Pharmaceutical Effect of Manganese Porphyrins on Manganese

Aug 21, 2012 - ABSTRACT: Mice lacking manganese-superoxide dismu- tase (Mn-SOD) activity exhibit typical pathology of dilated cardiomyopathy (DCM)...
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Pharmaceutical Effect of Manganese Porphyrins on Manganese Superoxide Dismutase Deficient Mice Natsumi Hayakawa,† Shoichiro Asayama,† Yoshihiro Noda,‡ Takahiko Shimizu,‡,§ and Hiroyoshi Kawakami*,† †

Department of Applied Chemistry, Tokyo Metropolitan University, Minami-Osawa, Hachioji, Tokyo 192-0397, Japan Research Team for Molecular Biomarkers, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Itabashi-ku, Tokyo 173-0015, Japan

‡

S Supporting Information *

ABSTRACT: Mice lacking manganese-superoxide dismutase (Mn-SOD) activity exhibit typical pathology of dilated cardiomyopathy (DCM). In the present study, the structure−activity relationship between the water-soluble manganese (Mn) porphyrin with SOD activity and the in vivo pharmaceutical effect on DCM is reported. The MnSOD-deficient mice were treated with Mn-porphyrins for 3 weeks. The treatment of a Mn-porphyrin, MnM2Py2P, suppressed the progression of cardiac dilation. These results suggest that the Mn-porphyrin MnM2Py2P treatment is proposed as a potential therapy for DCM. KEYWORDS: water-soluble manganese porphyrin, superoxide dismutase mimic, manganese superoxide dismutase deficient mice, dilated cardiomyopathy, antioxidative therapy

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ere we have reported the in vivo therapeutic effect of manganese (Mn) porphyrin antioxidants for Mn superoxide dismutase (Mn-SOD) deficient mice. The mice lacking Mn-SOD activity exhibit the typical pathology of dilated cardiomyopathy (DCM). The Mn-SOD (in mitochondria) catalyzing the reduction from superoxide radical anion (O2•−) to hydrogen peroxide (H2O2) is the key enzyme for the protection of oxidative stress.1 We have already reported that water-soluble Mn-porphyrins with SOD activity work as antioxidants in various biochemical fields.2−7 In the present study, Mn-SOD-deficient mice8 were treated with the SOD mimic Mn-porphyrins. If the dilated heart weight treated with the Mn-porphyrin antioxidants decreases, the radical approach to therapy would be promising. This paper describes the oxidative stress level, heart weight, and left ventricle cavity area after in vivo administration of Mn-porphyrins for DCM; detail studies on the mechanism of antioxidative activity for DCM are outside the scope of the present study. To administer SOD mimic Mn-porphyrins to Mn-SODdeficient mice, we have prepared three kinds of Mn-porphyrins, as shown in Figure 1. The structure−activity relationship of 5,15-bis(2-methylpyridyl)porphinatomanganese (MnM2Py2P),2 5-(1,3-dimethylimidazolium-2-yl)-10,15,20-triphenylporphinatomanganese (MnMImP3P),3 and 5,10,15,20tetrakis(4-methylpyridyl)porphinatomanganese (MnM4Py4P)9 as antioxidant for DCM has been demonstrated. To examine © 2012 American Chemical Society

Figure 1. Chemical structure of SOD mimic Mn-porphyrins: (A) MnM2Py2P, (B) MnMImP3P, (C) MnM4Py4P.

the antioxidative activity of these Mn-porphyrins, we first collected blood samples from the Mn-SOD-deficient mice as well as control subjects and analyzed the samples after their collection. A commercially available method (d-ROMs test10,11) was used for the assessment of the levels of oxidative stress in the blood. This is a spectrophotometric method that assesses overall oxidative stress by measuring total hydroperoxides, given that hydroperoxides are intermediate oxidative products of lipids, peptides, and amino acids. [A typical Received: Revised: Accepted: Published: 2956

March 14, 2012 June 18, 2012 August 21, 2012 August 21, 2012 dx.doi.org/10.1021/mp300147v | Mol. Pharmaceutics 2012, 9, 2956−2959

Molecular Pharmaceutics

Communication

actually also good substrates for ceruloplasm in the blood/ plasma, however, the ceruloplasm is an interferent in this assay. This may be why Figure 2 fails to show any true significance between the MnM2Py2P treatment and control. In spite of the assay's shortcoming, as compared among the Mn-porphyrins in this study, it can be said that the MnM2Py2P and MnM4Py4P work as antioxidants in the blood, but not the MnMImP3P. Accordingly, we evaluated the weight ratio of heart to body after the administration of SOD mimic Mn-porphyrins, as shown in Figure 3. [A typical procedure is as follows: After the

procedure is as follows: Each Mn-porphyrin suspended in saline at 2.0 mmol/L was injected intraperitoneally (20 mL/kg body weight) into the presymptomatic heart/muscle-specific MnSOD-deficient mice (H/M-Sod2−/−) once daily. The preventive administration began from 5 weeks of age and continued for 3 weeks. After the administration, the blood was collected and frozen at −80 °C until the d-ROMs test. The resulting blood was centrifuged at 3000 rpm for 15 min at 22 °C. The resulting supernatant was further centrifuged at 3000 rpm for 5 min at 22 °C, and 10 μL of the serum was diluted in acetate buffered solution (pH 4.8, R1 reagent). Hydroperoxide groups react with the transition metal ions liberated from the proteins in the acidic medium and are converted to alkoxyl and peroxyl radicals according to Fenton’s reaction. These newly formed radicals, the quantities of which are directly proportional to peroxides present in the serum, are trapped chemically with chromogen (N,N-diethyl-p-phenylenediamine, R2 reagent) leading to the formation of the radical cation of this chromogen. The purple color resulting from this reaction over time was monitored in a spectrophotometer at 505 nm. The results of this method are expressed in conventional units (Carratelli units, UCarr); 1 UCarr corresponds to 0.8 mg/L H2O2.] Figure 2 shows the

Figure 3. Effect of Mn-porphyrin administration on the normalized heart weight of the Mn-SOD-deficient mice (H/M-Sod2−/−) exhibiting the typical pathology of DCM. Representative images of hearts after the treatment period are indicated as upper panel. The heart weight per body weight (BW) of saline-treated wild-type (controls: n = 5), saline-treated H/M-Sod2−/− mice (+ saline: n = 4), MnM2Py2Ptreated H/M-Sod2−/− mice (+ MnM2Py2P: n = 4), MnMImP3Ptreated H/M-Sod2−/− mice (+ MnMImP3P: n = 4), and MnM4Py4Ptreated H/M-Sod2−/− mice (+ MnM4Py4P: n = 5) after the treatment period is indicated as vertical axis of the lower panel. Symbols and error bars represent the mean and standard deviation, n = 4−5 per group. *p < 0.001, as compared to saline-treated wild-type; **p < 0.005, ***p < 0.05, as compared to saline-treated H/M-Sod2−/− mice; Student’s t-test.

Figure 2. Effect of Mn-porphyrin administration on the oxidative stress level in the Mn-SOD-deficient mice (H/M-Sod2−/−) exhibiting the typical pathology of DCM. Oxidative stress of saline-treated wildtype (controls: n = 5), saline-treated H/M-Sod2−/− mice (+ saline: n = 5), MnM2Py2P-treated H/M-Sod2−/− mice (+ MnM2Py2P: n = 4), MnMImP3P-treated H/M-Sod2−/− mice (+ MnMImP3P: n = 4), and MnM4Py4P-treated H/M-Sod2−/− mice (+ MnM4Py4P: n = 5) after the treatment period is indicated as vertical axis. Symbols and error bars represent the mean and standard deviation, n = 4−5 per group. *p < 0.005, as compared to saline-treated wild-type; **p < 0.1, as compared to saline-treated H/M-Sod2−/− mice; Student’s t-test.

above preventive administration, isolated hearts were immersed in 10% buffered formalin. The resulting fixed heart tissues were imaged with a VHX-100 digital microscope (KEYENCE, Osaka, Japan).] The heart weight (HW) per body weight (BW) in the control group was 0.46 ± 0.03, whereas a significant (p < 0.001) increase in the HW/BW ratio was observed (1.13 ± 0.15) in the DCM group. The MnMImP3P treatment exhibited no significant difference in the HW/BW ratio (1.11 ± 0.07), as compared with the DCM groups. However, interestingly, the MnM2Py2P treatment (0.75 ± 0.23; p < 0.005), as well as the MnM4Py4P (0.86 ± 0.26; p < 0.05), significantly prevented macroscopic cardiac enlargement and elevation of the HW/BW ratio in the Mn-SOD-deficient mice. These results suggest that the SOD mimic MnM2Py2P is more effective than the MnM4Py4P for the DCM therapy. To examine further the in vivo therapeutic effect of the MnM2Py2P, we measured the left ventricle cavity area in the dilated heart. [A typical procedure is as follows: The above fixed heart tissues were dehydrated, embedded in paraffin, sectioned into 4 μm slices, and stained with hematoxylin−eosin (H&E). Images were obtained using a Pixera Pro600EX camera

oxidative stress level in the Mn-SOD-deficient mice exhibiting the typical pathology of DCM. The serum level of oxidative stress in the control group was 117 ± 12 UCarr. In the DCM group, a significant (p < 0.005) increase in serum level of oxidative stress was noted (179 ± 32 UCarr). When the MnMImP3P was administered in the DCM group, no significant difference in oxidative stress was observed (189 ± 58 UCarr). It should be noted that the MnM2Py2P (139 ± 48 UCarr) as well as the MnM4Py4P (148 ± 34 UCarr) significantly (p < 0.1) decreased the serum level of oxidative stress. Because the substrates used in this d-ROMs test are 2957

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(Figure S-2, Supporting Information). Accordingly, the MnM2Py2P is considered to be quickly excreted from heart or to be metabolized by heme oxygenase because the MnM2Py2P has two meso carbons with no substituent.12 Furthermore, we have already reported that the MnMImP3P is the highly amphiphilic Mn-porphyrin for the mitochondrial targeting SOD mimic in vitro,3 so that we expect the MnMImP3P to exhibit a therapeutic effect for the mice lacking Mn-SOD in mitochondria at first. Although the MnMImP3P was not excreted in urine, however, the amount of the accumulated MnMImP3P in heart was between the MnM2Py2P and the MnM4Py4P level (results not shown). In the presence of in vivo serum albumin, which was bound to the Mnporphyrin with phenyl groups,13 the MnMImP3P with three phenyl groups is therefore considered to be bound to albumin, resulting in the enhancement of its blood circulation. It is difficult for the resulting MnMImP3P bound to albumin to be excreted in urine and to be accumulated in heart. In conclusion, we have communicated the structure−activity relationship between the SOD mimic Mn-porphyrin and the in vivo pharmaceutical effect on DCM. Especially, the treatment of a Mn-porphyrin, MnM2Py2P, suppressed the progression of cardiac dilation. The SOD mimic Mn-porphyrin MnM2Py2P is expected to be a promising drug to cure DCM in vivo.

attached to a VANOX-S microscope (Olympus, Tokyo, Japan). The left ventricle cavity area was quantitatively analyzed with Qwin Plus V3 (Leica Co. Ltd.).] As shown in Figure 4, as well

Figure 4. Effect of Mn-porphyrin administration on the left ventricle cavity area of the Mn-SOD-deficient mice (H/M-Sod2−/−) exhibiting the typical pathology of DCM. Representative images of H&E stained left ventricular myocardium after the treatment period are indicated: saline-treated wild-type (controls); saline-treated H/M-Sod2−/− mice (+ saline); MnM2Py2P-treated H/M-Sod2−/− mice (+ MnM2Py2P); MnMImP 3 P-treated H/M-Sod2 −/ − mice (+ MnMImP 3 P); MnM4Py4P-treated H/M-Sod2−/− mice (+ MnM4Py4P).

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Table 1. Effect of Mn-Porphyrin Administration on the Left Ventricle Cavity Area of the Mn-SOD-Deficient Mice (H/MSod2−/−) Exhibiting the Typical Pathology of DCMa sample controls DCM + DCM + DCM + DCM +

saline MnM2Py2P MnMImP3P MnMPy4P

left ventricle cavity area [mm2] 1.54 7.80 1.96 5.67 4.22

± ± ± ± ±

ASSOCIATED CONTENT

S Supporting Information *

Figures S-1 and S-2 depicting the Mn-porphyrin accumulation in the heart and the Mn-porphyrin excretion in the urine, respectively, of the Mn-SOD-deficient mice (H/M-Sod2−/−) exhibiting the typical pathology of DCM. This material is available free of charge via the Internet at http://pubs.acs.org.

0.004 0.038 0.055 0.025 0.074

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a

The left ventricle cavity area of saline-treated wild-type (controls), saline-treated H/M-Sod2−/− mice (DCM + saline), MnM2Py2Ptreated H/M-Sod2−/− mice (DCM + MnM2Py2P), MnMImP3Ptreated H/M-Sod2−/− mice (DCM + MnMImP3P), and MnM4Py4Ptreated H/M-Sod2−/− mice (DCM + MnM4Py4P) after the treatment period is indicated. The mean and standard deviation of the measurements (n = 2) are indicated.

AUTHOR INFORMATION

Corresponding Author

*Tokyo Metropolitan University, Department of Applied Chemistry, 1-1 Minami-Osawa, Hachioji, Tokyo 192-0397, Japan. Tel: +81-42-677-1111 ext 4972. Fax: +81-42-677-2821. E-mail: [email protected]. Present Address §

Department of Advanced Aging Medicine, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.

as Table 1, the histological analyses of mutant hearts indicated dramatic left ventricle dilation. The left ventricle cavity area in the DCM group (7.80 ± 0.038 mm2) was larger than that in the control group (1.54 ± 0.004 mm2). Although not only the MnM4Py4P (4.22 ± 0.074 mm2) but also the MnMImP3P (5.67 ± 0.025 mm2) adequately decreased the left ventricle dilation, it is worth noting that the MnM2Py2P almost completely suppressed the dilation (1.96 ± 0.055 mm2) to the control level. These results suggest that the MnM2Py2P works as most effective antioxidant for the Mn-SOD-deficient mice exhibiting the typical pathology of DCM in this study. Although the SOD activity of the MnM2Py2P is less than that of the MnM4Py4P, in our previous study,2 the in vitro cellular uptake of the MnM2Py2P is higher than that of the MnM4Py4P, resulting in the rescue of lipopolysaccharide (LPS)-stimulated macrophage RAW 264.7 cells by the MnM2Py2P. Therefore, the cellular uptake of the MnM2Py2P is important for the complement of its lesser SOD activity to exhibit antioxidative activity. However, the in vivo accumulation of the MnM2Py2P in the heart is less than that of the MnM4Py4P (Figure S-1, Supporting Information), though both the MnM2Py2P and the MnM4Py4P were excreted in urine

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially supported by a Grant-in-Aid (No. 22300166) from Japan Society for the Promotion of Science. REFERENCES

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