was intravenously administered, the DOC pre-treatment (DOC-ADR) group showed significantly less toxic death than the ADR-alone group. We examined hepatopathy, nephropathy, leukopenia, and cardiotoxicity, all of which can cause toxic death. Of these toxicities, ADR-induced cardiotoxicity was significantly relieved in the DOC-ADR group. To elucidate the mechanism by which DOC pre-treatment relieved ADR-induced cardiotoxicity, lipid peroxidation as a proxy for the free radical level and the pharmacokinetics of ADR were measured. There was no difference in the pharmacokinetics of ADR between the ADR and DOC-ADR groups. On the other hand, the DOC-ADR group showed significantly inhibited lipid peroxidation in the heart compared with the ADR group. It was considered that DOC pre-administration inhibited ADR-induced free radicals and decreased cardiotoxicity.