Author Affiliations: Department of School Health Science, Faculty of Education, Hirosaki University, Hirosaki 036-8560, Japan (Tanaka H); Department of Pediatrics, Hirosaki University Hospital, Hirosaki 036-8563, Japan (Tanaka H, Tsuruga K); Department of Vacular Biology, Graduate School of Medicine, Hirosaki University, Hirosaki 036-8562, Japan (Imaizumi T)

 

Corresponding Author: Hiroshi Tanaka, MD, PhD, Department of School Health Science, Faculty of Education, Hirosaki University, 1 Bunkyo-cho, Hirosaki 036-8560, Japan (Email: hirotana@cc.hirosaki-u.ac.jp/hirotana@hirosaki-u.ac.jp)

 

doi: 10.1007/s12519-015-0013-7

 

 

 

 

 

 

 

 

Mizoribine (MZR), a purine synthesis inhibitor, was developed in Japan about 20 years ago. The mode of action of MZR is very similar to that of mycophenolate mofetil (MMF), involving selective inhibition of inosine monophosphate dehydrogenease (IMPD) in the pathway of de novo purine nucleotide synthesis, which results in the suppression of T and B lymphocyte proliferation.^[1,2] So far, MZR has been used successfully without serious adverse effects in the treatment of renal transplant recipients,^[3] nephrotic syndrome (NS),^[4,5] immunoglobulin (Ig)A  nephropathy,^[6,7] and lupus nephritis (LN),^[8,9] mainly in Japan and East Asia. In a previous experimental setting, MZR has been reported to reduce urinary protein excretion in rat models of nephrosis induced by puromycin aminonucleoside (PAN), in addition to its immunosuppressive effect.^[10] Also, it has been reported that 14-3-3 proteins, which bind to MZR, interact with glucocorticoid receptors after MZR administration, to enhance receptor transcriptional activity.^[11] These laboratory observations might explain the steroid-sparing effect of MZR.

From a clinical viewpoint, MZR inhibition of IMPD is competitive, and thus different from that induced by MMF.^[1,2] Therefore, the peak blood concentration of MZR required to effectively inhibit the human mixed-lymphocyte reaction is reported to be 3.0 to 6.0 μg/mL.^[3] In addition, the MZR concentration required to effectively enhance steroid receptor activity via 14-3-3 proteins is reportedly in excess of 10 μmol/L, which corresponds to a blood MZR level of approximately 2.6 μg/mL.^[11] Indeed, recent clinical reports have indicated that the efficacy of MZR may depend on the peak serum level of the drug.^[12-17] Accordingly, it is now thought that a peak serum MZR level of at least 2.5-3.0 μg/mL may be needed in order to sustain long-term efficacy for the treatment of patients with glomerular diseases.^[13,15] However, when using the conventional daily low-dose MZR protocol (3-4 mg/kg) in patients with NS or LN, the peak blood level of the drug usually remains at around 1.0 μg/mL,^[12] which may explain the previously reported relatively mild "immunosuppressive" efficacy of MZR in clinical practice.^[4,14] In this context, we and other groups have conducted trials of intermittent pulse therapy with oral MZR (up to 10 mg/kg; MZR-pulse) to attain increased peak blood levels of MZR in selective patients with NS and LN.^{[12,13,15-18]} The results suggested that this new treatment protocol was beneficial and resulted in higher efficacy and lower toxicity than the conventional protocol.^[15,17] The rationale for using MZR-pulse was as follows: 1) MZR is rapidly excreted into the urine: about 90% of the drug being completely eliminated from the circulation within about 12 hours after oral intake; therefore, accumulation of the drug is not considered to be a problem, at least under conditions of normal renal function.^[1,2] Considering this point, MZR-pulse may be relatively safe; 2) Higher doses of MZR increase the area under the serum concentration-time curve (AUC) in patients; thus, the efficacy of MZR may depend on the peak serum level of the drug, which, in turn, may be closely correlated with the AUC of the drug.^[19] Here, however, we do not intend to further discuss the relationship between MZR pharmacokinetics and clinical efficacy in the context of "immunosuppressive" treatment. With regard to these issues, the reader is referred to other general reviews of MZR.^[1,2,19]

 

 

Besides its immunosuppressive effects, MZR has recently been reported to suppress the progression of histologic chronicity in selected patients with pediatric-onset IgA nephropathy and LN.^[6,15,20-22] Previously, MZR has been reported to attenuate tubulointerstitial fibrosis in a dose-dependent manner in rat models of unilateral ureteral obstruction, non-insulin-dependent diabetes and peritoneal fibrosis via suppression of macrophage infiltration of the interstitium.^[23-25] Interestingly, MZR has been reported to bind specifically to heat shock protein (HSP) 60, thus interfering with the chaperone activity of HSP60 in vitro.^[26] This, in turn, may lead to suppression of the activity of α3β1-integrin, which is known to play a role in the development of interstitial fibrosis. Indeed, in a clinical setting, it has also been reported that posttreatment renal biopsy specimen from patients with severe IgA nephropathy treated with MZR showed marked attenuation of glomerular and interstitial lesions, and significantly reduced the number of activated macrophages, associated with expression of 14-3-3 proteins and HSP60, which are known to be MZR-binding proteins, in inflamed glomerular cells (typically seen as mesangial cell proliferation with cellular crescents and interstitial accumulation of leukocytes).^[20] Accordingly, it is speculated that MZR may bind directly to inflamed glomerular cells and prevent progressive damage through suppression of activated macrophages and intrinsic renal cells. Therefore, MZR itself may exert a favorable effect against the progression of interstitial fibrosis in the diseased kidney. These laboratory and clinical observations suggest another beneficial mechanism of action of MZR in the treatment of renal diseases. In a recent clinical study, we confirmed the reported beneficial histologic effects of MZR, noting significant suppression of intraglomerular macrophage infiltration accompanied by significant suppression of chronicity indices following MZR treatment,^[22] but, surprisingly, this was not the case with azathioprine.^[27] Interestingly, MZR treatment also tended to reduce interstitial macrophage infiltration and the expression of osteopontin, known to be a chemoattractant protein for macrophages, corroborating previous experimental observations in rat models.^[23] Since inflamed glomeruli reportedly express 14-3-3 proteins and HSP60,^[20] MZR may directly interact with inflamed glomerular cells, because MZR is directly excreted into the urine.^[28] Another recent experimental study has reported that MZR treatment reduced total glomerular macrophage accumulation with selective reduction of activated macrophages in rats with Thy-1 glomerulonephritis, whereas corticosteroid did not.^[29] Taken together, the data suggest that MZR binds directly to inflamed glomerular cells, subsequently attenuating the expression of osteopontin and reducing the accumulation of activated macrophages,^[22,23] thereby preventing any progressive damage by suppressing activated macrophages and intrinsic renal cells. Therefore, in addition to its immunosuppressive effects, MZR itself may slow the progression of interstitial fibrosis in the diseased kidney.

Since the proposal of a model of possible "pseudoviral" infection to explain the pathogenesis of LN,^[30] we have been examining the effect of polyinosinic-polycytidylic acid (poly IC), a synthetic analogue of viral dsRNA, on toll-like receptor (TLR) 3 signaling cascades involved in "pseudoviral" infection in cultured human mesangial cells (MCs).^[31-34] We found that activation of mesangial TLR3 upregulated the expression of monocyte/macrophage chemoattractants, such as monocyte chemoattractant protein-1 (MCP-1), CC chemokine ligand (CCL) 5 (or regulated on activation, normal T-cell expression and secretion), and fractalkine [or chemokine (C-X3-C motif) ligand 1], in cultured human MCs.^[31-34] Therefore, we examined the direct effects of MZR on the expression of MCP-1, CCL5 and fractalkine in cultured human MCs treated with poly IC.^[35] Pretreatment of cells with MZR partially, but significantly, attenuated the expression of MCP-1 mRNA and protein, whereas the poly IC-induced expression of both CCL5 and fractalkine was not influenced by MZR treatment (Fig.). On the other hand, pretreatment of cells with tacrolimus, a calcineurin inhibitor (CNI) did not suppress the expression of MCP-1 mRNA.^[35] Recently, it has been reported that MZR inhibits any increases in MCP-1 mRNA and protein dose-dependently within the range 1-100 μg/mL in thrombin-treated rat glomerular epithelial cells.^[36] These experimental observations suggest that MZR, besides its immunosuppressive effect, directly inhibits MCP-1 in inflamed glomerular cells of humans as well as rats, although this theory remains preliminary. The urinary concentration of MZR reportedly reaches 400 μg/mL in pediatric patients with glomerular diseases. Even if they do not receive a high-dose of the drug,^[28,37] a concentration of 100 μg/mL in the region of residual glomerular cells is not so irrelevant in a clinical setting.^[35,36] Therefore, it is thought that these experimental studies further support a possible benefit of MZR in the treatment of glomerulonephritis.^{[23-25,29,35,36]}

 

 

For more than 15 years, it has been reported that MZR reduces urinary protein excretion in PAN-induced rat nephrosis models.^[10] However, the precise mechanism responsible remains to be determined. It has recently been reported that MZR directly prevents podocyte injury in rat PAN-induced nephropathy.^[38] This protective effect is independent of IMPD, which is the target of the drug when it acts as an immunosuppressant, whereas MZR reduces PAN-induced integrin-linked kinase activation and phosphorylation of glycogen synthase kinase-3β, thereby preserving nephrin structure in vivo and in vitro, but this is not the case for MMF.^[38] Moreover, it has been reported that MZR, through a mechanism that is likely dependent on inhibition of IMPD activity in podocytes, restores the intracellular energy balance by increasing the levels of adenosine triphosphate and corrects the posttranslational processing of nephrin in cultured podocyte.^[39] Thus, MZR may protect podocytes against loss of actin structure and cell death, leading to preservation of nephrin distribution.^[38] In a clinical setting also, MZR monotherapy has been reported to exert an anti-proteinuric effect, albeit it remains temporarily, in patients with membranous glomerulopathy.^[40] These experimental and clinical observations suggest a direct anti-proteinuric effect of MZR via prevention of podocyte injury, besides its immunosuppressive effects.

 

 

MZR also appears to exert a beneficial protective effect against CNI nephropathy, CNI-induced intimal hyperplasia and perivascular inflammatory cell infiltration in rat models.^[41,42] These studies demonstrated that MZR administration markedly attenuated the lesions of CNI nephropathy, such as macrophage accumulation and interstitial fibrosis.^[42] These findings also indicate that MZR has beneficial effects on several processes, helping to prevent interstitial fibrogenesis due to CNIs-induced nephropathy. Clinically, we have recently treated children with refractory NS associated with CNI nephropathy due to previous aggressive cyclosporine treatment, in whom long-term renal function was preserved and complete remission achieved after MZR treatment.^[43,44] Although the precise nature of the "renoprotective" effect of MZR in a clinical setting remains speculative, these beneficial actions of MZR would warrant its additional use in the treatment of patients with refractory NS, especially those who have undergone long-term CNI treatment.^[44]

 

 

It is speculated that MZR may bind directly to inflamed glomerular cells and prevent progressive damage though suppression of activated macrophages and intrinsic renal cells. MZR also acts to protect podocytes through prevention of nephrin distribution. Therefore, MZR itself may exert a favorable effect against the progression of glomerular lesions. Although further detailed studies remain to be done, we believe that MZR may be an attractive treatment of choice for children with glomerular diseases from a histologic as well as clinical standpoint.

 

 

Funding: Part of this work was supported by grants-in-aid for Science from the Ministry of Education, Culture, Sports, Science and Technology of Japan (TH and IT).

Ethical approval: Our research involving human subjects was approved by the ethics committee of Hirosaki University Graduate School of Medicine.

Competing interest: The authors have no conflicts of interest to declare.

Contributors: TH wrote the first draft of this paper. All authors contributed to the intellectual contentand approved the final version.

 

 

1   Yokota S. Mizoribine: mode of action and effects in clinical use. Pediatr Int 2002;44:196-198.

2   Kawasaki Y. Mizoribine: a new approach in the treatment of renal disease. Clin Dev Immunol 2009;2009:681482.

3   Sonda K, Takahashi K, Tanabe K, Funchinoue S, Hayasaka Y, Kawaguchi H, et al. Clinical pharmacokinetic study of mizoribine in renal transplantation patients. Transplant Proc 1996;28:3643-3648.

4   Yoshioka K, Ohashi Y, Sakai T, Ito H, Yoshikawa N, Nakamura H, et al. A multicenter trial of mizoribine compared with placebo in children with frequently relapsing nephrotic syndrome. Kidney Int 2000;58:317-324.

5   Honda M. Nephrotic syndrome and mizoribine in children. Pediatr Int 2002;44:210-216.

6   Kawasaki Y, Hosoya M, Suzuki J, Onishi N, Takahashi A, Isome M, et al. Efficacy of multidrug therapy combined with mizoribine in children with diffuse IgA nephropathy in comparison with multidrug therapy without mizoribine and with methylprednisolone pulse therapy. Am J Nephrol 2004;24:576-581.

7   Yoshikawa N, Nakanishi K, Ishikura K, Hataya H, Iijima K, Honda M. Combination therapy with mizoribine for severe childhood IgA nephropathy: a pilot study. Pediatr Nephrol 2008;23:757-763.

8   Tanaka H, Tsugawa K, Tsuruga K, Suzuki K, Nakahata T, Ito E, et al. Mizoribine for the treatment of lupus nephritis in children and adolescents. Clin Nephrol 2004;62:412-417.

9   Yumura W, Suganuma S, Uchida K, Moriyama T, Otsubo S, Takei T, et al. Effects of long-term treatment with mizoribine in patients with proliferative lupus nephritis. Clin Nephrol 2005;64:28-34.

10 Shibasaki T, Matsuda H, Gomi H, Usui M, Ishimoto F, Sakai O. Mizoribine reduces urinary protein excretion in rats given puromycin aminonucleoside. Am J Nephrol 1996;16:167-172.

11 Takahashi S, Wakui H, Gustafsson JA, Zilliacus J, Itoh H. Functional interaction of the immunosuppressant mizoribine with the 14-3-3 protein. Biochem Biophys Res Commun 2000;274:87-92.

12 Tanaka H, Suzuki K, Nakahata T, Tsugawa K, Ito E, Waga S. Mizoribine oral pulse therapy for patients with disease flare of lupus nephritis. Clin Nephrol 2003;60:390-394.

13 Tanaka H, Tsugawa K, Suzuki K, Nakahata T, Ito E. Long-term mizoribine intermittent pulse therapy for young patients with flare of lupus nephritis. Pediatr Nephrol 2006;21:962-966.

14 Kuroda T, Hirose S, Tanabe N, Sato H, Nakatsue T, Ajiro J, et al. Mizoribine therapy for patients with lupus nephritis: the association between peak mizoribine concentration and clinical efficacy. Mod Rheumatol 2007;17:206-212.

15 Tanaka H, Oki E, Tsuruga K, Sato N, Matsukura H, Matsunaga A, et al. Mizoribine treatment of young patients with severe lupus nephritis: a clinicopathologic study by the tohoku pediatric study group. Nephron Clin Pract 2008;110:c73-c79.

16 Ohtomo Y, Fujinaga S, Takada M, Murakami H, Akashi S, Shimizu T, et al. High-dose mizoribine therapy for childhood-onset frequently relapsing steroid-dependent nephrotic syndrome with cyclosporin nephrotoxicity. Pediatr Nephrol 2005;20:1744-1749.

17 Kawasaki Y, Takano K, Isome M, Suzuki J, Suyama K, Kanno H, et al. Efficacy of single dose of oral mizoribine pulse therapy two times per week for frequently relapsing nephrotic syndrome. J Nephrol 2007;20:52-56.

18 Doi T, Masaki T, Shiraki N, Kawai T, Yorioka N. Oral mizoribine pulse therapy for steroid-dependent focal segmental glomerulosclerosis. Clin Nephrol 2008;69:433-435.

19 Fuke T, Abe Y, Hibino S, Takeshi M, Saito T, Sakurai S, et al. Mizoribine requires individual dosing due to variation of bioavailability. Pediatr Int 2012;54:885-891.

20 Ikezumi Y, Suzuki T, Karasawa T, Kawachi H, Nikolic-Paterson DJ, Uchiyama M. Use of mizoribine as a rescue drug for steroid-resistant pediatric IgA nephropathy. Pediatr Nephrol 2008;23:645-650.

21 Tanaka H, Oki ES, Tsugawa K, Suzuki K, Tsuruga K, Ito E. Long-term intermittent pulse therapy with mizoribine attenuates histologic progression in young patients with severe lupus nephritis: report of two patients. Nephrology (Carlton) 2007;12:376-379.

22 Tanaka H, Oki E, Tsuruga K, Aizawa-Yashiro T, Ito Y, Sato N, et al. Mizoribine attenuates renal injury and macrophage infiltration in patients with severe lupus nephritis. Clin Rheumatol 2010;29:1049-1054.

23 Sato N, Shiraiwa K, Kai K, Watanabe A, Ogawa S, Kobayashi Y, et al. Mizoribine ameliorates the tubulointerstitial fibrosis of obstructive nephropathy. Nephron 2001;89:177-185.

24 Kikuchi Y, Imakiire T, Yamada M, Saigusa T, Hyodo T, Hyodo N, et al. Mizoribine reduces renal injury and macrophage infiltration in non-insulin-dependent diabetic rats. Nephrol Dial Transplant 2005;20:1573-1581.

25 Takahashi S, Taniguchi Y, Nakashima A, Arakawa T, Kawai T, Doi S, et al. Mizoribine suppresses the progression of experimental peritoneal fibrosis in a rat model. Nephron Exp Nephrol 2009;112:e59-e69.

26 Itoh H, Komatsuda A, Wakui H, Miura AB, Tashima Y. Mammalian HSP60 is a major target for an immunosuppressant mizoribine. J Biol Chem 1999;274:35147-35151.

27 Tanaka H, Tsugawa K, Oki E, Suzuki K, Waga S, Ito E. Long-term mizoribine intermittent pulse therapy, but not azathioprine therapy, attenuated histologic progression in a patient with severe lupus nephritis. Clin Nephrol 2007;68:198-200.

28 Stypinski D, Obaidi M, Combs M, Weber M, Stewart AJ, Ishikawa H. Safety, tolerability and pharmacokinetics of higher-dose mizoribine in healthy male volunteers. Br J Clin Pharmacol 2006;63:459-468.

29 Ikezumi Y, Suzuki T, Karasawa T, Hasegawa H, Kawachi H, Nikolic-Paterson DJ, et al. Contrasting effects of steroids and mizoribine on macrophage activation and glomerular lesions in rat thy-1 mesangial proliferative glomerulonephritis. Am J Nephrol 2010;31:273-282.

30 Anders HJ. Pseudoviral immunity-a novel concept for lupus. Trends Mol Med 2009;15:553-561.

31 Imaizumi T, Tanaka H, Matsumiya T, Yoshida H, Tanji K, Tsuruga K, et al. Retinoic acid-inducible gene-I is induced by double-stranded RNA and regulates the expression of CC chemokine ligand (CCL) 5 in human mesangial cells. Nephrol Dial Transplant 2010;25:3534-3539.

32 Imaizumi T, Aizawa-Yashiro T, Tsuruga K, Tanaka H, Matsumiya T, Yoshida H, et al. Melanoma differentiation-associated gene 5 regulates the expression of a chemokine CXCL10 in human mesangial cells: implications for chronic inflammatory renal diseases. Tohoku J Exp Med 2012;228:17-26.

33 Aizawa-Yashiro T, Imaizumi T, Tsuruga K, Watanabe S, Matsumiya T, Hayakari R, et al. Glomerular expression of fractalkine is induced by polyinosinic-polycytidylic acid in human mesangial cells: possible involvement of fractalkine after viral infection. Pediatr Res 2013;73:180-186.

34 Tanaka H, Imaizumi T. Inflammatory chemokine expression via Toll-like receptor 3 signaling in normal human mesangial cells. Clin Dev Immunol 2013;2013:984708.

35 Aizawa T, Imaizumi T, Tsuruga K, Watanabe S, Chiba Y, Matsumiya T, et al. Mizoribine selectively attenuates monocyte chemoattractant protein-1 production in cultured human glomerular mesangial cell: a possible benefit of its use in the treatment of lupus nephritis. Nephrology (Carlton) 2014;19:47-52.

36 Yamabe H, Shimada M, Murakami R, Fujita T, Shimaya Y, Nakamura N. Mizoribine suppresses proliferation of rat glomerular epithelial cells in culture and inhibits increase of monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 stimulated by thrombin. Biol Pharm Bull 2012;35:705-708.

37 Uemura O, Ushijima K, Yamada T, Kimpara Y. Blood concentration and urinary excretion of mizoribine for internal use in pediatric kidney disease patients. Jpn J Pediatr Nephrol 2007;20:9-13. [In Japanese]

38 Takeuchi S, Hiromura K, Tomioka M, Takahashi S, Sakairi T, Maeshima A, et al. The immunosuppressive drug mizoribine directly prevents podocyte injury in puromycin aminonucleoside nephrosis. Nephron Exp Nephrol 2010;116:e3-e10.

39 Nakajo A, Khoshnoodi J, Takenaka H, Hagiwara E, Watanabe T, Kawakami H, et al. Mizoribine corrects defective nephrin biogenesis by restoring intracellular energy balance. J Am Soc Nephrol 2007;18:2554-2564.

40 Fujita T, Shimizu C, Ito K, Fuke Y, Satomura A, Matsumoto K. Anti-proteinuric effect of mizoribine monotherapy in three patients with membranous nephropathy. Clin Nephrol 2010;74:81-82.

41 Shimizu H, Takahashi M, Takeda S, Tahara K, Inoue S, Hakamata Y, et al. Effect of conversion from cyclosporine A to mizoribine on transplant arteriosclerosis in rat aortic allograft models. Microsurgery 2003;23:454-457.

42 Hara S, Umino D, Someya T, Fujinaga S, Ohtomo Y, Murakami H, et al. Protective effects of Mizoribine on Cyclosporine A nephropathy in rats. Pediatr Res 2009;66:524-527.

43 Aizawa-Yashiro T, Tsuruga K, Watanabe S, Oki E, Ito E, Tanaka H. Novel multidrug therapy for children with cyclosporine-resistant or -intolerant nephrotic syndrome. Pediatr Nephrol 2011;26:1255-1261.

44 Hirose C, Aizawa T, Watanabe S, Tsuruga K, Ito E, Tanaka H. Efficacy of long-term multidrug therapy in a patient with focal segmental glomerulosclerosis. Pediatr Int 2014;56:129-130.