Givosiran: A Review in Acute Hepatic Porphyria.

Givosiran (Givlaari®) is an δ-aminolevulinic acid synthase 1 (ALAS1)-directed small interfering RNA (siRNA) approved for the treatment of acute hepatic porphyria (AHP). In the phase 3 ENVISION trial, givosiran significantly reduced the annualized rate of composite porphyria attacks (i.e. attacks requiring hospitalization, urgent healthcare visit or intravenous hemin administration at home) compared with placebo in patients with recurrent acute intermittent porphyria (the most common type of AHP) attacks. Givosiran also improved several other outcomes, including hemin use and pain (the cardinal symptom of AHP). While generally well tolerated with an acceptable safety profile, the drug may increase the risk of hepatic and kidney adverse events. Givosiran offers the convenience of once-monthly subcutaneous administration. Available evidence indicates that givosiran is an important newer therapeutic option for patients with AHP and severe recurrent attacks.

Nanoscale coordination polymers induce immunogenic cell death by amplifying radiation therapy mediated oxidative stress.

Radiation therapy can potentially induce immunogenic cell death, thereby priming anti-tumor adaptive immune responses. However, radiation-induced systemic immune responses are very rare and insufficient to meet clinical needs. Here, we demonstrate a synergetic strategy for boosting radiation-induced immunogenic cell death by constructing gadolinium-hemin based nanoscale coordination polymers to simultaneously perform X-ray deposition and glutathione depletion. Subsequently, immunogenic cell death is induced by sensitized radiation to potentiate checkpoint blockade immunotherapies against primary and metastatic tumors. In conclusion, nanoscale coordination polymers-sensitized radiation therapy exhibits biocompatibility and therapeutic efficacy in preclinical cancer models, and has the potential for further application in cancer radio-immunotherapy.

5-Aminolevulinate dehydratase porphyria: Update on hepatic 5-aminolevulinic acid synthase induction and long-term response to hemin.

BACKGROUND: 5-Aminolevulinic acid dehydratase (ALAD) porphyria (ADP) is an ultrarare autosomal recessive disease, with only eight documented cases, all of whom were males. Although classified as an acute hepatic porphyria (AHP), induction of the rate limiting hepatic enzyme 5-aminolevulinic acid synthase-1 (ALAS1) has not been demonstrated, and the marrow may also contribute excess 5-aminolevulinic acid (ALA). Two patients have died and reported follow up for the others is limited, so the natural history of this disease is poorly understood and treatment experience limited. METHODS: We report new molecular findings and update the clinical course and treatment of the sixth reported ADP patient, now 31 years old and the only known case in the Americas, and review published data regarding genotype-phenotype correlation and treatment. RESULTS: Circulating hepatic 5-aminolevulinic acid synthase-1 (ALAS1) mRNA was elevated in this case, as in other AHPs. Gain of function mutation of erythroid specific ALAS2 - an X-linked modifying gene in some other porphyrias - was not found. Seven reported ADP cases had compound heterozygous ALAD mutations resulting in very low residual ALAD activity and symptoms early in life or adolescence. One adult with a germline ALAD mutant allele developed ADP in association with a clonal myeloproliferative disorder, polycythemia vera. CONCLUSIONS: Elevation in circulating hepatic ALAS1 and response to treatment with hemin indicate that the liver is an important source of excess ALA in ADP, although the marrow may also contribute. Intravenous hemin was effective in most reported cases for treatment and prevention of acute attacks of neurological symptoms.

miR-331-3p Inhibits Inflammatory Response after Intracerebral Hemorrhage by Directly Targeting NLRP6.

BACKGROUND: The mechanism of inflammatory reaction after intracerebral hemorrhage remains unclear, which to some extent restrains the therapeutic development of hemorrhagic stroke. The present study attempts to verify whether NLRP6 plays an important role in inflammatory reaction after intracerebral hemorrhage and identify the critical microRNA during the process. METHODS: Suitable simulated cerebral hemorrhage environments were established in vitro and in vivo. BV2 cells were treated with hemin to induce cell damage. Collagenase was used to establish a model of mouse cerebral hemorrhage. The relationship among NLRP6, miR-331-3p, and the corresponding inflammatory expression was closely observed during this process. Techniques, such as western blot, real-time quantitative PCR, immunofluorescence, and immunocytochemistry, were used to detect the expression of relative genes and molecules in the in vitro and in vivo models. RESULTS: Downregulated miR-331-3p increased the expression of NLRP6 and alleviated the expression of TNF-α and IL-6. The neurological function recovery of mice was promoted after intracerebral hemorrhage. CONCLUSION: miR-331-3p regulated the inflammatory response after cerebral hemorrhage by negatively regulating the expression of NLRP6.

Heme oxygenase-1 alleviated non-alcoholic fatty liver disease via suppressing ROS-dependent endoplasmic reticulum stress.

AIMS: The endoplasmic reticulum (ER) stress response plays a crucial role in the development of nonalcoholic steatohepatitis (NASH). Heme oxygenase-1 (HO-1) exerts beneficial effects against oxidative injury in NASH. This study is aimed to clarify whether HO-1 is an effective therapeutic strategy for NASH via regulation of ER stress. METHODS: The C57BL/6J mice were fed with methionine-choline deficient (MCD) for 4 weeks and high fat-high carbohydrate-high cholesterol (HFD) diet for 16 weeks, with hemin or zinc protoporphyrin IX (ZnPP-IX), respectively. The LO-2 cells were cultured in palmitic medium, with transfected pEX-HO-1 or sh-HO-1 plasmid for 24 h. Meanwhile, thirty NASH patients and 15 health controls were enrolled. The ER ultrastructure was observed by transmission electron microscopy (TEM) and confocal microscopy. The expressions of mRNAs and proteins of HO-1, ER stress related genes were detected by real time PCR, western blot and immunohistochemical staining, respectively. RESULTS: The swelled and broken rough endoplasmic reticulums were observed in MCD and HFD fed mice. The reactive hepatic expression of HO-1 was related with the increased ROS production and ER stress, companied with upregulation of GRP78, p-IRE1, PERK, ATF6. Through hemin administration, hepatocyte apoptosis was suppressed companied down-regulation of CHOP, caspase12 and up-regulation of BCL2. Conserved results were exhibited in ZnPP-IX administrated mice and HO-1 silent cells. Consistent results were observed in the NASH Patients. CONCLUSIONS: HO-1 could serve as a protective factor in the progression of nutritional steatohepatitis by suppresses hepatocyte excessive ER stress and might be a potential target for therapy of nonalcoholic steatohepatitis.

Real-world annualized healthcare utilization and expenditures among insured US patients with acute intermittent porphyria (AIP) treated with hemin.

Background and aims: Patients with acute intermittent porphyria (AIP) may suffer from acute non-specific attacks that often result in hospitalizations or emergency room (ER) visits. Prior to the recent approval of givosiran (November 2019), hemin was the only FDA-approved therapy for AIP attacks in the US. Our aim was to estimate the annual healthcare utilization and expenditures for AIP patients treated with hemin using real-world data.Methods: Patients with ≥1 hemin claim and confirmed AIP diagnosis - 1 inpatient claim or 2 outpatient claims ≥30 d apart for AIP (2015-2017) or acute porphyria (prior to 2015) - were identified in MarketScan administrative claims dataset between 2007 and 2017. Continuous enrolment for ≥6 months from confirmed diagnosis was required. A secondary analysis ("active disease population") limited the sample to adult patients with ≥3 attacks or 10 months of prophylactic use of hemin within a 12-month pre-index period. AIP-related care was defined by hemin use during an attack (daily glucose and/or hemin use) or prophylaxis (non-attack hemin use). Outcomes were annualized and expenditures were inflated to 2017.Results: Across 10 years, patients with a confirmed AIP diagnosis (N = 8,877) and ≥1 hemin claim (N = 164) were restricted by ≥6 months continuous follow-up (N = 139). AIP patients were mostly female (N = 112; 81%), had median age of 40 and 3 years average follow-up. Annualized average total expenditures for AIP-related care were $113,477. Annualized average all-cause (any diagnosis) hospitalizations were statistically significantly lower for patients treated with hemin prophylaxis vs. acute treatment (1.0 vs. 2.1; p < .001). In the secondary analysis (N = 27), annualized average total expenditures for AIP-related care were higher ($187,480).Conclusions: For AIP patients treated with hemin, patients treated for acute attacks may use a greater number of resources compared to patients treated prophylactically.

Acute intermittent porphyria: analgesia can be dangerous.

Acute intermittent porphyria (AIP) is a rare condition, a metabolic disorder of the haem biosynthesis. An acute crisis of AIP can present as a combination of symptoms, such as abdominal pain, autonomic dysfunction, hyponatremia, muscle weakness and neurological symptoms in the absence of others obvious causes. We report the case of a 53-year-old woman, who was previously diagnosed with AIP 5 weeks after therapeutic suspension has developed an acute disease exacerbation. During hospitalisation, further exacerbation has occurred after analgesia with metamizole. Glucose and hemin infusions resulted in slow improvement. Physical rehabilitation was crucial to peripheral polyneuropathy recovery. Taking into account the porphyrinogenic effect described for metamizole, this drug might have triggered the second attack. Clinical history was sufficient to suspect the diagnosis and to start the treatment immediately, preventing important sequelae.

Hemin attenuated oxidative stress and inflammation to improve wound healing in diabetic rats.

Oxidative stress and persistent inflammation play crucial role in the progression of diabetic wound complications. Hemeoxgenase-1 (HO-1) by degrading hemin has been shown to display anti-oxidant and anti-inflammatory effects. Further, hemin is a potent HO-1 inducer. Thus, the current study was aimed to evaluate the effect of topical application of hemin on diabetic wound in rats. Four hundred square millimeter open excision wound were created 2 weeks after induction of diabetes with single intraperitoneal injection of streptozotocin (60 mg/kg), and the diabetic rats were divided into three groups namely diabetic control, hemin, and tin protoporphyrin (SnPPIX). Ointment base, hemin (0.5% in ointment base), and SnPPIX (0.5% in ointment base) were applied topically to wounded area in diabetic control, hemin, and SnPPIX group rats, respectively, twice daily for 19 days. Hemin significantly increased the wound contraction in comparison to control and SnPPIX-treated rats. Time-dependent analysis revealed significant increase in anti-oxidants with concomitant decrease in oxidants in hemin-treated rats as compared to diabetic control rats. Further, mRNA expression decreased for inflammatory cytokine and increased for anti-inflammatory cytokine in hemin group as compared to diabetic control rats. Expression of HO-1 also increased in hemin group as compared to diabetic control rats. However, SnPPIX group results were in disagreement with results of hemin which is clearly reflected in histopathology. Results indicate the ability of hemin to accelerate wound healing in diabetic rats by combating inflammation and oxidative stress probably via HO-1.

Acute hepatic porphyrias: Current diagnosis & management.

Each of the four acute hepatic porphyrias is due to mutation of an enzyme in the heme biosynthetic pathway. The accumulation of pathway intermediates that occur most notably when these diseases are active is the basis for screening and establishing a biochemical diagnosis of these rare disorders. Measurement of enzyme activities and especially DNA testing also are important for diagnosis. Suspicion of the diagnosis and specific testing, particularly measurement of urinary porphobilinogen, are often delayed because the symptoms are nonspecific, even when severe. Urinary porphyrins are also measured, but their elevation is much less specific. If porphobilinogen is elevated, second line testing will establish the type of acute porphyria. DNA testing identifies the familial mutation and enables screening of family members. Management includes removal of triggering factors whenever possible. Intravenous hemin is the most effective treatment for acute attacks. Carbohydrate loading is sometimes used for mild attacks. Cyclic attacks, if frequent, can be prevented by a GnRH analogue. Frequent noncyclic attacks are sometime preventable by scheduled (e.g. weekly) hemin infusions. Long term complications may include chronic pain, renal impairment and liver cancer. Other treatments, including RNA interference, are under development.

The interaction of Hemin and Sestrin2 modulates oxidative stress and colon tumor growth.

Several large epidemiological and animal studies demonstrate a direct correlation between dietary heme iron intake and/or systemic iron levels and cancer risk in several cancers including colorectal cancer (CRC). However, the precise mechanisms for how heme iron contributes to CRC and how cancer cells respond to heme iron-induced stress are still unclear. Previously we have shown that one of the stress-inducible proteins, Sestrin2 (SESN2), is a novel tumor suppressor in colon by limiting endoplasmic reticulum stress and mammalian target of rapamycin complex 1 (mTORC1) signaling and tumor growth. But the relationship between heme iron and SESN2, especially in the context of colon carcinogenesis, was not investigated previously. Here, we found that hemin dose-dependently increased SESN2 expression in an oxidative stress and nuclear factor (erythroid-derived 2)-like 2 (NFE2L2, NRF2)-dependent manner. Since SESN2 overexpression reduced hemin-induced oxidative stress, SESN2 could be an important target of NRF2 exerting antioxidant function. Indeed, expression of several oxidative stress responsive proteins such as NRF2 and its target genes was reduced by SESN2. Although we formerly reported that SESN2 expression was reduced after p53 mutation in colon tumors, mouse colon tumors, which have intact p53 and NRF2, induced SESN2 expression in response to iron stimulus. Although SESN2 overexpression decreased murine colon tumor cell growth both in vitro and in vivo, it rendered colon cancer cells more resistant to hemin-induced apoptosis and therefore promoted tumor growth during hemin treatment. Taken together, although SESN2 generally suppresses tumorigenesis, it can produce tumor-promoting role in iron-rich environment by suppressing oxidative stress-associated cancer cell death.

Oral DhHP-6 for the Treatment of Type 2 Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is associated with pancreatic ß-cell dysfunction which can be induced by oxidative stress. Deuterohemin-ßAla-His-Thr-Val-Glu-Lys (DhHP-6) is a microperoxidase mimetic that can scavenge reactive oxygen species (ROS) in vivo. In our previous studies, we demonstrated an increased stability of linear peptides upon their covalent attachment to porphyrins. In this study, we assessed the utility of DhHP-6 as an oral anti-diabetic drug in vitro and in vivo. DhHP-6 showed high resistance to proteolytic degradation in vitro and in vivo. The degraded DhHP-6 product in gastrointestinal (GI) fluid retained the enzymatic activity of DhHP-6, but displayed a higher permeability coefficient. DhHP-6 protected against the cell damage induced by H2O2 and promoted insulin secretion in INS-1 cells. In the T2DM model, DhHP-6 reduced blood glucose levels and facilitated the recovery of blood lipid disorders. DhHP-6 also mitigated both insulin resistance and glucose tolerance. Most importantly, DhHP-6 promoted the recovery of damaged pancreas islets. These findings suggest that DhHP-6 in physiological environments has high stability against enzymatic degradation and maintains enzymatic activity. As DhHP-6 lowered the fasting blood glucose levels of T2DM mice, it thus represents a promising candidate for oral administration and clinical therapy.

Heme oxygenase-1 prevents heart against myocardial infarction by attenuating ischemic injury-induced cardiomyocytes senescence.

BACKGROUND: Cellular senescence is a stable cell-cycle arrest induced by telomere shortening and various types of cellular stress including oxidative stress, oncogene activation, DNA damage etc. Heme oxygenase-1 (HO-1) is an inducible stress-response protein that plays antioxidant and anti-apoptotic effects. However, the role and underlying mechanisms of HO-1 in cellular senescence in heart are largely unknown. METHODS: Echocardiography was employed to detect the effect of HO-1 on heart function in adult mice with myocardial infarction (MI) and aged mice. The senescence markers, p53, p16 and LaminB, were analyzed by western blot. The immunofluorescence and immunohistochemical staining were applied to analyze the expression level of p16. SA-ß-Gal staining showed the level of cardiomyocyte senescence. FINDINGS: We found that hemin significantly induced the expression of HO-1, which notably suppressed cardiomyocyte senescence containing the secretion of senescence-associated secretory phenotype. Further studies showed that systemic HO-1 transgenic overexpression improved heart function by inhibiting aging-induced extracellular matrix deposition and fibrogenesis. More importantly, treatment of hemin improved heart function in MI mice. Furthermore, forced expression of HO-1 blunted cardiomyocyte senescence in natural aged mice and in primary cultured neonatal mouse cardiomyocytes. INTERPRETATION: Our study revealed that HO-1 improved heart function and attenuated cardiomyocyte senescence triggered by ischemic injury and aging. In addition, HO-1 induction alleviated H2O2-induced cardiomyocyte senescence. Finally, our study suggested a novel mechanism of HO-1 to play cardioprotective effect. FUND: This study was supported by the National Natural Science Foundation of China (81770284 to Hongli Shan); and the National Natural Science Foundation of China (81673425, 81872863 to Yuhong Zhou). The National Natural Science Foundation of China (81473213 to Chaoqian Xu). National Key R&D Program of China (2017YFC1307403 to Baofeng Yang), National Natural Science Foundation of China (81730012 to Baofeng Yang).

Recurrent attacks of acute hepatic porphyria: major role of the chronic inflammatory response in the liver.

BACKGROUND: Acute intermittent porphyria (AIP) is an inherited disorder of haem metabolism characterized by life-threatening acute neurovisceral attacks due to the induction of hepatic δ-aminolevulinic acid synthase 1 (ALAS1) associated with hydroxymethylbilane synthase (HMBS) deficiency. So far, the treatment of choice is hemin which represses ALAS1. The main issue in the medical care of AIP patients is the occurrence of debilitating recurrent attacks. OBJECTIVE: The aim of this study was to determine whether chronic hemin administration contributes to the recurrence of acute attacks. METHODS: A follow-up study was conducted between 1974 and 2015 and included 602 French AIP patients, of whom 46 had recurrent AIP. Moreover, we studied the hepatic transcriptome, serum proteome, liver macrophage polarization and oxidative and inflammatory profiles of Hmbs-/- mice chronically treated by hemin and extended the investigations to five explanted livers from recurrent AIP patients. RESULTS: The introduction of hemin into the pharmacopeia has coincided with a 4.4-fold increase in the prevalence of chronic patients. Moreover, we showed that both in animal model and in human liver, frequent hemin infusions generate a chronic inflammatory hepatic disease which induces HO1 remotely to hemin treatment and maintains a high ALAS1 level responsible for recurrence. CONCLUSION: Altogether, this study has important impacts on AIP care underlying that hemin needs to be restricted to severe neurovisceral crisis and suggests that alternative treatment targeting the liver such as ALAS1 and HO1 inhibitors, and anti-inflammatory therapies should be considered in patients with recurrent AIP.

An Unusual Case of Abdominal Pain and Hyponatremia in a 16-Year-Old Girl With Disordered Eating.

A previously healthy 16-year-old girl presented to the emergency department with 1 week of severe, diffuse abdominal pain and constipation, as well as several episodes of nonbloody, nonbilious emesis. Her symptoms began several days after she decreased her caloric intake in an attempt to lose weight. She had been drinking 48 to 60 oz of water per day for several days before admission in an attempt to ameliorate her constipation. She also admits to drinking alcohol the night before her pain began. She had visited several other emergency departments before her presentation to our hospital, and she had been sent home on a bowel regimen without amelioration of her symptoms. On arrival to our emergency department, she described severe diffuse abdominal pain. Her abdomen was tender to palpation throughout but soft with no rebound tenderness or peritoneal signs. The remainder of her physical examination yielded normal results. She was found to have hyponatremia with a sodium level of 122 and no neurologic sequelae. Abdominal radiograph showed moderate constipation but her abdominal pain continued even after bowel cleanout. The home, education, activities, drugs, sex, suicide, and safety assessment revealed several stressors, including a recent suicide in the family and a history of disordered eating and anxiety. Here, we present her case, diagnostic evaluation, ultimate diagnosis, and complications.

Enhancement of colon carcinogenesis by the combination of indole-3 carbinol and synbiotics in hemin-fed rats.

The risk of developing colorectal cancer (CRC) could be associated with red and processed meat intake. Experimental data supports that hemin iron, found abundantly in red meat, promotes CRC in mice and rats, while indole-3 carbinol (I3C) and synbiotics (syn) exert anti-carcinogenic activities in most studies of colon carcinogenesis. This study aimed to investigate the modifying effects of I3C and syn (inulin + Bifidobacterium lactis), given separately or together, on dimethylhidrazine (DMH)-induced colon carcinogenesis in hemin-fed rats. All animals were given four subcutaneous DMH injections and then, two weeks after carcinogen exposure, they began a basal diet containing hemin, hemin + I3C, hemin + syn, or hemin + I3C + syn for 23 weeks. The combination of I3C + syn significantly increased fecal water genotoxicity, tumor volume and invasiveness when compared to the hemin-fed control group. The groups fed I3C or syn alone had a significant reduction in the number of preneoplastic aberrant crypt foci (ACF) lesions compared to the hemin-fed group. Dietary I3C also reduced fecal water genotoxicity. Gene expression analysis of colorectal tumors demonstrated that the combination of dietary I3C + syn increased transcript levels for Raf1 and decreased tumor progression and invasiveness related to the genes Cdh1 and Appl1. This analysis also revealed that the Tnf and Cdh1 genes were significantly up- and down-regulated, respectively, in tumors of rats that received I3C, in comparison with the hemin-fed group. These findings reveal that the joint administration of I3C and syn enhanced the development of colon tumors induced by DMH in hemin-fed rats, while they potentially reduced ACF development when given alone.

In Vivo Hemin Conditioning Targets the Vascular and Immunologic Compartments and Restrains Prostate Tumor Development.

Purpose: Conditioning strategies constitute a relatively unexplored and exciting opportunity to shape tumor fate by targeting the tumor microenvironment. In this study, we assessed how hemin, a pharmacologic inducer of heme oxygenase-1 (HO-1), has an impact on prostate cancer development in an in vivo conditioning model.Experimental Design: The stroma of C57BL/6 mice was conditioned by subcutaneous administration of hemin prior to TRAMP-C1 tumor challenge. Complementary in vitro and in vivo assays were performed to evaluate hemin effect on both angiogenesis and the immune response. To gain clinical insight, we used prostate cancer patient-derived samples in our studies to assess the expression of HO-1 and other relevant genes.Results: Conditioning resulted in increased tumor latency and decreased initial growth rate. Histologic analysis of tumors grown in conditioned mice revealed impaired vascularization. Hemin-treated human umbilical vein endothelial cells (HUVEC) exhibited decreased tubulogenesis in vitro only in the presence of TRAMP-C1-conditioned media. Subcutaneous hemin conditioning hindered tumor-associated neovascularization in an in vivo Matrigel plug assay. In addition, hemin boosted CD8+ T-cell proliferation and degranulation in vitro and antigen-specific cytotoxicity in vivo A significant systemic increase in CD8+ T-cell frequency was observed in preconditioned tumor-bearing mice. Tumors from hemin-conditioned mice showed reduced expression of galectin-1 (Gal-1), key modulator of tumor angiogenesis and immunity, evidencing persistent remodeling of the microenvironment. We also found a subset of prostate cancer patient-derived xenografts and prostate cancer patient samples with mild HO-1 and low Gal-1 expression levels.Conclusions: These results highlight a novel function of a human-used drug as a means of boosting the antitumor response. Clin Cancer Res; 23(17); 5135-48. ©2017 AACR.

Plasma heme-induced renal toxicity is related to a capillary rarefaction.

Severe hypertension can lead to malignant hypertension (MH) with renal thrombotic microangiopathy and hemolysis. The role of plasma heme release in this setting is unknown. We aimed at evaluating the effect of a mild plasma heme increase by hemin administration in angiotensin II (AngII)-mediated hypertensive rats. Prevalence of MH and blood pressure values were similar in AngII and AngII + hemin groups. MH rats displayed a decreased renal blood flow (RBF), increased renal vascular resistances (RVR), and increased aorta and interlobar arteries remodeling with a severe renal microcirculation assessed by peritubular capillaries (PTC) rarefaction. Hemin-treated rats with or without AngII displayed also a decreased RBF and increased RVR explained only by PCT rarefaction. In AngII rats, RBF was similar to controls (with increased RVR). PTC density appeared strongly correlated to tubular damage score (rho = -0.65, p < 0.0001) and also renal Heme Oygenase-1 (HO-1) mRNA (rho = -0.67, p < 0.0001). HO-1 was expressed in PTC and renal tubules in MH rats, but only in PTC in other groups. In conclusion, though increased plasma heme does not play a role in triggering or aggravating MH, heme release appears as a relevant toxic mediator leading to renal impairment, primarily through PTC endothelial dysfunction rather than direct tubular toxicity.

Targeting and modulating infarct macrophages with hemin formulated in designed lipid-based particles improves cardiac remodeling and function.

Uncontrolled activation of pro-inflammatory macrophages after myocardial infarction (MI) accelerates adverse left ventricular (LV) remodeling and dysfunction. Hemin, an iron-containing porphyrin, activates heme oxygenase-1 (HO-1), an enzyme with anti-inflammatory and cytoprotective properties. We sought to determine the effects of hemin formulated in a macrophage-targeted lipid-based carrier (denoted HA-LP) on LV remodeling and function after MI. Hemin encapsulation efficiency was ~100% at therapeutic dose levels. In vitro, hemin/HA-LP abolished TNF-α secretion from macrophages, whereas the same doses of free hemin and drug free HA-LP had no effect. Hemin/HA-LP polarized peritoneal and splenic macrophages toward M2 anti-inflammatory phenotype. We next induced MI in mice and allocated them to IV treatment with hemin/HA-LP (10mg/kg), drug free HA-LP, free hemin (10mg/kg) or saline, one day after MI. Active in vivo targeting to infarct macrophages was confirmed with HA-LP doped with PE-rhodamine. LV remodeling and function were assessed by echocardiography before, 7, and 30days after treatment. Significantly, hemin/HA-LP effectively and specifically targets infarct macrophages, switches infarct macrophages toward M2 anti-inflammatory phenotype, improves angiogenesis, reduces scar expansion and improves infarct-related regional function. In conclusion, macrophage-targeted lipid-based drug carriers with hemin switch macrophages into an anti-inflammatory phenotype, and improve infarct healing and repair. Our approach presents a novel strategy to modulate inflammation and improve infarct repair.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation.

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1ß and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1ß secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1ß secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.