Lipophilicity is a critical parameter that dominates the efficacy of metalloporphyrins in blocking the development of morphine antinociceptive tolerance through peroxynitrite-mediated pathways

doi:10.1016/j.freeradbiomed.2008.09.037

Free Radical Biology and Medicine

Volume 46, Issue 2, 15 January 2009, Pages 212-219

https://doi.org/10.1016/j.freeradbiomed.2008.09.037 Get rights and content

Abstract

Severe pain syndromes reduce the quality of life of patients with inflammatory and neoplastic diseases, partly because reduced analgesic effectiveness with chronic opiate therapy (i.e., tolerance) leads to escalating doses and distressing side effects. Peroxynitrite-mediated nitroxidative stress in the dorsal horn of the spinal cord plays a critical role in the induction and development of antinociceptive tolerance to morphine. This provides a valid pharmacological basis for developing peroxynitrite scavengers as potent adjuncts to opiates in the management of pain. The cationic Mn(III) ortho-N-alkylpyridylporphyrins MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺ are among the most potent peroxynitrite scavengers, with nearly identical scavenging rate constants (~10⁷ M^− 1 s^− 1). Yet, MnTnHex-2-PyP⁵⁺ is significantly more lipophilic and more bioavailable and, in turn, was 30-fold more effective in blocking the development of morphine antinociceptive tolerance than MnTE-2-PyP⁵⁺ using the hot-plate test in a well-characterized murine model. The hydrophilic MnTE-2-PyP⁵⁺ and the lipophilic MnTnHex-2-PyP⁵⁺ were 10- and 300-fold, respectively, more effective in inhibiting morphine tolerance than the hydrophilic Fe(III) porphyrin FeTM-4-PyP⁵⁺. Both Mn porphyrins decreased levels of TNF-α, IL-1β, and IL-6 to normal values. Neither of them affected acute morphine antinociceptive effects nor caused motor function impairment. Also neither was able to reverse already established morphine tolerance. We have recently shown that the anionic porphyrin Mn(III) tetrakis(4-carboxylatophenyl)porphyrin is selective in removing ONOO⁻ over O₂⁻, but at ∼ 2 orders of magnitude lower efficacy than MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺, which in turn parallels up to 100-fold lower ability to reverse morphine tolerance. These data (1) support the role of peroxynitrite rather than superoxide as a major mechanism in blocking the development of morphine tolerance and (2) show that lipophilicity is a critical parameter in enhancing the potency of such novel peroxynitrite scavengers.

Section snippets

Mn porphyrins

MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺ were synthesized and characterized as previously described [23], [46], [48].

Induction of morphine-induced antinociceptive tolerance in mice

Male CD-1 mice (24–30 g; Charles River) were housed and cared for in accordance with the guidelines of the Institutional Animal Care and Use Committee of the St. Louis University Health Science Center and in accordance with the National Institutes of Health guidelines on laboratory animals and the University of Messina, in compliance with Italian regulations on the protection of animals

The development of morphine-induced tolerance is blocked by the Mn porphyrin-based peroxynitrite decomposition catalysts MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺

Compared to animals receiving an equivalent injection of vehicle (naïve group), acute injection of morphine (3 mg/kg) in animals that received saline over 4 days (vehicle group) produced a significant and near-maximal antinociceptive response (%MPE ranging between 90 and 95%) (Figs. 2A and 2B). In contrast, compared to the antinociceptive response to acute morphine in animals that received saline over 4 days, repeated administrations of morphine over the same time course (morphine group) led to

Discussion

Chronic administration of morphine promotes neuroimmune activation as evidenced by activation of spinal cord glial cells; production of proinflammatory cytokines such as TNF-α, IL-1β, and IL-6; and spinal sensitization [50], [51], [52]. Thus, inhibitors of glial cell metabolism and anti-cytokine approaches block morphine-induced antinociceptive tolerance and hyperalgesia [50], [51], [52]. The possible mechanisms for chronic morphine-induced glial cell activation are not known with certainty.

Concluding remarks

In summary, we have: (1) provided evidence that ONOO⁻ rather than O₂⁻ is a predominant player in the development of morphine antinociceptive tolerance and (2) pointed to the lipophilicity as a critical parameter that determines the efficacy of the drug in blocking morphine tolerance and decreasing the levels of the inflammatory cytokines TNF-α, IL-1β, and IL-6. With high ability to eliminate ONOO⁻ along with enhanced lipophilicity, MnTnHex-2-PyP⁵⁺ (0.1 mg/kg/day) is 300-fold more effective in

Acknowledgments

Ines Batinić-Haberle and Júlio S. Rebouças are grateful for financial support from NIH U19 AI67798-01/Pilot Project and The Wallace H. Coulter Translational Partners Grant Program. Ivan Spasojević acknowledges NIH/NCI Duke Comprehensive Cancer Center Core Grant 5-P30-CA14236-29. Daniela Salvemini and Ines Batinić-Haberle acknowledge support from NIH 1 R01 DA24074-01A1.

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NOX activity is elevated by morphine, and genetic or pharmacological disruption of these enzymes attenuates tolerance and hyperalgesia [171–173]. Superoxide and peroxynitrite have been implicated as downstream mediators because decomposition catalysts also attenuate tolerance and hyperalgesia [174–176]. It remains unclear how morphine engages these enzymes, but it may be mediated by classical μ-opioid receptors and/or TLR4 [168].
Tissue injury can initiate bidirectional signaling between neurons, glia, and immune cells that creates and amplifies pain. While the ability for neurotransmitters, neuropeptides, and cytokines to initiate and maintain pain has been extensively studied, recent work has identified a key role for reactive oxygen and nitrogen species (ROS/RNS; nitroxidative species), including superoxide, peroxynitrite, and hydrogen peroxide. In this review we describe how nitroxidative species are generated after tissue injury and the mechanisms by which they enhance neuroexcitability in pain pathways. Finally, we discuss potential therapeutic strategies for normalizing nitroxidative signaling, which may also enhance opioid analgesia, to help to alleviate the enormous burden of pathological pain.
A comprehensive evaluation of catalase-like activity of different classes of redox-active therapeutics
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Because of the increased insight into the biological role of hydrogen peroxide (H₂O₂) under physiological and pathological conditions and the role it presumably plays in the action of natural and synthetic redox-active drugs, there is a need to accurately define the type and magnitude of reactions that may occur with this intriguing and key species of redoxome. Historically, and frequently incorrectly, the impact of catalase-like activity has been assigned to play a major role in the action of many redox-active drugs, mostly SOD mimics and peroxynitrite scavengers, and in particular MnTBAP^3– and Mn salen derivatives. The advantage of one redox-active compound over another has often been assigned to the differences in catalase-like activity. Our studies provide substantial evidence that Mn(III) N-alkylpyridylporphyrins couple with H₂O₂ in actions other than catalase-related. Herein we have assessed the catalase-like activities of different classes of compounds: Mn porphyrins (MnPs), Fe porphyrins (FePs), Mn(III) salen (EUK-8), and Mn(II) cyclic polyamines (SOD-active M40403 and SOD-inactive M40404). Nitroxide (tempol), nitrone (NXY-059), ebselen, and MnCl₂, which have not been reported as catalase mimics, were used as negative controls, while catalase enzyme was a positive control. The dismutation of H₂O₂ to O₂ and H₂O was followed via measuring oxygen evolved with a Clark oxygen electrode at 25 °C. The catalase enzyme was found to have k_cat(H₂O₂)=1.5×10⁶ M^–1 s^–1. The yield of dismutation, i.e., the maximal amount of O₂ evolved, was assessed also. The magnitude of the yield reflects an interplay between the k_cat(H₂O₂) and the stability of compounds toward H₂O₂-driven oxidative degradation, and is thus an accurate measure of the efficacy of a catalyst. The k_cat(H₂O₂) values for 12 cationic Mn(III) N-substituted (alkyl and alkoxyalkyl) pyridylporphyrin-based SOD mimics and Mn(III) N,N′-dialkylimidazolium porphyrin, MnTDE-2-ImP⁵⁺, ranged from 23 to 88 M^–1 s^–1. The analogous Fe(III) N-alkylpyridylporphyrins showed ~10-fold higher activity than the corresponding MnPs, but the values of k_cat(H₂O₂) are still ~4 orders of magnitude lower than that of the enzyme. While the k_cat(H₂O₂) values for Fe ethyl and n-octyl analogs were 803.5 and 368.4 M^–1 s^–1, respectively, the FePs are more prone to H₂O₂-driven oxidative degradation, therefore allowing for similar yields in H₂O₂ dismutation as analogous MnPs. The k_cat(H₂O₂) values are dependent on the electron deficiency of the metal site as it controls the peroxide binding in the first step of the dismutation process. SOD-like activities depend on electron deficiency of the metal site also, as it controls the first step of $O_{2}^{● -}$ dismutation. In turn, the k_cat( $O_{2}^{● -}$ ) parallels the k_cat(H₂O₂). Therefore, the electron-rich anionic non-SOD mimic MnTBAP^3– has essentially very low catalase-like activity, k_cat(H₂O₂)=5.8 M^–1 s^–1. The catalase-like activities of Mn(III) and Fe(III) porphyrins are at most, 0.0004 and 0.05% of the enzyme activity, respectively. The k_cat(H₂O₂) values of 8.2 and 6.5 M^–1 s^–1 were determined for electron-rich Mn(II) cyclic polyamine-based compounds, M40403 and M40404, respectively. The EUK-8, with modest SOD-like activity, has only slightly higher k_cat(H₂O₂)=13.5 M^–1 s^–1. The biological relevance of k_cat(H₂O₂) of MnTE-2-PyP⁵⁺, MnTDE-2-ImP⁵⁺, MnTBAP^3–, FeTE-2-PyP⁵⁺, M40403, M40404, and Mn salen was evaluated in wild-type and peroxidase/catalase-deficient E. coli.
NADPH-oxidase 2 activation promotes opioid-induced antinociceptive tolerance in mice
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We have reported that in the spinal cord, peroxynitrite (PN) formation, the diffusion-limited product of superoxide and nitric oxide (NO) (Beckman et al., 1990), is central to these processes (Dang and Christie, 2012). Systemic delivery of PN decomposition catalysts (PNDCs) in mice and rats not only blocks morphine-induced antinociceptive tolerance (Muscoli et al., 2007; Batinic-Haberle et al., 2009), but does so by attenuating the activation of redox-sensitive transcription factors (NFκB) and MAPK kinases (ERK, p38 kinase) and the increased formation of various proinflammatory cytokines (Muscoli et al., 2007). PNDCs also block post-translational nitration and inactivation of glutamate transporters, GLAST and GLT-1, and glutamine synthetase (Salvemini and Neumann, 2009).
The analgesic effectiveness of long-term opioid therapies is compromised by the development of antinociceptive tolerance linked to the overt production of peroxynitrite (ONOO⁻, PN), the product of the interaction between superoxide (O₂⁻, SO) and nitric oxide (NO), and to neuroinflammatory processes. We have recently reported that in addition to post-translational nitration and inactivation of mitochondrial manganese superoxide dismutase (MnSOD), activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase holoenzyme (NOX) in the spinal cord is a major source for the overt production of superoxide-derived PN during the development of morphine-induced antinociceptive tolerance. However, the NOX complex involved in these processes is not known. The objective of these studies is to identify a potential role for the NOX2 complex, an enzyme involved in inflammation. Mice lacking the catalytic subunit of NOX2 (Nox2^−/−) or its regulatory subunit, p47^phox (p47^phox^−/−), developed antinociceptive tolerance similar to wildtype (wt) mice after 3 days of continuous morphine. However, while wt mice continue to develop tolerance by day six, morphine analgesia was restored in both Nox2^−/− and p47^phox^−/− mice. Moreover, the loss of Nox2 or p47 did not affect acute morphine analgesia in naïve mice. In wt mice, antinociceptive tolerance was associated with increased activation of NOX, nitration of MnSOD, and proinflammatory cytokines production in the spinal cord. These events were markedly attenuated in Nox2^−/− and p47^phox^−/− mice and instead, there was enhanced formation of antiinflammatory cytokine (IL4 and IL10) production. These results suggest that NOX2 activity provides a significant source of superoxide-derived PN to undertake post-translational modifications of mitochondrial MnSOD and to engage neuroinflammatory signaling in the spinal cord associated with opioid-induced antinociceptive tolerance. Thus, NOX2 may provide a potential target for adjuvant therapy to protect opioid analgesia.
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Thermal stability of the prototypical Mn porphyrin-based superoxide dismutase mimic and potent oxidative-stress redox modulator Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride, MnTE-2-PyP5+
2013, Journal of Pharmaceutical and Biomedical Analysis
Citation Excerpt :
These therapeutics, initially developed as catalytic antioxidant agents, are now regarded as potent modulators that are able to redox-cycle with reactive oxygen and nitrogen species (e.g., superoxide and peroxynitrite) and modulate several redox-dependent transcription factors (e.g., NF-κB, HIF-1α, VEGF, AP-1, and p53) [1–4], affecting, thus, a variety of inflammatory, angiogenic, and oncogenic pathways [3,6]. The prototypical cationic Mn porphyrin most studied in vivo, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl5, Fig. 1) has been efficacious in many animal models of oxidative stress-related diseases and injuries [1–4], such as diabetes [7] and radiation injury [8], and as an adjunct in cancer pain management (reversal of morphine tolerance) [9] as well as cancer therapy [4,6]. Master drug file for MnTE-2-PyPCl5 has been filed with the U.S. Food and Drug Administration (FDA) and the toxicity studies needed for Investigational New Drug filing with FDA are finalized [3].
Cationic Mn porphyrins are among the most potent catalytic antioxidants and/or cellular redox modulators. Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin chloride (MnTE-2-PyPCl₅) is the Mn porphyrin most studied in vivo and has successfully rescued animal models of a variety of oxidative stress-related diseases. The stability of an authentic MnTE-2-PyPCl₅ sample was investigated hereon by thermogravimetric, derivative thermogravimetric, and differential thermal analyses (TG/DTG/DTA), under dynamic air, followed by studies at selected temperatures to evaluate the decomposition path and appropriate conditions for storage and handling of these materials. All residues were analyzed by thin-layer chromatography (TLC) and UV–vis spectroscopy. Three thermal processes were observed by TG/DTG. The first event (endothermic) corresponded to dehydration, and did not alter the MnTE-2-PyPCl₅ moiety. The second event (endothermic) corresponded to the loss of EtCl (dealkylation), which was characterized by gas chromatography–mass spectrometry. The residue at 279 °C had UV–vis and TLC data consistent with those of the authentic, completely dealkylated analog, MnT-2-PyPCl. The final, multi-step event corresponded to the loss of the remaining organic matter to yield Mn₃O₄ which was characterized by IR spectroscopy. Isothermal treatment at 188 °C under static air for 3 h yielded a mixture of partially dealkylated MnPs and traces of the free-base, dealkylated ligand, H₂T-2-PyP, which reveals that dealkylation is accompanied by thermal demetallation under static air conditions. Dealkylation was not observed if the sample was heated as a solid or in aqueous solution up to ∼100 °C. Whereas moderate heating changes sample composition by loss of H₂O, the dehydrated sample is indistinguishable from the original sample upon dissolution in water, which indicates that catalytic activity (on Mn basis) remains unaltered. Evidently, dealkylation at high temperature compromises sample activity.
A new SOD mimic, Mn(III) ortho N-butoxyethylpyridylporphyrin, combines superb potency and lipophilicity with low toxicity
2012, Free Radical Biology and Medicine
Citation Excerpt :
Among the lipophilic analogs, MnTnHex-2-PyP5+ has been the most frequently studied porphyrin [1,3,6]. The remarkable efficacy of MnTE-2-PyP5+ and MnTnHex-2-PyP5+ in numerous animal models of oxidative stress diseases [1,3,8–10] may be at least in part attributed to their ability to accumulate in mitochondria, mimicking therefore the site and the action of critical mitochondrial enzyme, MnSOD [7]. Mitochondrial accumulation of lipophilic MnTnHex-2-PyP5+ was significantly enhanced when compared to a more hydrophilic MnTE-2-PyP5+ [7].
The Mn porphyrins of k_cat(O₂^.-) as high as that of a superoxide dismutase enzyme and of optimized lipophilicity have already been synthesized. Their exceptional in vivo potency is at least in part due to their ability to mimic the site and location of mitochondrial superoxide dismutase, MnSOD. MnTnHex-2-PyP⁵⁺ is the most studied among lipophilic Mn porphyrins. It is of remarkable efficacy in animal models of oxidative stress injuries and particularly in central nervous system diseases. However, when used at high single and multiple doses it becomes toxic. The toxicity of MnTnHex-2-PyP⁵⁺ has been in part attributed to its micellar properties, i.e., the presence of polar cationic nitrogens and hydrophobic alkyl chains. The replacement of a CH₂ group by an oxygen atom in each of the four alkyl chains was meant to disrupt the porphyrin micellar character. When such modification occurs at the end of long alkyl chains, the oxygens become heavily solvated, which leads to a significant drop in the lipophilicity of porphyrin. However, when the oxygen atoms are buried deeper within the long heptyl chains, their excessive solvation is precluded and the lipophilicity preserved. The presence of oxygens and the high lipophilicity bestow the exceptional chemical and physical properties to Mn(III) meso-tetrakis(N-n-butoxyethylpyridinium-2-yl)porphyrin, MnTnBuOE-2-PyP⁵⁺. The high SOD-like activity is preserved and even enhanced: log k_cat(O₂^.-) = 7.83 vs 7.48 and 7.65 for MnTnHex-2-PyP⁵⁺ and MnTnHep-2-PyP⁵⁺, respectively. MnTnBuOE-2-PyP⁵⁺ was tested in an O₂^.- -specific in vivo assay, aerobic growth of SOD-deficient yeast, Saccharomyces cerevisiae, where it was fully protective in the range of 5–30 μM. MnTnHep-2-PyP⁵⁺ was already toxic at 5 μM, and MnTnHex-2-PyP⁵⁺ became toxic at 30 μM. In a mouse toxicity study, MnTnBuOE-2-PyP⁵⁺ was several-fold less toxic than either MnTnHex-2-PyP⁵⁺ or MnTnHep-2-PyP⁵⁺.

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¹: Present address: VA Medical Center, 915 N. Grand Boulevard, St. Louis, MO 63106, USA.

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Original ContributionLipophilicity is a critical parameter that dominates the efficacy of metalloporphyrins in blocking the development of morphine antinociceptive tolerance through peroxynitrite-mediated pathways

Abstract

Section snippets

Mn porphyrins

Induction of morphine-induced antinociceptive tolerance in mice

The development of morphine-induced tolerance is blocked by the Mn porphyrin-based peroxynitrite decomposition catalysts MnTE-2-PyP5+ and MnTnHex-2-PyP5+

Discussion

Concluding remarks

Acknowledgments

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Neurosci. Res.

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Brain Behav. Immun.

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Eur. J. Pharmacol.

Pain

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Free Radic. Biol. Med.

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J. Biol. Chem.

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Nat. Rev. Drug Discovery

Clinical experience of long-term treatment with epidural and intrathecal opioids—a nationwide survey

Acta Anaesthesiol. Scand.

Antinociceptive and nociceptive actions of opioids

J. Neurobiol.

Misconceptions and controversies regarding the use of opioids in cancer pain

Anticancer Drugs

Original Contribution
Lipophilicity is a critical parameter that dominates the efficacy of metalloporphyrins in blocking the development of morphine antinociceptive tolerance through peroxynitrite-mediated pathways

The development of morphine-induced tolerance is blocked by the Mn porphyrin-based peroxynitrite decomposition catalysts MnTE-2-PyP⁵⁺ and MnTnHex-2-PyP⁵⁺