Journal of Perinatology (2011) 31, S35–S41 r 2011 Nature America, Inc. All rights reserved. 0743-8346/11 www.nature.com/jp ORIGINAL ARTICLE In vitro inhibition of isoenzymes by metalloporphyrins

RJ Wong, HJ Vreman, S Schulz, FS Kalish, NW Pierce and DK Stevenson Department of Pediatrics, Stanford University School of Medicine, Stanford, CA, USA

Introduction Objective: Neonatal jaundice results from an increased bilirubin Neonatal jaundice results from an imbalance between bilirubin production and decreased hepatic bilirubin conjugation and excretion. production and bilirubin elimination, the latter reflecting mainly Severe hyperbilirubinemia is currently treated with phototherapy or a transient impairment of conjugation in the newborn after birth. exchange transfusion; however, its prevention by inhibiting bilirubin It is exacerbated in a number of clinical conditions such as formation is a more logical strategy. (HO), with inducible prematurity,1 but the most severe hyperbilirubinemia is usually (HO-1) and constitutive (HO-2) isoenzymes, is the rate-limiting in secondary to hemolytic conditions such as ABO or Rh heme catabolism, producing equimolar amounts of bilirubin and carbon incompatibilities and glucose-6-phosphate dehydrogenase monoxide (CO). Metalloporphyrins (Mps) are heme derivatives that deficiency.2–4 Hemolysis is more often associated with brain injury competitively inhibit HO and thereby suppress hyperbilirubinemia. No (kernicterus).5 Currently, excessive hyperbilirubinemia is being systematic studies have been reported evaluating whether the HO treated with phototherapy6,7 and/or exchange transfusion in isoenzymes are inhibited differentially by various Mps. Identification of most serious and acute cases.8,9 These procedures, however, only Mps that selectively inhibit the inducible HO-1 without affecting the eliminate bilirubin after it has been produced and accumulated ‘housekeeping’ HO-2 isoenzyme might be desirable in the clinical setting of in circulation to a level that could portend even higher, more hemolytic disease, in which the Hmox1 is greatly induced. Although dangerous, levels. A rational and perhaps more desirable approach bilirubin production is due to the activity of both HO-1 and HO-2, the would be to prevent the formation of excessive bilirubin. inhibition of HO-1 with a relative sparing of HO-2 activity might provide the Heme oxygenase (HO) is the rate-limiting enzyme in the most selective approach for the treatment of hemolytic disease. catabolism of heme to bilirubin.10 HO enzyme activity is primarily Study Design: We determined for the deutero-, proto-, meso- and bis- due to the action of two isoenzymes, HO-1 and HO-2, both of which glycol porphyrins with zinc, tin and chromium as central atoms, are isolated and purified.11 HO-1 has a molecular weight of 32 kDa, respectively, the concentration needed for 50% inhibition (I50) of HO-1 is a gene product of a single mRNA and can be induced by and HO-2 activities in rat spleen and brain tissue. a variety of stimuli such as heme itself, heat shock, heavy Result: For a given Mp, HO-1 activity was less inhibited than that of HO-2. metals, glutathione depletion, radiation, ischemia, hypoxia and 12,13 The order of inhibitor potency of each Mp was nearly identical for both hyperoxia. It is the predominant form in the spleen, the isoenzymes. Tin mesoporphyrin was the most potent inhibitor for both primary organ responsible for the sequestration and degradation of isoenzymes. HO-2 selectivity was greatest for tin protoporphyrin. Conversely, the senescent erythrocytes. Because of this role, HO activity in the Zn compounds were least inhibitory toward HO-2. No Mp preferentially spleen is higher than in any other tissue and the HO-1 isoenzyme 14 inhibited HO-1. is believed to be greatly upregulated at all times. In conditions in which the splenic HO system is saturated (such as in hemolysis), Conclusion: Mps that produce a less inhibitory effect on HO-2, while the excessive heme load is degraded to a greater extent by the limiting the response of the inducible HO-1, such as ZnPP, may be hepatic HO system. In contrast, HO-2, which is considered to be the a useful clinical tool. constitutive form, has a molecular weight of 36 kDa, is the product Journal of Perinatology (2011) 31, S35–S41; doi:10.1038/jp.2010.173 of two or more transcripts, is not induced by the above stress Keywords: bilirubin; carbon monoxide; hyperbilirubinemia; isozyme; conditions (except by glucocorticoids) and is the predominant form jaundice found in the brain.12,13,15 A third isoenzyme, HO-3, has been identified, but its contribution to heme degradation is minimal because of its low level of activity.16 Nevertheless, it is the combined Correspondence: Dr RJ Wong, Department of Pediatrics, Stanford University School of Medicine, 300 Pasteur Drive, Room S230, Stanford, CA 94305-5208, USA. activity of all HO isoenzymes that contributes to bodily heme E-mail: [email protected] catabolism and is measured as HO activity in vitro. Metalloporphyrin selectivity RJ Wong et al S36

CH3 CH3

HOOCCH2CH2 R NN

Metal α

NN

HOOCCH2CH2 CH3

CH3 R

Porphyrin Type Based on Ring Substituent

Deuteroporphyrin Mesoporphyrin Protoporphyrin Bis Glycol Porphyrin Metal (R = -H) (R = -CH2-CH3) (R = -CH=CH2) (R = -CHOH-CH2OH) Iron (Fe2+) FeDP FeMP FePP (Hemin) FeBG Zinc (Zn2+) ZnDP ZnMP ZnPP ZnBG Tin (Sn4+) SnDP SnMP SnPP SnBG Chromium (Cr2+) CrDP CrMP CrPP CrBG Figure 1 (Metallo)porphyrin chemical structure, types and abbreviations. (Modified from Vreman et al.,37 with permission).

The endoplasmic reticulum-bound HO cleaves and mediates the isoenzyme, which may have a role in the production of CO necessary oxidation of the a-methene bridge of the heme molecule, yielding for physiological functioning,25 and of bilirubin for the maintenance of equimolar amounts of iron, carbon monoxide (CO) and biliverdin. tissue antioxidant levels.26,27 Alternatively, the selective inhibition of Bilverdin is sub-sequently reduced to yield bilirubin.10 This HO-2 could allow the study of the function of HO-2, as well as the stoichiometry, the insolubility of CO in water and the absence physiological roles for CO and bilirubin. of other significant CO-generating reactions allow the use of Therefore, we determined the I50 concentrations of 12 Mps and measurements of CO to serve as indices of bilirubin formation. Because four Mf porphyrins in the in vitro enzyme activity of HO-1, the HO is the rate-limiting enzyme in CO and bilirubin production, its predominant isoenzyme in rat spleen, and of HO-2, the predominant inhibition results in decreased CO and bilirubin production rates. In isoenzyme in rat brain. Data from studies conducted on mice are the context of inhibition of heme catabolism, non-metabolized heme is also provided for comparison of the four most attractive Mps. excreted with the bile into the intestine.17 Natural and synthetic derivatives of heme (iron protoporphyrin) or metalloporphyrins (Mps) Methods have been found to inhibit competitively in vitro and in vivo HO Animals 18 activity. In vivo administration of Mps results in the suppression of The study was approved by the Stanford University Department of 19 20 plasma bilirubin, total body CO excretion and biliary heme Comparative Medicine. Animal use and care were in accordance 17,21 excretion. As a result, these compounds have potential use for the with the guidelines established by Stanford University’s prevention of neonatal jaundice. Institutional Animal Care and Use Committee. Adult male Wistar A number of these compounds have been studied in vitro and rats (Simonsen Laboratories, Gilroy, CA, USA) weighing 253–371 g 18,20,22–24 in vivo for efficacy, safety and route of administration. These (316±33, n ¼ 12) and adult FVB mice (Jackson Laboratory, Bar include the metal-free (Mf) and metal-containing porphyrins with ring Harbor, ME, USA) weighing 20–25 g (23±1, n ¼ 9) were used as modifications to yield deutero-, meso-, proto- and bis-glycol porphyrins sources of tissue. Food and water were available ad libitum. All (Figure 1). However, no comprehensive or systematic study has yet animals were killed by decapitation. Spleen and brain were been reported that examines whether the two major HO isoenzymes immediately harvested and processed. are differentially sensitive to inhibition by Mps. A strategic approach to the treatment of neonatal jaundice would be to selectively inhibit HO-1 Tissue preparation activity induced by hemolysis in order to prevent excessive bilirubin Spleen (serving as the source of HO-1 isoenzyme) and brain formation. However, it would be desirable, if not important, to (serving as the source of HO-2 isoenzyme) were diced in nine maintain differentially the activity of the ‘housekeeping’ HO-2 (10% w/w) and four (20% w/w) volumes of iced buffer,

Journal of Perinatology Metalloporphyrin selectivity RJ Wong et al S37 respectively, homogenized with an iced Biohomogenizer 140 1C. Analyzer response to CO, which had a retention time of (Biospec Products, Bartelsville, OK, USA) and then centrifuged for B30 s, was recorded with an integrating recorder (CR-3A, Shimadzu 60 s at 13 000  g. The supernatant was decanted and analyzed Scientific Instruments, Columbia, MD, USA) by measuring peak area. for HO activity. The gas chromatography was standardized daily with 0–250 ml volumes of 10.8 mlofCOlÀ1 air (482 nmol CO lÀ1). Reagents Buffer was prepared by dissolving 0.1 mole of KH2PO4KH2Oinwater, Calculations titrating the pH to 7.4 with KOH, and adjusting the volume to 1 l. HO activity is defined as the difference in CO production in the All Mps and Mf porphyrins (Frontier Scientific, Logan, UT, USA) total reaction vial (in the presence of nicotinamide adenine were dissolved with ethanolamine and carefully neutralized with dinucleotide phosphate) minus that of the blank reaction vial. 1-N HCl to a pH of 7.4 under vortex mixing. The concentration was HO activity is expressed as mean±s.d. pmol of CO produced per 18 adjusted to 1.2 mM. Concentrations were then varied through hour per mg fresh weight. Inhibition is defined as the suppression appropriate dilutions with buffer. All experiments were conducted of HO activity due to the presence of Mp or Mf porphyrin. under subdued light. Percentage of HO activity is defined as the amount of CO formed at Stock methemalbumin (1.5 mM heme with 0.15 mM bovine serum each porphyrin concentration divided by the amount of CO formed by albumin) solutions were prepared weekly.28,29 The working the control vial, that is, with no porphyrin added, times 100. methemalbumin or HO substrate was prepared daily by diluting one CO produced at each ½mMŠ volume of the stock heme solution with nine volumes of buffer. % HO Activity ¼ Â100 CO produced by control Reduced nicotinamide adenine dinucleotide phosphate (Sigma/Aldrich, St Louis, MO, USA) was dissolved in buffer to 4.5 mM and prepared fresh daily. The concentration of each porphyrin needed to inhibit CO production A volume of 6 g of sulfosalicylic acid (Sigma/Aldrich) was by 50% (the I50 value) was determined by interpolation. dissolved in distilled water and the volume adjusted to 10 ml to The HO-1 Selectivity Index (SI) is defined as the ratio of the I50 yield a 60% (w/v) solution. value of each porphyrin for HO-2 relative to the I50 value of All solutions were stored at 4 1C in the dark. each porphyrin for HO-1, where an HO-1 SI >1.0 denotes the selectivity of an Mp for HO-1 and an HO-1 SI p1.0 denotes HO activity equal or greater selectivity for HO-2. 28,29 HO activity was determined essentially as described previously. I50 of Mp for HO-2 A volume of 20 ml of tissue supernatant, representing 4- (for brain) HO-1 Selectivity Index ¼ I50 of Mp for HO-1 or 2-mg (for spleen) fresh weight of tissue, was mixed in 2-ml amber glass vials containing 20 ml of 150 mM methemalbumin and All data are presented as means±s.d. 20 ml of 4.5 mM nicotinamide adenine dinucleotide phosphate for the total reaction vials (control). For the blank reaction vials, the nicotinamide adenine dinucleotide phosphate was replaced with an Results equal volume of buffer. The vials were sealed with septum caps For this study, the proto-, deutero-, meso- and bis-glycol porphyrins and placed in a 37 1C water bath for 5 min of temperature with zinc, tin and chromium as the central metal atom, and equilibration. The vials were then purged with CO-free air and the porphyrins without metal, a total of 16 compounds were evaluated incubation was continued. After exactly 15.0 min post purging, the at concentrations of 0–48 mM. Each concentration was tested reactions were terminated by the addition of 2 ml 60% (w/v) with tissue preparations from three different rats. sulfosalicylic acid.30 To determine the concentration needed to Figure 2 presents examples of typical dose–response curves for tin inhibit HO activity by 50% (I50) for each Mp and Mf porphyrin, protoporphyrin (SnPP), comparing inhibition of HO-1 (spleen) and varying concentrations of each inhibitor were added to additional HO-2 (brain). Mean HO activity levels for control spleen and brain total reaction vials in minute (p1.0 ml) volumes. were 553±85 and 85±11 pmol CO hr-1 mgÀ1 fresh weight (n ¼ 12), respectively. The I50 value for HO-1 was interpolated to be CO determination 0.47 mM in contrast to 0.10 mM for HO-2. Thus, the HO-1 SI is 0.21. CO generated into the vial headspace during each reaction was Figure 3, in contrast, illustrates the dose–response curves for the quantitated by gas chromatography with a Reduction Gas Analyzer less-potent inhibitor, zinc protoporphyrin (ZnPP). The I50 values for (Peak Laboratories, LLC, Mountain View, CA, USA). The headspace both isozymes (5.45 and 2.65 mM, for HO-1 and HO-2, respectively) gas was passed with CO-free carrier air (B55 ml minÀ1) through resulted in an HO-1 SI of 0.49. a stainless steel column (68  0.53 cm i.d.) packed with 13  In Table 1, the inhibitory potency of each Mp is ranked on molecular sieve (Alltech Associates, Deerfield, IL, USA) and operated at the basis of increasing I50 concentrations for each isoenzyme.

Journal of Perinatology Metalloporphyrin selectivity RJ Wong et al S38

100 Table 1 Order of decreasing potency of metalloporphyrins and Mf porphyrins Brain (HO-2) toward inhibition of HO-1 (spleen) and HO-2 (brain) isoenzyme activities

75 I50 (mM) HO-1 HO-2 I50 (mM) Spleen (HO-1) 0.07 (0.08) SnMP SnMP 0.02 (0.03) 50 0.08 (0.05) CrMP CrMP 0.04 (0.05) 0.14 (0.12) ZnBG ZnBG 0.04 (0.08) 0.30 CrPP SnPP 0.10 25 0.42 CrDP SnBG 0.15

% Inhibition of HO Activity 0.47 SnPP CrPP 0.18 0.80 SnBG CrDP 0.25 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 1.60 ZnMP ZnMP 0.70 SnPP (M) 2.80 CrBG SnDP 0.80 0.10 0.47 3.15 SnDP CrBG 2.10 Figure 2 Representative concentration response curves for tin protoporphyrin 5.25 ZnDP ZnDP 2.10 5.45 (6.00) ZnPP ZnPP 2.65 (3.45) (SnPP). The I50 value was interpolated to be 0.10 for the heme oxygenase (HO)-2 isoenzyme (brain tissue, ) and 0.47 mM for the inducible HO isoenzyme >48 MfBG MfBG 17.30 (HO-1; spleen tissue, K). Data are shown as percentage inhibition of HO activity >48 MfMP MfMP 23.40 of control levels as a function of SnPP concentration (n ¼ 3 for each concentration). Abbreviations: BG, bis-glycol; Cr, chromium; DP, deuteroporphyrin; HO-1, inducible heme oxygenase isoenzyme; HO-2, constitutive heme oxygenase isoenzyme; I50, 50% inhibition; Mf, metal-free; MP, mesoporphyrin; PP, protoporphyrin; Sn, tin; Zn, zinc. The I (mM) denotes the concentration of each Mp and Mf porphyrin needed to 100 50 inhibit activity of each isoenzyme by 50%. Mouse isoenzyme values are shown in bold (n ¼ 3 for each Mp). Brain (HO-2) 75 Spleen (HO-1)

50 Table 2 HO-1 SI of metalloporphyrins and Mf porphyrin toward inhibition of HO-1 and HO-2 activity

Mp I HO-1 (mM)IHO-2 (mM) HO-1 SI 25 50 50

% Inhibition of HO Activity SnPP 0.47 0.10 0.21 SnDP 3.15 0.80 0.25 0 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 ZnBG 0.14 (0.12) 0.04 (0.08) 0.29 (0.67) ZnPP (M) SnMP 0.07 (0.08) 0.02 (0.03) 0.29 (0.38) 2.65 5.45 ZnMP 1.60 0.70 0.44 Figure 3 Representative concentration response curve for zinc protoporphyrin SnBG 0.80 0.15 0.19 (ZnPP). The I50 value was interpolated to be 2.65 for the heme oxygenase (HO)-2 CrPP 0.30 0.18 0.60 isoenzyme (brain tissue, ) and 5.45 mM for the inducible HO-1 isoenzyme CrMP 0.08 (0.05) 0.04 (0.05) 0.50 (1.00) (HO-1; spleen tissue, K). Data are shown as percentage inhibition of HO activity of CrBG 2.80 2.10 0.75 control levels as a function of ZnPP concentration (n ¼ 3 for each concentration). ZnDP 5.25 2.10 0.40 CrDP 0.42 0.25 0.60 The order for each Mp is nearly identical for both isoenzymes. ZnPP 5.45 (6.00) 2.65 (3.45) 0.49 (0.58) Inhibition of HO-1 and HO-2 isoenzymes was found to occur most Abbreviations: BG, bis-glycol; Cr, chromium; DP, deuteroporphyrin; HO-1, inducible heme effectively with tin mesoporphyrin. Some cross-over of the potency oxygenase isoenzyme; HO-2, constitutive heme oxygenase isoenzyme; I50, 50% inhibition; Mf, metal-free; MP, mesoporphyrin; PP, protoporphyrin; SI, Selectivity Index; Sn, tin; order of (metallo)porphyrins does, however, occur. The least Zn, zinc. effective inhibition of both HO-1 and HO-2 isoenzymes was I50 (mM) denotes the concentration of each (metallo)porphyrin needed to inhibit activity observed with the Mps zinc deuteroporphyrin (5.25 and 2.10 mM, of each isoenzyme by 50%. HO-2/HO-1 denotes the ratio of I50 of each Mp and Mf porphyrin for HO-2 and HO-1, respectively, and represents the relative selectivity of a respectively) and ZnPP (5.45 and 2.65 mM, respectively) and the Mf porphyrin to inhibit HO-1 over HO-2, where a HO-1 SI >1.0 denotes the selectivity of porphyrins (>48 mM). Because there may be species differences in Mp for HO-1, and a HO-1 SI p1.0 denotes equal or more selectivity for HO-2. their sensitivity to Mps, we also determined the I50 value of four Mouse isoenzyme values (n ¼ 3 for each Mp) are shown in bold. Mps in adult FVB mouse spleen and brain tissues. Mean HO activity Other metalloporphyrins or Mf porphyrins studied did not inhibit HO-1 or HO-2 to 50% ± ± of control levels at concentrations <48 mM.I50 for HO-2 isoenzyme inhibition for MfBG levels for control spleen and brain were 429 93 and 60 5 pmol and MfMP were 17.30 and 23.40 mM, respectively, and therefore the HO-1 SIs were À1 CO hr-1 mg fresh weight (n ¼ 9). We found that for tin not calculated.

Journal of Perinatology Metalloporphyrin selectivity RJ Wong et al S39 mesoporphyrin, CrMP, ZnBG and ZnPP the I50 values for HO-1 compounds have found that they are selective in inhibiting HO-1 were 0.08, 0.05, 0.12 and 6.00 mM, and for HO-2 the values were over HO-2. These non-porphyrin HO inhibitors have comparable 0.03, 0.05, 0.08 and 3.45 mM, respectively, compared with those for inhibitory potencies toward HO-1 as the Mps, ranging from 0.6 to the rat (Tables 1 and 2). 20 mM for HO-1 and from 16 to >100 mM for HO-2. These Table 2 summarizes the I50 values with respect to the Mp compounds also, similar to some Mps, have little effect on nitric discriminatory potential toward the two isoenzymes. The most oxide synthase or soluble guanylate cyclase. Some dioxolanes have striking finding is that, of all the Mps, the HO-1 SI for SnPP is been found to affect nitric oxide synthas and soluble guanylate 0.21, and therefore this compound is the most specific in inhibiting cyclase as well as cytochrome P450 in rat tissues.32 We have HO-2 over HO-1. Interestingly, Mf bis-glycol and Mf mesoporphyrin performed in vivo studies using azalanstat, a dioxolane compound, appeared to favor inhibition of only HO-2 at the concentration and found that it can inhibit in vivo HO activity, but only at a range tested. ZnPP and chromium deuteroporphyrin displayed the high dose (500 mmol kgÀ1 body weight), and also can induce smallest indices. However, none of the HO-1 SIs are >1.0, HO-1.34 Because these compounds were first designed as inhibitors indicating that not one of the tested Mps selectively inhibits of cholesterol production by lanosterol 14-a-demethylase, the HO-1 over HO-2. effects of inhibiting this important pathway may preclude their use in a developing newborn, and as such have been proposed as only pharmacological tools for studying HO-1 and CO.32,33 Discussion Although major differences exist between HO-1 and HO-2, each The HO isoenzymes, HO-1 and HO-2, are different gene products isoenzyme is evolutionarily conserved in the primary amino-acid having little similarity in amino-acid or nucleotide sequences, and nucleotide sequences, with homology between HO-1 and HO-2 amino-acid composition, transcript number or size. They also differ being B42%. As mentioned above, the Hmox1 gene responds to in gene organization and structure as well as in chromosomal all types of stimuli, many of which have in common the localization. The , however, have similar mechanisms of association with oxidative stress; in contrast, HO-2 is responsive heme catalysis, substrate specificity and /coenzyme mainly to adrenal glucocorticoids. The consensus sequences requirements. The mechanism of action of the isoenzymes arises necessary for binding several regulatory factors are present and from the presence of a conserved sequence of 23 amino acids that functional in the HO-1 . Only a single glucocorticoid forms the ‘heme pocket’, which is the catalytic site of the enzymes. response element is present and functional in the Hmox2 gene. The ‘heme pocket’ recognizes the porphyrin ring of the Mps, but The glucocorticoid response element is not a strong transcriptional not the chelated metal.31 Therefore, various synthetic Mps can bind promoter, and glucocorticoids exert a prominent regulatory effect to prevent access to the pocket by the natural substrate, heme. on HO-2 expression at the protein level. On the basis of the inhibitory potency of each Mp for each Specific activity of HO in different organs varies greatly, the isoenzyme (Table 1), even though the isoenzymes differ vastly in highest HO activity being found in the spleen, testes and the brain. molecular and biochemical properties, they appear to respond The spleen is the only organ in which, under normal, unstressed similarly to inhibition by each Mp. The most striking finding was conditions, HO-1 is the predominant form. In addition to the that, of all the Mps, the HO-1 SI for SnPP was 0.21, and therefore function of HO isoenzymes in directly regulating heme and the most specific in inhibiting HO-2 over HO-1. Surprisingly, Mf hemoprotein levels, as well as generating CO, HO-2 may have bis-glycol and Mf mesoporphyrin inhibited only HO-2, suggesting another possible function: that of the ‘heme/oxygen sensor’ for the that a central metal is needed for conferring HO-1 inhibitory cell, with two copies of a sequence 100% identical to the oxygen- properties. There also appeared to be no apparent pattern of sensing consensus sequence of erythropoietic 50-TTTTGCA-30 that is inhibitory potency based on the central metal and porphyrin ring, induced by hypoxia.35,36 Moreover, in addition to the catalytic which indicates that the whole molecule confers the degree of ‘fit’ heme pocket in the HO-2 isoenzyme, two additional high-affinity into the heme pocket allowing for its potency for inhibiting HO as heme-binding sites, named heme regulatory motifs, are present, well as its selectivity of HO-2 over HO-1. Tin mesoporphyrin and which have conserved cysteine–proline core residues that bind CrMP most potently inhibit both HO isoenzymes, but ZnMP had heme with high affinity but are not involved in heme catalysis. fairly low potency. ZnPP and CrBG displayed the largest HO-1 SI These characteristics of the Hmox2 gene, the presence of the values (Table 2). In addition, none of the HO-1 SI values are >1.0, consensus sequence of oxygen sensor elements and the high- indicating that no Mp selectively inhibits HO-1 over HO-2. affinity heme-binding motifs in HO-2 are consistent with its role as Nevertheless, CrMP appears to have the best HO-1 selectivity of all a major gene regulator in the cell.31 Thus, there is good rationale Mps with an HO-1 SI of 1.0 in mouse tissues. No species-specific for not perturbing the HO-2 system while trying to control the sensitivities to Mps were observed between the rat and mouse. HO-1 system. Previous studies conducted by us32 and our colleagues33 It is intriguing to speculate that, because of these heme investigating the efficacy and selectivity of imidazole–dioxolane regulatory motifs, Mps may also bind to these regions in addition

Journal of Perinatology Metalloporphyrin selectivity RJ Wong et al S40 to the heme pocket and thus may explain the greater sensitivity of 12 Cruse I, Maines MD. Evidence suggesting that the two forms of heme oxygenase HO-2 to Mps. Elucidating the contribution of the non-competitive are products of different . J Biol Chem 1988; 263: 3348–3353. and competitive inhibition of HO-2 to the total inhibitory potency 13 Shibahara S, Yoshizawa M, Suzuki H, Takeda K, Meguro K, Endo K. Functional of an Mp is beyond the scope of this paper. analysis of cDNAs for two types of human heme oxygenase and evidence for their separate regulation. J Biochem 1993; 113: 214–218. We conclude that SnPP appears to be the most selective Mp in 14 Vreman HJ, Wong RJ, Stevenson DK. Sources, sinks, and measurements of carbon inhibiting HO-2 activity while minimally affecting that of HO-1 monoxide. Carbon Monoxide and Cardiovascular Functions. CRC Press: Boca in vitro. In general, the HO-2 isoenzyme is more sensitive to Raton, FL, 2001. inhibition by Mps than is HO-1. The inhibitory potency of each Mp 15 Raju VS, McCoubrey Jr WK, Maines MD. Regulation of heme oxygenase-2 is nearly identical for both isoenzymes. It appears that none of the by glucocorticoids in neonatal rat brain: characterization of a functional glucocorticoid response element. Biochim Biophys Acta 1997; Mps studied preferentially inhibits HO-1. However, the natural Mp, 1351: 89–104. ZnPP, effectively inhibits HO-1 with a relative sparing of HO-2 16 McCoubrey Jr WK, Huang TJ, Maines MD. Isolation and characterization of a cDNA activity. In other words, depending on the potency required for from the rat brain that encodes hemoprotein heme oxygenase-3. Eur J Biochem 1997; clinical efficacy, less-potent Mps, such as ZnPP, would produce a 247: 725–732. less inhibitory effect on HO-2, while limiting the response of the 17 Hintz SR, Kwong LK, Vreman HJ, Stevenson DK. Recovery of exogenous heme as inducible HO-1, and may be useful as a clinical tool. carbon monoxide and biliary heme in adult rats after tin protoporphyrin treatment. J Pediatr Gastroenterol Nutr 1987; 6: 302–306. 18 Vreman HJ, Ekstrand BC, Stevenson DK. Selection of metalloporphyrin heme oxygenase inhibitors based on potency and photoreactivity. Pediatr Res 1993; 33: Conflict of interest 195–200. 19 Qato MK, Maines MD. Prevention of neonatal hyperbilirubinaemia in non-human The authors declare no conflict of interest. primates by Zn-protoporphyrin. Biochem J 1985; 226: 51–57. 20 Vreman HJ, Lee OK, Stevenson DK. In vitro and in vivo characteristics of a heme oxygenase inhibitor: ZnBG. Am J Med Sci 1991; 302: 335–341. Acknowledgments 21 Kappas A, Simionatto CS, Drummond GS, Sassa S, Anderson KE. The liver excretes large amounts of heme into bile when heme oxygenase is This work was supported in part by the National Institutes of Health (Grants HD inhibited competitively by Sn protoporphyrin. Proc Natl Acad Sci USA 1985; 82: 14426, HD/HL 58013, HL 68703), the Hess Research Fund, the Mary L Johnson 896–900. Research Fund and the HM Lui Research Fund. 22 Vallier HA, Rodgers PA, Stevenson DK. Oral administration of zinc deuteroporphyrin IX 2,4 bis glycol inhibits heme oxygenase in neonatal rats. Dev Pharmacol Ther 1991; 17: 220–222. 23 Vallier HA, Rodgers PA, Stevenson DK. Inhibition of heme oxygenase after References oral vs intraperitoneal administration of chromium porphyrins. Life Sci 1993; 52: 1 Vreman HJ, Mahoney JJ, Stevenson DK. Carbon monoxide and carboxyhemoglobin. Adv L79–L84. Pediatr 1995; 42: 303–325. 24 Vreman HJ, Cipkala DA, Stevenson DK. Characterization of porphyrin heme oxygenase 2 Coburn RF, Wallace HW, Abboud R. Redistribution of body carbon monoxide after inhibitors. Can J Physiol Pharmacol 1996; 74: 278–285. hemorrhage. Amer J Physiol 1971; 220: 868–873. 25 Marks GS, Brien JF, Nakatsu K, McLaughlin BE. Does carbon monoxide have a 3 Landaw SA, Winchell HS, Boone RF. Measurement of endogenous carbon monoxide physiological function? Trends Pharmacol Sci 1991; 12: 185–188. production in hemolytic disease of the inborn. Clin Res 1971; 19: 208A. 26 Stocker R, Yamamoto Y, McDonagh AF, Glazer AN, Ames BN. Bilirubin is an 4 Valaes T, Drummond GS, Kappas A. Control of hyperbilirubinemia in glucose-6- antioxidant of possible physiological importance. Science 1987; 235: phosphate dehydrogenase-deficient newborns using an inhibitor of bilirubin 1043–1046. production, Sn mesoporphyrin. Pediatrics 1998; 101: E1. 27 Dennery PA, McDonagh AF, Spitz DR, Rodgers PA, Stevenson DK. Hyperbilirubinemia 5 Lucey JF. Bilirubin and brain damageFa real mess. Pediatrics 1982; 69: 381–382. results in reduced oxidative injury in neonatal Gunn rats exposed to hyperoxia. 6 Ennever JF. Phototherapy for neonatal jaundice. Yearly review. Photochem Photobiol Free Radic Biol Med 1995; 19: 395–404. 1988; 47: 871–876. 28 Vreman HJ, Stevenson DK. Heme oxygenase activity as measured by carbon monoxide 7 Vreman HJ, Wong RJ, Stevenson DK, Route RK, Reader SD, Fejer MM et al. production. Anal Biochem 1988; 168: 31–38. Light emitting diodes: a novel light source for phototherapy. Pediatr Res 1998; 44: 29 Vreman HJ, Stevenson DK. Detection of heme oxygenase activity by 804–809. measurement of CO. In: Maines MD, Costa LG, Reed DJ, Sassa S, Sipes IG (eds). 8 Lee KS, Gartner LM. Management of unconjugated hyperbilirubinemia in the newborn. Current Protocols in Toxicology. John Wiley & Sons, Inc.: New York, 1999, Semin Liver Dis 1983; 3: 52–63. pp 9.2.1–9.2.10. 9 Mahoney JJ, Wong RJ, Vreman HJ, Stevenson DK. Fetal hemoglobin of transfused 30 Vreman HJ, Wong RJ, Kim EC, Nabseth DC, Marks GS, Stevenson DK. Haem neonates and spectrophotometric measurements of oxyhemoglobin and oxygenase activity in human umbilical cord and rat vascular tissues. Placenta 2000; carboxyhemoglobin. J Clin Monit 1991; 7: 154–160. 21: 337–344. 10 Tenhunen R, Marver HS, Schmid R. The enzymatic conversion of heme to 31 McCoubrey Jr WK, Huang TJ, Maines MD. Heme oxygenase-2 is a hemoprotein and bilirubin by microsomal heme oxygenase. Proc Natl Acad Sci USA 1968; 61: binds heme through heme regulatory motifs that are not involved in heme catalysis. 748–755. J Biol Chem 1997; 272: 12568–12574. 11 Maines MD, Trakshel GM, Kutty RK. Characterization of two constitutive forms of rat 32 Kinobe RT, Vlahakis JZ, Vreman HJ, Stevenson DK, Brien JF, Szarek WA et al. Selectivity liver microsomal heme oxygenase. Only one molecular species of the enzyme is of imidazole-dioxolane compounds for in vitro inhibition of microsomal haem inducible. J Biol Chem 1986; 261: 411–419. oxygenase isoforms. Br J Pharmacol 2006; 147: 307–315.

Journal of Perinatology Metalloporphyrin selectivity RJ Wong et al S41

33 Vlahakis JZ, Kinobe RT, Bowers RJ, Brien JF, Nakatsu K, Szarek WA. Imidazoledioxolane lacking heme oxygenase-2: evidence for a novel role of heme oxygenase-2 compounds as isozyme-selective heme oxygenase inhibitors. J Med Chem 2006; 49: as an oxygen sensor. Adv Exp Med Biol 2006; 580: 161–166; discussion 4437–4441. 351–169. 34 Morisawa T, Wong RJ, Bhutani VK, Vreman HJ, Stevenson DK. Inhibition of heme 36 Kemp PJ. Hemeoxygenase-2 as an O2 sensor in K+ channel-dependent oxygenase activity in newborn mice by azalanstat. Can J Physiol Pharmacol 2008; 86: chemotransduction. Biochem Biophys Res Commun 2005; 338: 651–659. 648–652. 35 Zhang Y, Furuyama K, Adachi T, Ishikawa K, Matsumoto H, Masuda T et al. 37 Vreman HJ, Wong RJ, Stevenson DK. Alternative metalloporphyrins for the treatment of Hypoxemia and attenuated hypoxic ventilatory responses in mice neonatal jaundice. J Perinatol 2001; 21(Suppl 1): S108–S113.

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