A Novel Therapeutic for the Treatment of Inflammatory Bowel Disease

biomedicines

Review Adrenomedullin: A Novel Therapeutic for the Treatment of Inﬂammatory Bowel Disease

Shinya Ashizuka 1,* , Toshihiro Kita 2 , Haruhiko Inatsu 1 and Kazuo Kitamura 2

1 Division of Gastroenterology and Hepatology, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Miyazaki 889-1692, Japan; [email protected] 2 Department of Projects Research, Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan; [email protected] (T.K.); [email protected] (K.K.) * Correspondence: [email protected]

Abstract: Adrenomedullin (AM) is a bioactive peptide with various physiological functions, including vasodilation, angiogenesis, anti-inflammation, organ protection, and tissue repair. AM suppresses inflammatory cytokine production in the intestinal mucosa, improves vascular and lymphatic regeneration and function, mucosal epithelial repair, and immune function in the intestinal bacteria of animal models with intestinal inflammation. We have been promoting translational research to develop novel therapeutic agents for inflammatory bowel disease (IBD) using AM and have started clinical research for IBD patients since 2010. A multicenter clinical trial is currently underway in Japan for patients with refractory ulcerative colitis and Crohn’s disease. Moreover, since current AM administration is limited to continuous intravenous infusion, the development of a subcutaneous

formulation using long-acting AM is underway for outpatient treatment.

Citation: Ashizuka, S.; Kita, T.; Keywords: adrenomedullin; inflammatory bowel disease; ulcerative colitis; Crohn’s disease; transla- Inatsu, H.; Kitamura, K. tional research; drug discovery research Adrenomedullin: A Novel Therapeutic for the Treatment of Inflammatory Bowel Disease. Biomedicines 2021, 9, 1068. https:// 1. Introduction doi.org/10.3390/biomedicines9081068 Ulcerative colitis (UC) and Crohn’s disease (CD) are the most common types of chronic inflammatory bowel disease (IBD). The number of people with IBD worldwide reached Academic Editor: Giovanni Pallio over 6.8 million in 2017 [1]. The cause of IBD is unknown but is thought to be caused by abnormalities in intestinal immunity, a genetic predisposition, and environmental factors, Received: 28 July 2021 such as diet and intestinal microflora. Accepted: 20 August 2021 Although there is no cure for IBD, there have been remarkable therapeutic develop- Published: 23 August 2021 ments that contribute to the induction and maintenance of remission over the past 20 years, including anti-TNF-alpha antibodies, tacrolimus, anti-IL-12/23p40 antibodies, Janus kinase Publisher’s Note: MDPI stays neutral (JAK) inhibitors, and integrin inhibitors. While these drugs have shown excellent efficacy, with regard to jurisdictional claims in published maps and institutional affil- they also negatively affect patients’ immune systems, leading some patients to develop iations. infections and malignant lymphomas. Severe infections such as tuberculosis and Pneumocystis pneumonia require serious attention, particularly in elderly patients and patients with underlying diseases, such as diabetes mellitus. Further, the above treatments demonstrate a diminishing therapeutic response, as they are immunogenic and targets of anti-drug antibodies. Therefore, the de- Copyright: © 2021 by the authors. velopment of new therapeutic agents for IBD requires efficacy, safety, and immunogenicity. Licensee MDPI, Basel, Switzerland. Adrenomedullin (AM), an endogenous vasodilatory peptide, was isolated from hu- This article is an open access article man pheochromocytoma by Kitamura et al. in 1993 [2]. AM was initially studied as a distributed under the terms and conditions of the Creative Commons circulatory agonist but was later found to promote angiogenesis, organ protection, and Attribution (CC BY) license (https:// anti-inflammatory immune activity. AM is also widely expressed in the gastrointestinal creativecommons.org/licenses/by/ tract epithelia and effectively treats gastric ulcers and enteritis in animal models. Therefore, 4.0/). we began translational research on the clinical application of AM in IBD [3].

Biomedicines 2021, 9, 1068. https://doi.org/10.3390/biomedicines9081068 https://www.mdpi.com/journal/biomedicines Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 11 Biomedicines 2021, 9, x FOR PEER REVIEW 2 of 11

Biomedicines 2021, 9, 1068 2 of 12 epithelia and effectively treats gastric ulcers and enteritis in animal models. Therefore, we epithelia and effectively treats gastric ulcers and enteritis in animal models. Therefore, we began translational research on the clinical application of AM in IBD [3]. began translational research on the clinical application of AM in IBD [3]. Since AM is an endogenous bioactive peptide, it has low immunogenicity, is consid- Since AM is an endogenous bioactive peptide, it has low immunogenicity, is considered relatively safe, and is expected to be developed into a novel IBD treatment. In this ered relativelyrelatively safe,safe, andand isis expectedexpected toto bebe developeddeveloped intointo aa novelnovel IBDIBD treatment.treatment. InIn thisthis article, we review the basics of AM research findings, including its effects on enteritis in article, we review thethe basicsbasics ofof AMAM researchresearch ﬁndings,findings, includingincluding itsits effectseffects onon enteritisenteritis inin animal models, and present the current status and potential of clinical research findings animal models,models, andand present present the the current current status status and and potential potential of clinicalof clinical research research ﬁndings findings for for IBD patients. IBDfor IBD patients. patients. 2. Structure and Biosynthesis of Adrenomedullin 2. Structure and Biosynthesis of Adrenomedullin AM is composed of 52 amino acids [2] and has a ring structure consisting of six amino AM is is composed composed of of 52 52 amino amino acids acids [2] [and2] and has hasa ring a structure ring structure consisting consisting of six amino of six acids and a C-terminal amide structure (Figure 1). These two structural features are essen- aminoacids and acids a C-terminal and a C-terminal amide structure amide structure (Figure 1). (Figure These1 ).two These structural two structural features are features essential for their biological activity. It also shares homology with calcitonin gene-related pep- aretial essentialfor their biological for their biologicalactivity. It activity.also shares Italso homology shares with homology calcitonin with gene-related calcitonin gene- pep- tide (CGRP), amylin, and adrenomedullin 2/intermedin (AM2/IMD) to form the calcitonin relatedtide (CGRP), peptide amylin, (CGRP), and amylin, adrenomedullin and adrenomedullin 2/intermedin 2/intermedin (AM2/IMD) to (AM2/IMD) form the calcitonin to form peptide superfamily. thepeptide calcitonin superfamily. peptide superfamily.

Figure 1. Amino-acid sequence of human Adrenomedullin. Figure 1.1. Amino-acid sequence of human Adrenomedullin. AM is widely expressed throughout the blood vessels, heart, lungs, kidneys, and gas- AM is widely expressed throughout the blood vessels, heart, lungs, kidneys, and gastrointestinal tract and is highly concentrated in the adrenal medulla. The pro-adrenome- trointestinaltrointestinal tracttract and and is is highly highly concentrated concentrated in thein the adrenal adrenal medulla. medulla. The The pro-adrenomedullin pro-adrenomedullin N-terminal 20 peptide (PAMP) has a shorter duration of antihypertensive activity N-terminaldullin N-terminal 20 peptide 20 peptide (PAMP) (PAMP) has a shorter has a duration shorter duration of antihypertensive of antihypertensive activity than activity AM than AM and cooperatively regulates blood circulation with AM (Figure 2) [4]. Although andthancooperatively AM and cooperatively regulates regulates blood circulation blood circulation with AM with (Figure AM2 )[(Figure4]. Although 2) [4]. Although mid re- mid regional pro-adrenomedullin (MR-pro ADM) has no biological activity, it has been gionalmid regional pro-adrenomedullin pro-adrenomedullin (MR-pro (MR-pro ADM) has ADM) no biological has no biological activity, it activity, has been it attracting has been attracting attention as a biomarker for the prognosis of heart failure [5], myocardial in- attentionattracting asattention a biomarker as a biomarker for the prognosis for the ofprognosis heart failure of heart [5], failure myocardial [5], myocardial infarction [in-5], farction [5], community-acquired pneumonia [6], septic shock [7], and COVID-19 [8,9]. community-acquiredfarction [5], community-acquired pneumonia [pneumonia6], septic shock [6], septic [7], and shock COVID-19 [7], and [8 COVID-19,9]. [8,9].

Figure 2. Schematic representations of the processingprocessing of AM, MR-proAM, andand PAMPPAMP fromfrom proAM. proAM.Figure 2. Schematic representations of the processing of AM, MR-proAM, and PAMP from 3.proAM. Adrenomedullin Receptors 3. AdrenomedullinThe AM receptor Receptors consists of a complex of calcitonin receptor-like receptors (CRLR; 3. Adrenomedullin Receptors a seven-transmembraneThe AM receptor consists G protein of a complex receptor) of andcalcitonin a receptor receptor-like activity-modifying receptors (CRLR; protein a The AM receptor consists of a complex of calcitonin receptor-like receptors (CRLR; a (RAMP;seven-transmembrane a single transmembrane G protein receptor). receptor) Thereand a are receptor three subtypes activity-modifying of RAMPs, RAMP1,protein seven-transmembrane G protein receptor) and a receptor activity-modifying protein (RAMP;RAMP2, aand single RAMP3, transmembrane and the CRLR/RAMP1receptor). Therecomplex are three hassubtypes a high of afﬁnity RAMPs, for RAMP1, CGRP, (RAMP; a single transmembrane receptor). There are three subtypes of RAMPs, RAMP1, whileRAMP2, the and CRLR/RAMP2 RAMP3, and andthe CRLR/RAMP1 CRLR/RAMP3 comp complexeslex has havea high high affinity afﬁnity for CGRP, for AM. while The RAMP2, and RAMP3, and the CRLR/RAMP1 complex has a high affinity for CGRP, while theCRLR/RAMP2 CRLR/RAMP2 complex and comprisesCRLR/RAMP3 theAM1 complexes receptor, have and thehigh CRLR/RAMP3 affinity for AM. complex The the CRLR/RAMP2 and CRLR/RAMP3 complexes have high affinity for AM. The CRLR/RAMP2constitutes the AM2complex receptor comprises (Figure the3). AM1 receptor, and the CRLR/RAMP3 complex CRLR/RAMP2 complex comprises the AM1 receptor, and the CRLR/RAMP3 complex constitutes the AM2 receptor (Figure 3). constitutes the AM2 receptor (Figure 3). Biomedicines 2021, 9, x FOR PEER REVIEW 3 of 11

AM contributes to the development and homeostasis of blood vessels and lymphatic vessels; AM knockout mice develop defective embryos due to defective blood and lymphatic vessel formation. Furthermore, studies using RAMP2 and RAMP3 gene knockout mice report that the AM1 receptor is involved in angiogenesis and vascular homeostasis, Biomedicines 2021, 9, 1068 3 of 12 while the AM2 receptor regulates lymph vessel function [10]. Thus, we speculate that AM influences vascular and lymphatic system ecological functions through two receptors.

Figure 3. CGRP and AM Receptors. Figure 3. CGRP and AM Receptors. AM contributes to the development and homeostasis of blood vessels and lymphatic vessels;4. General AM knockout Physiological mice develop Effects defective of Adrenomedullin embryos due to defective blood and lymphatic vessel formation. Furthermore, studies using RAMP2 and RAMP3 gene knockout mice reportAM thatcirculates the AM1 the receptor blood is involvedconstitutively in angiogenesis and is detected and vascular in homeostasis,healthy individuals; its ex- whilepression the AM2 is enhanced receptor regulates by mechanical lymph vessel stimuli, function such [10]. Thus,as: we speculate that AM influences• Myocardial vascular and and lymphatic vascular system wall ecological stretching functions through two receptors. 4.• General Organ Physiological ischemia Effects and hypoxia of Adrenomedullin • AMInflammatory circulates the bloodfactors, constitutively such as andinflammatory is detected in cytokines, healthy individuals; angiotensin its II, oxidative expressionstress, is enhanced and various by mechanical tissue stress. stimuli, such as: • MyocardialAM promotes and vascular various wall pathophysiological stretching effects, such as (Table 1) [11]: • Organ ischemia and hypoxia • • InflammatoryVasodilation factors, such as inflammatory cytokines, angiotensin II, oxidative stress, • andAngiogenesis various tissue stress. • AMCardioprotection promotes various pathophysiological effects, such as (Table1)[11]: • • VasodilationNephroprotection • • AngiogenesisAnti-oxidation • • Cardioprotection • Anti-apoptosis • Nephroprotection • Anti-oxidationTissue repair and regeneration • Anti-apoptosis • TableTissue 1. Diverse repair and physiological regeneration effects of adrenomedullin.

Table 1. Diverse physiological effects of adrenomedullin.Hemodynamic Improvement Vasodilation Hemodynamic Improvement Diuresis VasodilationDiuresisInotropic effectImprovement of intestinal blood flow Inotropic effect Organ protection Improvement of intestinal blood flow Suppression of oxidative stressInhibition of cardiac hypertrophy and fibrosisInhibition of vascular smooth muscle proliferationResistanceOrgan to ischemia protection in heart, kidney and brain Anti-inflammatorySuppression effect of oxidative stress Inhibition of pro-inflammatoryInhibition cytokinesAntibacterial of cardiac hypertrophy effectOrgan protection and fibrosis in SIRSImprovementInhibition of gastrointestinalof vascular mucosalsmooth injury muscle proliferation ResistanceTissue repair to and ischemia regeneration in heart, kidney and brain Vascular regeneration and stabilizationInhibition of apoptosisPromotes intestinal epithelial growth and remodelingRepairAnti-inflammatory of epithelial junction moleculeseffect Inhibition of pro-inflammatory cytokines Antibacterial effect Organ protection in SIRS Improvement of gastrointestinal mucosal injury Biomedicines 2021, 9, 1068 4 of 12

In a study of blood levels in patients with diseases, increases in blood (AM) were observed in cardiovascular diseases such as essential hypertension, heart failure, and renal failure [12]. Elevated blood (AM) is associated with the development of inﬂammatory diseases such as pancreatitis and sepsis [13] in patients with IBD [14], the severity of which increases in a concentration-dependent manner.

5. Anti-Inflammatory Effects of Adrenomedullin Blood (AM) is markedly elevated during severe inflammation, such as burns, pancreatitis, and systemic inflammatory response syndrome (SIRS) associated with sepsis [12]. In vitro studies using cultured vascular smooth muscle cells (VSMCs) and endothelial cells have shown that pro-inflammatory cytokines such as IL-1, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) stimulate AM expression in smooth muscle and endothelial cells [15]. In addition, the monocyte/macrophage cell line (RAW 264.7), murine celiac macrophages, and peripheral blood-derived monocytes have shown that monocyte- macrophage differentiation enhances AM production. Further, AM inhibits monocyte and macrophage TNF-α and IL-6 secretion following LPS-stimulation [16]. In an in vivo mouse study of sepsis treated with LPS and D-galactosamine, AM-overexpressing mice had demonstrated milder liver failure than wild-type mice, demonstrating that endogenous AM protects against SIRS [17]. Further, septic mice models treated with AM demonstrated improved hemodynamics and decreased pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [18] than control mice. Thus, AM ameliorates inflammatory conditions and is expected to be applied clinically to treat various inflammatory diseases.

6. Physiological Functions of Adrenomedullin in the Gastrointestinal Tract AM is widely expressed throughout the mucosal epithelium, glandular duct cells, neuroendocrine cells, and smooth muscle cells of the gastrointestinal tract, between the oral cavity and the large intestine [19]. The physiological effects of AM include suppressed gastric acid secretion via somatostatin in the stomach, enhanced electrolyte secretion in the colon, suppressed gastrointestinal motility, and changes to microcirculation ﬂux. Addition- ally, AM has similar physiological and antibacterial effects as defensins and may contribute to the mucosal defense system by regulating the oral and intestinal microbiome [20].

7. Pathophysiological Function of Adrenomedullin in Inﬂammatory Diseases of the Gastrointestinal Tract The pathophysiological function of AM in gastrointestinal diseases has been reported in many studies of stomach, small and large intestine mucosal injury.

7.1. Effect of AM on Gastric Mucosal Injury AM is strongly expressed in the tissues surrounding ulcers in patients with gastric ulcers, and the expression of AM tends to increase with ulcer healing, suggesting that AM may be involved in mucosal regeneration [21]. Animal models of gastric mucosal injury have demonstrated that AM ameliorates mucosal injury. It has been reported that AM inhibits gastric acid secretion [22], maintains mucosal blood ﬂow by inhibiting gastric artery contraction [23], and promotes proliferation [24] and restructuring of mucosal epithelial cells [25]. However, speciﬁc mechanisms are under active investigation.

7.2. Effect of AM on Enteritis Since AM inhibits systemic inﬂammation and protects the gastric mucosa, there have been many studies on the effects of AM on animal models of intestinal inﬂammation. We administered AM enterically to acetic acid-induced colonic ulcer model rats. We found that AM dose-dependently ameliorated colitis [26], which was in mice with dextran sulfate sodium (DSS)-induced colitis, which is a general model for IBD research [27]. Several research groups in Europe and Japan have reported that AM ameliorates trini- trobenzene sulfonic acid (TNBS) and DSS-induced colitis in various animal models [28,29]. Biomedicines 2021, 9, x FOR PEER REVIEW 5 of 11

Since AM inhibits systemic inflammation and protects the gastric mucosa, there have been many studies on the effects of AM on animal models of intestinal inflammation. We administered AM enterically to acetic acid-induced colonic ulcer model rats. We found that AM dose-dependently ameliorated colitis [26], which was in mice with dextran sulfate sodium (DSS)-induced colitis, which is a general model for IBD research [27]. Biomedicines 2021, 9, 1068 Several research groups in Europe and Japan have reported that AM ameliorates5 of 12 tri- nitrobenzene sulfonic acid (TNBS) and DSS-induced colitis in various animal models [28,29]. Further, TNBS enteritis is more severe in AM knockout mice than in control mice [30]. Further,Administering TNBS enteritis AM antagonists is more severe to in indomethacin-induced AM knockout mice than in small control intestinal mice [30]. ulcer rat modelsAdministering increased AMthe antagonistsseverity of to enteritis indomethacin-induced [31], suggesting small intestinalthat endogenous, ulcer rat models physiological increased the severity of enteritis [31], suggesting that endogenous, physiological AM may AM mayalso relievealso relieve enteritis. enteritis. AM AMis reported is reported to to ameliorate ameliorate intestinalintestinal inﬂammation inflammation by regulating by regulating mucosal mucosal immune immune responses,responses, enhancing enhancing vascular, vascular, lymphatic, lymphatic, regenerative, regenerative, barrier, andbarrier, antimicrobial and antimicrobial functions functions (Figure(Figure4)[ 4)32 ].[32].

FigureFigure 4. Pathophysiology 4. Pathophysiology of of IBD IBD and and therapeutictherapeutic targets targets of AM of therapy.AM therapy.

7.2.1.7.2.1. Effects Effects on onIntestinal Intestinal Immunity Immunity AnimalAnimal enteritis enteritis models models havehave shown shown that that AM administrationAM administration decreases thedecreases production the produc- of the pro-inflammatory cytokines; TNF-α, IL-6, and IFN-γ, and increases the production tion ofof thethe immunosuppressive pro-inflammatory cytokines; cytokines; IL-10 andTNF- TGF-α,β IL-6,in intestinal and IFN- mucosaγ, and and mesentericincreases the pro- ductionlymph of nodethe immunosuppressive mononuclear cells [27–31 cytokines;]. IL-10 and TGF-β in intestinal mucosa and mesentericWe lymph found that node AM mononuclear inhibited STAT3 cells and STAT1[27–31]. phosphorylation in colonic epithelial Wecells found in mice that with AM DSS-induced inhibited colitis. STAT3 Kinoshita and STAT et al. recently1 phosphorylation showed that AM in colonic inhibits epithelial κ cells NFin miceBp65, with and STAT3 DSS-induced phosphorylation, colitis. suggesting Kinoshita AM et regulates al. recently cytokine showed activity that via theAM inhibits STAT3-NFκB pathway [33]. In addition, MacManus et al. reported that AM exerts intestinal NFκBp65,epithelial and anti-inflammatory STAT3 phosphorylation, effects by fine-tuning suggesting and stabilizing AM regulates hypoxia-inducible cytokine activity factor via the STAT3-NF(HIF) activityκB pathway in Caco2 [33]. cells In [34 ].addition, MacManus et al. reported that AM exerts intestinal epithelial anti-inflammatory effects by fine-tuning and stabilizing hypoxia-inducible factor (HIF) activity in Caco2 cells [34].

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7.2.2. Actions on the Intestinal Vasculature AM contributes to the development and homeostasis of blood vessels and lymphatic vessels. Talero et al. reported that AM influences intestinal vascular function by regulating the activity of COX2 and iNOS-derived vasoactive substances in the small mesenteric arteries of TNBS enteritis rats [29]. Recently, Davis et al. generated a mouse model with a lymphatic-specific knockout of the CRLR gene (CALCRL) of the AM receptor and found that the mice developed intestinal lymphangiectasia. Further, administration of indomethacin prolonged intestinal inflammation [35], suggesting that AM may ameliorate intestinal inflammation through its effects on lymphatic function.

7.2.3. Repair of Intestinal Epithelium and Restoration of Barrier Function We reported that AM treatment preserved tight and adherence junctions [27], increased Klf4 expression [33], ameliorated goblet cell loss, and preserved intestinal tissue integrity in a mouse DSS enteritis model. Hayashi et al. reported that AM promoted the regeneration of injured epithelial cells via cell proliferation and wound healing assays using Caco- 2 cells [36]. In addition, Yi et al. showed that AM restores intestinal epithelial barrier dysfunction by regulating myosin light chain phosphorylation [37], which is a major agonist of intestinal barrier dysfunction [38].

7.2.4. Antimicrobial Effects of AM Abnormalities in the function of the entire bacterial flora associated with dysbiosis in the intestinal microbiota have been reported in IBD patients [39]. It has been reported that Th17 cells are induced when there is an increased representation of Proteobacteria, including adherent/invasive E. coli (AIEC), in the gut microflora. Th17 cell hyperactivity is involved in the pathogenesis of IBD [40,41], which may be compounded by an accumulation of regulatory T cells with functional abnormalities, due to a decrease in butyric acid producing bacteria [42]. Marutsuka et al. reported that AM was distributed throughout the colonic mucosal surface layer and demonstrated dose-dependent antibacterial activity against E. coli [43]. Furthermore, AM administration to DSS colitis mice significantly reduced the number of anaerobic bacteria in the colon than control mice [3]. These results suggest that the antimicrobial activity of AM may mechanistically contribute to the improvement of colitis. AM treatment can restore function to dysfunctional epithelial cells and treat intestinal dysbiosis. The relationship between the antimicrobial activity of AM and the improvement of colitis requires further investigation.

8. Translational Research with Adrenomedullin Since its discovery, AM has been studied as a cardiovascular agonist. Clinical trials for AM therapeutics have been initiated in Japan for patients with acute myocardial infarction, pulmonary hypertension, and peripheral arterial occlusive diseases [44]. Based on the ﬁndings of these studies, we initiated clinical trials for the development of a therapeutic drug for IBD in 2010. We conducted a preliminary single-center clinical trial of patients with refractory IBD. Intravenous infusion of AM 9 ng/kg/min, 8 h/day, for 14 days to patients with steroid-refractory UC resulted in 71% (n = 5/7) remission at 12 weeks. We observed a decrease in serum IL-6 and TNF-α 8 h after AM administration (Figure5, case A–C) [ 45]. In patients with refractory Crohn’s disease with secondary failure of inﬂiximab, continuous intravenous infusion of AM resulted in marked improvement in clinical symptoms and mucosal healing of longitudinal ulcers of the colon, as well as a re-elevation of IFX levels in the blood (Figure5, case D) [46]. BiomedicinesBiomedicines 20212021, 9, 9, ,x 1068 FOR PEER REVIEW 7 of 12 7 of 11

Figure 5. 5.Improvement Improvement of colonoscopicof colonoscopic ﬁndings findin ings adrenomedullin in adrenomedullin therapy. therapy.

WideWide and and deep deep ulcers ulcers were were observed observed in the in colon the colon before before the AM the therapy. AM therapy. Significant Significant mucosal regeneration regeneration and and reddening reddening with with neovascularization neovascularization were observedwere observed after AM after AM therapytherapy (middle(middle figures). figures). Eight Eight to to 12 12 weeks weeks after after treatment treatment with with AM, AM, the the ulcers ulcers had had dis- disappeared, and ulcer scars were observed (each right figure) (modified and redrawn appeared, and ulcer scars were observed (each right figure) (modified and redrawn from from [32,45,46]). UC, ulcerative colitis; CD, Crohn’s disease. [32,45,46]).We positioned UC, ulcerative these preliminary colitis; CD, study Crohn’s results asdisease. proof of concept, developed a GMP- compliantWe positioned investigational these new preliminary drug in 2015, study and resu beganlts investigator-initiated as proof of concept, clinical developed trials a GMP- incompliant 2016. In investigational a phase I clinical new trial drug in healthy in 2015, adults, and began we performed investigator-initiated a single, 12-h clinical 3, 9, trials andin 2016. 15 ng/kg/mL In a phase AM I clinical dose study trial (24in healthy patients) adults, and a seven-daywe performed repeated-dose a single, study12-h 3, 9, and (1215 ng/kg/mL patients) with AM a dose placebo study as the(24 controlpatients) condition. and a seven-day The plasma repeated-dose AM concentration study (12 pa- increasedtients) with in a a dose-dependent placebo as the manner,control conditio reached Cn.max Theat theplasma end ofAM treatment, concentration and rapidly increased in returned to baseline after AM administration, with a T1/2 < 60 min [47]. We confirmed a dose-dependent manner, reached Cmax at the end of treatment, and rapidly returned to thatbaseline continuous after AM intravenous administration, AM infusion with was a T1/2 safe < and 60 min tolerable [47]. forWe patients confirmed in this that trial. continuous We conducted two multicenter RCTs in Japan: intravenous AM infusion was safe and tolerable for patients in this trial. 1. “Effects of adrenomedullin in steroid-resistant patients with ulcerative colitis: ran- We conducted two multicenter RCTs in Japan: domized, double-blind, placebo-controlled phase 2a clinical trial” and 2.1. “Effects“Effects of of adrenomedullin adrenomedullin in biologics-resistant in steroid-resistant patients patients with with Crohn’s ulcerative disease: ran-colitis: randomized,domized, double-blind, double-blind, placebo-controlled placebo-controlled phase phase 2a clinical 2a clinical trial”. trial” and 2. In“Effects the phase of 2adrenomedullin study in UC patients, in biologics-re 28 patients withsistant steroid-resistant patients with ulcerative Crohn’s colitis disease: ran- were divideddomized, into double-blind, AM15, 10, and placebo-controlled 5 ng/kg/min and placebo. phase 2a There clinical were trial”. no differences in the clinicalIn the remission phase 2 study rates at in week UC patients, two, or the 28 primary patients endpoint with steroid-resistant among the four groups.ulcerative colitis were divided into AM15, 10, and 5 ng/kg/min and placebo. There were no differences in the clinical remission rates at week two, or the primary endpoint among the four groups. However, the Mayo score at week eight was significantly lower in the high-dose AM group (15 ng/kg/min) than in the placebo group (−9.3 ± 1.2 vs. −3.0 ± 2.8, p = 0.035). The clinical remission rate at week eight was significantly higher than that of the placebo Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 11

Biomedicines 2021, 9, 1068 8 of 12

(3/3, 100% vs. 0/2, 0%, p = 0.025), and 50% of patients in the high AM dose group achieved

mucosalHowever, healing, the Mayo Mayo score atendoscop week eightic was subscore significantly = 0. lowerThe adverse in the high-dose events AM caused group by the vasodilatory(15 ng/kg/min) effect of than AM in thewere placebo mild, group and (− 9.3none± 1.2 were vs. − 3.0serious.± 2.8, p This= 0.035). study The clinicaldemonstrated the efficacyremission and rate safety at week of eightintravenous was significantly AM therapy higher thanin patients that of the with placebo steroid-resistant (3/3, 100% UC [48]. vs.The 0/2, 0%,clinicalp = 0.025), trial andresults 50% offor patients Crohn’s in the disease high AM patients dose group are achieved currently mucosal not available for publication,healing, Mayo and endoscopic the detai subscorels have =been 0. The withheld. adverse events caused by the vasodilatory effect of AM were mild, and none were serious. This study demonstrated the efficacy and safetyAM of therapies intravenous are AM expected therapy in to patients be developed with steroid-resistant into new UC IBD [48 ].treatments, as its mechanism ofThe action clinical is trialnot resultsdemonstrated for Crohn’s by disease existing patients drugs, are currentlyand it has not a availablehigh safety for profile. On thepublication, other hand, and due the details to its haveshort been half-life withheld. of <60 min, prolonged intravenous administration was requiredAM therapies in the are aforementioned expected to be developed clinical into trials. new IBD treatments, as its mechanism of action is not demonstrated by existing drugs, and it has a high safety profile. On the otherAlthough hand, due continuous to its short half-life intravenous of <60 min, infusion prolonged is acceptable intravenous administrationfor remission was induction ther- apyrequired for patients in the aforementioned requiring hospitalization, clinical trials. it is not convenient for outpatient treatment or remissionAlthough maintenance continuous intravenoustherapy. Therefore, infusion is acceptable we prepared for remission polyethylene induction ther-glycol-modified (PEGylated)apy for patients AM requiring for subcutaneous hospitalization, injection it is not in convenient outpatient for outpatientclinics. We treatment succeeded or in extend- ingremission the half-life maintenance of AM therapy. significantly. Therefore, Further, we prepared the polyethylenerapid decrease glycol-modified in blood (PE-pressure, which Gylated) AM for subcutaneous injection in outpatient clinics. We succeeded in extending is thea side half-life effect of AMof native significantly. AM administra Further, the rapidtion, decreasewas suppressed, in blood pressure, enabling which the is adevelopment of sidea hemodynamically effect of native AM administration,safe treatment was [49]. suppressed, enabling the development of a hemodynamicallyFurthermore, safe Nagata treatment et al. [49 created]. PEGylated AM with a larger molecular weight of 60 kDaFurthermore, and a longer Nagata half-life. et al. createdThey confirmed PEGylated AM that with 60 akDa larger PEGylated molecular weight AM has of the physio- 60 kDa and a longer half-life. They confirmed that 60 kDa PEGylated AM has the physiological logicaland therapeutic and therapeutic functions functions of native AM of innative animal AM enteritis in animal models enteritis (Figures6 andmodels7) [ 50 (Figures]. 6 and 7) Soon,[50]. Soon, we plan we to plan initiate to a initiate phase I studya phase using I study 60 kDa using PEGylated 60 kDa AM. PEGylated In addition, AM. we In addition, wehave have successfully successfully developed developed IgG Fc regionIgG Fc fusion region AM fusion [51], and AM human [51], albumin-modified and human albumin-mod- ifiedAM AM [52] and[52] are and currently are currently conducting conducting preclinical studiespreclinical using studies these modified using AMs.these modified AMs.

FigureFigure 6. 6. PEGylatedPEGylated humanhuman adrenomedullin adrenomedullin (PEG-hAM) (PEG-hAM) has a prolonged has a prolonged half-life in thehalf-life blood. in the blood. PlasmaPlasma disappearance disappearance ofof 5 5 kDa kDa PEG-hAM PEG-hAM (open (open circles) circles) and 60and kDa 60 PEG-hAM kDa PEG-hAM (closed circles)(closed circles) over time.over Five time. kDa Five PEG-AM kDa PEG-AM (30 nmol/ (30 nmol/kg)kg) and and 60 60kDa kDa PEG-AM PEG-AM (10 (10 nmol/kg) werewere injected injected subcutane- ouslysubcutaneously into anesthetized into anesthetized rats, and rats, blood and blood samples samples we werere taken taken at at the the indicatedindicated time time points points (modified and(modiﬁed redrawn and from redrawn [50]). from [50]).

Figure 7. Anti-Inflammatory Effects of 60 kDa PEG-hAM on DSS-Induced Colitis. A comparison of the total inflammation score in the DSS-induced colitis model after treatment with 60 kDa PEG- hAM (n = 10). In mice with DSS-induced colitis, treatment with 60 kDa PEG-hAM reduced bloody Biomedicines 2021, 9, x FOR PEER REVIEW 8 of 11

(3/3, 100% vs. 0/2, 0%, p = 0.025), and 50% of patients in the high AM dose group achieved mucosal healing, Mayo endoscopic subscore = 0. The adverse events caused by the vasodilatory effect of AM were mild, and none were serious. This study demonstrated the efficacy and safety of intravenous AM therapy in patients with steroid-resistant UC [48]. The clinical trial results for Crohn’s disease patients are currently not available for publication, and the details have been withheld. AM therapies are expected to be developed into new IBD treatments, as its mechanism of action is not demonstrated by existing drugs, and it has a high safety profile. On the other hand, due to its short half-life of <60 min, prolonged intravenous administration was required in the aforementioned clinical trials. Although continuous intravenous infusion is acceptable for remission induction therapy for patients requiring hospitalization, it is not convenient for outpatient treatment or remission maintenance therapy. Therefore, we prepared polyethylene glycol-modified (PEGylated) AM for subcutaneous injection in outpatient clinics. We succeeded in extending the half-life of AM significantly. Further, the rapid decrease in blood pressure, which is a side effect of native AM administration, was suppressed, enabling the development of a hemodynamically safe treatment [49]. Furthermore, Nagata et al. created PEGylated AM with a larger molecular weight of 60 kDa and a longer half-life. They confirmed that 60 kDa PEGylated AM has the physiological and therapeutic functions of native AM in animal enteritis models (Figures 6 and 7) [50]. Soon, we plan to initiate a phase I study using 60 kDa PEGylated AM. In addition, we have successfully developed IgG Fc region fusion AM [51], and human albumin-modified AM [52] and are currently conducting preclinical studies using these modified AMs.

Figure 6. PEGylated human adrenomedullin (PEG-hAM) has a prolonged half-life in the blood. Plasma disappearance of 5 kDa PEG-hAM (open circles) and 60 kDa PEG-hAM (closed circles) over time. Five kDa PEG-AM (30 nmol/kg) and 60 kDa PEG-AM (10 nmol/kg) were injected subcutane- Biomedicines 2021, 9, 1068 ously into anesthetized rats, and blood samples were taken at the indicated time9 of 12points (modified and redrawn from [50]).

FigureFigure 7.7.Anti-Inflammatory Anti-Inflammatory Effects Effects of 60 kDa of 60 PEG-hAM kDa PEG-hAM on DSS-Induced on DSS-Induced Colitis. A comparison Colitis. of A comparison of thethe total inflammation inflammation score score in the DSS-inducedin the DSS-induce colitis modeld colitis after treatment model withafter 60 treatment kDa PEG-hAM with 60 kDa PEG- hAM(n = 10). (n In= 10). mice In with mice DSS-induced with DSS-induced colitis, treatment colitis, with treatment 60 kDa PEG-hAM with 60 reduced kDa PEG-hAM bloody stool reduced bloody and body weight reduction scores. The total inflammation scores were lower in the group treated with 60 kDa PEG-hAM than in controls. Results are shown as the mean ± SE. * p < 0.05, ** p < 0.01 versus control and †† p < 0.01 versus 1.0 nmol/kg 60 kDa PEG-hAM (modified and drawn from [50]).

9. Future Perspectives on the Development of Adrenomedullin Therapies for IBD As described above, the safety and efficacy of AM therapy was confirmed in a relatively small number of UC patients. We are now considering the following strategies for future development. (1) Phase 3 trials should be conducted to evaluate the safety and efficacy of AM therapy in patients with IBD refractory to steroids and biologics. (2) The efficacy and safety of remission maintenance by the intermittent administration of native AM and PEGylated AM in patients who have achieved remission with AM therapy should be evaluated (Phase 1–2). (3) The safety of long-term repeated administration of AM should be evaluated.

10. Conclusions In this review, we reviewed the biosynthesis, receptor characteristics, and physiopatho- logical effects of AM and reported on the current status of drug discovery research for novel IBD therapies. In particular, results of clinical research on AM therapy have demonstrated that a signiﬁcant efﬁcacy can be achieved at relatively low doses, where the side effect of hypotension caused by AM is not a clinical problem. Although further validation of effective doses, dosages, and administration methods of AM therapy for IBD patients is needed, AM is considered to be a promising drug for IBD, acting through a novel mechanism.

Author Contributions: Conceptualization, K.K.; original draft preparation, S.A.; writing—review and editing, T.K., H.I. and K.K. All authors have read and agreed to the published version of the manuscript. Funding: The author’s clinical trials on the effect of AM for IBD have most recently been supported by AMED grants (JP17ek0109089 and JP19Im0203058). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Informed consent was obtained from all subjects involved in the study. Data Availability Statement: All the data provided in the review article. Conﬂicts of Interest: The authors declare that S. Ashizuka, T. Kita and K. Kitamura have stock in Himuka AM Pharma Corporation. Biomedicines 2021, 9, 1068 10 of 12

References 1. GBD 2017 Inflammatory Bowel Disease Collaborators. The Global, Regional, and National Burden of Inflammatory Bowel Disease in 195 Countries and Territories, 1990–2017: A Systematic Analysis for the Global Burden of Disease Study 2017. Lancet Gastroenterol. Hepatol. 2020, 5, 17–30. [CrossRef] 2. Kitamura, K.; Kangawa, K.; Kawamoto, M.; Ichiki, Y.; Nakamura, S.; Matsuo, H.; Eto, T. Adrenomedullin: A novel hypotensive peptide isolated from human pheochromocytoma. Biochem. Biophys. Res. Commun. 1993, 192, 553–560. [CrossRef][PubMed] 3. Ashizuka, S.; Inatsu, H.; Inagaki-Ohara, K.; Kita, T.; Kitamura, K. Adrenomedullin as a potential therapeutic agent for inflammatory bowel disease. Curr. Protein Pept. Sci. 2013, 14, 246–255. [CrossRef][PubMed] 4. Tsuruda, T.; Kato, J.; Kuwasako, K.; Kitamura, K. Adrenomedullin: Continuing to Explore Cardioprotection. Peptides 2019, 111, 47–54. [CrossRef] 5. Self, W.H.; Storrow, A.B.; Hartmann, O.; Barrett, T.W.; Fermann, G.J.; Maisel, A.S.; Struck, J.; Bergmann, A.; Collins, S.P. Plasma Bioactive Adrenomedullin as a Prognostic Biomarker in Acute Heart Failure. Am. J. Emerg. Med. 2016, 34, 257–262. [CrossRef] [PubMed] 6. Liu, D.; Xie, L.; Zhao, H.; Liu, X.; Cao, J. Prognostic Value of Mid-Regional Pro-Adrenomedullin (MR-ProADM) in Patients with Community-Acquired Pneumonia: A Systematic Review and Meta-Analysis. BMC Infect. Dis. 2016, 16, 232. [CrossRef] 7. Elke, G.; Bloos, F.; Wilson, D.C.; Brunkhorst, F.M.; Briegel, J.; Reinhart, K.; Loeffler, M.; Kluge, S.; Nierhaus, A.; Jaschinski, U.; et al. The Use of Mid-Regional Proadrenomedullin to Identify Disease Severity and Treatment Response to Sepsis-a Secondary Analysis of a Large Randomised Controlled Trial. Crit. Care 2018, 22, 79. [CrossRef][PubMed] 8. Montrucchio, G.; Sales, G.; Rumbolo, F.; Palmesino, F.; Fanelli, V.; Urbino, R.; Filippini, C.; Mengozzi, G.; Brazzi, L. Effectiveness of Mid-Regional Pro-Adrenomedullin (MR-ProADM) as Prognostic Marker in COVID-19 Critically Ill Patients: An Observational Prospective Study. PLoS ONE 2021, 16, e0246771. [CrossRef] 9. Sozio, E.; Tascini, C.; Fabris, M.; D’Aurizio, F.; De Carlo, C.; Graziano, E.; Bassi, F.; Sbrana, F.; Ripoli, A.; Pagotto, A.; et al. MR-ProADM as Prognostic Factor of Outcome in COVID-19 Patients. Sci. Rep. 2021, 11, 5121. [CrossRef] 10. Yamauchi, A.; Sakurai, T.; Kamiyoshi, A.; Ichikawa-Shindo, Y.; Kawate, H.; Igarashi, K.; Toriyama, Y.; Tanaka, M.; Liu, T.; Xian, X.; et al. Functional Differentiation of RAMP2 and RAMP3 in Their Regulation of the Vascular System. J. Mol. Cell. Cardiol. 2014, 77, 73–85. [CrossRef][PubMed] 11. Kitamura, K. Developmental Research of Adrenomedullin. Adrenomedullin No Tenkaikenkyu. Shinzo 2013, 45, 1496–1502. (In Japanese) 12. Eto, T.; Kitamura, K. Adrenomedullin and Its Role in Renal Diseases. Nephron 2001, 89, 121–134. [CrossRef] 13. Ueda, S.; Nishio, K.; Minamino, N.; Kubo, A.; Akai, Y.; Kangawa, K.; Matsuo, H.; Fujimura, Y.; Yoshioka, A.; Masui, K.; et al. Increased Plasma Levels of Adrenomedullin in Patients with Systemic Inflammatory Response Syndrome. Am. J. Respir. Crit. Care Med. 1999, 160, 132–136. [CrossRef][PubMed] 14. Ashizuka, S.; Kawakami, H.; Kitamura, K. Plasma Adrenomedullin Levels as a Novel Biomarker in Inflammatory Bowel Disease. Enshouseichousikkan Ni Okeru Shinki Biomarker to Shiteno Kecchu Adrenomedullin Noudo No Kentou. J. Jpn. Soc. Gastroenterol. 2019, 116, A688. (In Japanese) 15. Minamino, N.; Kikumoto, K.; Isumi, Y. Regulation of Adrenomedullin Expression and Release. Microsc. Res. Tech. 2002, 57, 28–39. [CrossRef] 16. Kubo, A.; Minamino, N.; Isumi, Y.; Katafuchi, T.; Kangawa, K.; Dohi, K.; Matsuo, H. Production of adrenomedullin in macrophage cell line and peritoneal macrophage. J. Biol. Chem. 1998, 273, 16730–16738. [CrossRef] 17. Shindo, T.; Kurihara, H.; Maemura, K.; Kurihara, Y.; Kuwaki, T.; Izumida, T.; Minamino, N.; Ju, K.H.; Morita, H.; Oh-Hashi, Y.; et al. Hypotension and Resistance to Lipopolysaccharide-Induced Shock in Transgenic Mice Overexpressing Adrenomedullin in Their Vasculature. Circulation 2000, 101, 2309–2316. [CrossRef] 18. Yang, S.; Zhou, M.; Fowler, D.E.; Wang, P. Mechanisms of the Beneficial Effect of Adrenomedullin and Adrenomedullin-Binding protein-1 in Sepsis: Down-Regulation of Proinflammatory Cytokines. Crit. Care Med. 2002, 30, 2729–2735. [CrossRef] 19. Marutsuka, K.; Hatakeyama, K.; Sato, Y.; Yamashita, A.; Sumiyoshi, A.; Asada, Y. Immunohistological Localization and Possible Functions of Adrenomedullin. Hypertens. Res. 2003, 26, S33–S40. [CrossRef] 20. Martínez-Herrero, S.; Martínez, A. Adrenomedullin Regulates Intestinal Physiology and Pathophysiology. Domest. Anim. Endocrinol. 2016, 56, S66–S83. [CrossRef] 21. Hashimoto, H.; Akimoto, M.; Maeda, A.; Shigemoto, M.; Yamashita, K.; Yokoyama, I. Changes in Vasoactive Substances During Gastric Ulcer Healing. J. Cardiovasc. Pharmacol. 2000, 36 (Suppl. 1), S278–S281. [CrossRef][PubMed] 22. Clementi, G.; Caruso, A.; Cutuli, V.M.; Mangano, N.G.; Salomone, S.; Lempereur, L.; Prato, A.; Matera, M.; Amico-Roxas, M. Gastroprotective Effect of Adrenomedullin Administered Subcutaneously in the Rat. Peptides 2002, 23, 1149–1153. [CrossRef] 23. Salomone, S.; Caruso, A.; Cutuli, V.M.; Mangano, N.G.; Prato, A.; Amico-Roxas, M.; Bianchi, A.; Clementi, G. Effects of Adrenomedullin on the Contraction of Gastric Arteries During Reserpine-Induced Gastric Ulcer. Peptides 2003, 24, 117–122. [CrossRef] 24. Wang, H.; Tomikawa, M.; Jones, M.K.; Pai, R.; Sarfeh, I.J.; Tarnawski, A.S. Sequential Expression of Adrenomedullin and Its Receptor During Gastric Ulcer Healing in Rats. Dig. Dis. Sci. 2000, 45, 591–598. [CrossRef][PubMed] 25. Fukuda, K.; Tsukada, H.; Oya, M.; Onomura, M.; Kodama, M.; Nakamura, H.; Hosokawa, M.; Seino, Y. Adrenomedullin Promotes Epithelial Restitution of Rat and Human Gastric Mucosa In Vitro. Peptides 1999, 20, 127–132. [CrossRef] Biomedicines 2021, 9, 1068 11 of 12

26. Ashizuka, S.; Ishikawa, N.; Kato, J.; Yamaga, J.; Inatsu, H.; Eto, T.; Kitamura, K. Effect of Adrenomedullin Administration on Acetic Acid-Induced Colitis in Rats. Peptides 2005, 26, 2610–2615. [CrossRef] 27. Ashizuka, S.; Inagaki-Ohara, K.; Kuwasako, K.; Kato, J.; Inatsu, H.; Kitamura, K. Adrenomedullin Treatment Reduces Intestinal Inflammation and Maintains Epithelial Barrier Function in Mice Administered Dextran Sulphate Sodium. Microbiol. Immunol. 2009, 53, 573–581. [CrossRef] 28. Gonzalez-Rey, E.; Fernandez-Martin, A.; Chorny, A.; Delgado, M. Therapeutic Effect of Urocortin and Adrenomedullin in a Murine Model of Crohn’s Disease. Gut 2006, 55, 824–832. [CrossRef] 29. Talero, E.; Sánchez-Fidalgo, S.; de la Lastra, C.A.; Illanes, M.; Calvo, J.R.; Motilva, V. Acute and Chronic Responses Associated with Adrenomedullin Administration in Experimental Colitis. Peptides 2008, 29, 2001–2012. [CrossRef] 30. Martínez-Herrero, S.; Larrayoz, I.M.; Narro-Íñiguez, J.; Rubio-Mediavilla, S.; Martínez, A. Lack of Adrenomedullin Aggravates Acute TNBS-Induced Colitis Symptoms in Mice, Especially in Females. Front. Physiol. 2017, 8, 1058. [CrossRef] 31. Kani, K. A Review of the Mechanism of Action of Adrenomedullin in the Process of Indomethacin-Induced Small Intestinal Ulcera- tion. Indometacin kiinsei shouchoukaiyou keiseikatei ni okeru adrenomedullin no sayoukijo ni tsuiteno kentou. Saitamaikadaigaku zasshi (J. Saitama Med. Univ.) 2013, 40, T1–T11. (In Japanese) 32. Ashizuka, S.; Inatsu, H.; Kita, T.; Kitamura, K. Adrenomedullin Therapy in Patients with Inflammatory bowel disease. Adrenomedullin ni yoru IBD chiryou. J. IBD Res. 2020, 14, 183–191. (In Japanese) 33. Kinoshita, Y.; Arita, S.; Murazoe, H.; Kitamura, K.; Ashizuka, S.; Inagaki-Ohara, K. Subcutaneously Administered Adrenomedullin Exerts a Potent Therapeutic Effect in a Murine Model of Ulcerative Colitis. Hum. Cell 2019, 32, 12–21. [CrossRef][PubMed] 34. MacManus, C.F.; Campbell, E.L.; Keely, S.; Burgess, A.; Kominsky, D.J.; Colgan, S.P. Anti-Inflammatory Actions of Adrenomedullin Through Fine Tuning of HIF Stabilization. FASEB. J. 2011, 25, 1856–1864. [CrossRef][PubMed] 35. Davis, R.B.; Kechele, D.O.; Blakeney, E.S.; Pawlak, J.B.; Caron, K.M. Lymphatic Deletion of Calcitonin Receptor-Like Receptor Exacerbates Intestinal Inflammation. JCI Insight 2017, 2, e92465. [CrossRef][PubMed] 36. Hayashi, Y.; Narumi, K.; Tsuji, S.; Tsubokawa, T.; Nakaya, M.A.; Wakayama, T.; Zuka, M.; Ohshima, T.; Yamagishi, M.; Okada, T. Impact of Adrenomedullin on Dextran Sulfate Sodium-Induced Inflammatory Colitis in Mice: Insights from In Vitro and In Vivo Experimental Studies. Int. J. Colorectal Dis. 2011, 26, 1453–1462. [CrossRef] 37. Yi, Z.; Fan, H.; Liu, X.; Tang, Q.; Zuo, D.; Yang, J. Adrenomedullin Improves Intestinal Epithelial Barrier Function by Downregu- lating Myosin Light Chain Phosphorylation in Ulcerative Colitis Rats. Mol. Med. Rep. 2015, 12, 3615–3620. [CrossRef] 38. Graham, W.V.; He, W.; Marchiando, A.M.; Zha, J.; Singh, G.; Li, H.S.; Biswas, A.; Ong, M.L.D.M.; Jiang, Z.H.; Choi, W.; et al. Intracellular MLCK1 Diversion Reverses Barrier Loss to Restore Mucosal Homeostasis. Nat. Med. 2019, 25, 690–700. [CrossRef] [PubMed] 39. Matsuoka, K.; Kanai, T. The Gut Microbiota and Inflammatory Bowel Disease. Semin. Immunopathol. 2015, 37, 47–55. [CrossRef] [PubMed] 40. Atarashi, K.; Tanoue, T.; Ando, M.; Kamada, N.; Nagano, Y.; Narushima, S.; Suda, W.; Imaoka, A.; Setoyama, H.; Nagamori, T.; et al. Th17 Cell Induction by Adhesion of Microbes to Intestinal Epithelial Cells. Cell 2015, 163, 367–380. [CrossRef] 41. Kostic, A.D.; Xavier, R.J.; Gevers, D. The Microbiome in Inflammatory Bowel Disease: Current Status and the Future Ahead. Gastroenterology 2014, 146, 1489–1499. [CrossRef][PubMed] 42. Atarashi, K.; Tanoue, T.; Shima, T.; Imaoka, A.; Kuwahara, T.; Momose, Y.; Cheng, G.; Yamasaki, S.; Saito, T.; Ohba, Y.; et al. Induction of Colonic Regulatory T Cells by Indigenous Clostridium species. Science 2011, 331, 337–341. [CrossRef][PubMed] 43. Marutsuka, K.; Nawa, Y.; Asada, Y.; Hara, S.; Kitamura, K.; Eto, T.; Sumiyoshi, A. Adrenomedullin and Proadrenomudullin N-Terminal 20 Peptide (PAMP) Are Present in Human Colonic Epithelia and Exert an Antimicrobial Effect. Exp. Physiol. 2001, 86, 543–545. [CrossRef][PubMed] 44. Kataoka, Y.; Miyazaki, S.; Yasuda, S.; Nagaya, N.; Noguchi, T.; Yamada, N.; Morii, I.; Kawamura, A.; Doi, K.; Miyatake, K.; et al. The First Clinical Pilot Study of Intravenous Adrenomedullin Administration in Patients with Acute Myocardial Infarction. J. Cardiovasc. Pharmacol. 2010, 56, 413–419. [CrossRef][PubMed] 45. Ashizuka, S.; Inatsu, H.; Kita, T.; Kitamura, K. Adrenomedullin Therapy in Patients with Refractory Ulcerative Colitis: A Case Series. Dig. Dis. Sci. 2016, 61, 872–880. [CrossRef] 46. Ashizuka, S.; Kuroishi, N.; Nakashima, K.; Inatsu, H.; Kita, T.; Kitamura, K. Adrenomedullin: A Novel Therapy for Intractable Crohn’s Disease with a Loss of Response to Infliximab. Intern. Med. 2019, 58, 1573–1576. [CrossRef] 47. Kita, T.; Kaji, Y.; Kitamura, K. Safety, Tolerability, and Pharmacokinetics of Adrenomedullin in Healthy Males: A Randomized, Double-Blind, Phase 1 Clinical Trial. Drug Des. Dev. Ther. 2020, 14, 1–11. [CrossRef] 48. Kita, T.; Ashizuka, S.; Ohmiya, N.; Yamamoto, T.; Kanai, T.; Motoya, S.; Hirai, F.; Nakase, H.; Moriyama, T.; Nakamura, M.; et al. Adrenomedullin for Steroid-Resistant Ulcerative Colitis: A Randomized, Double-Blind, Placebo-Controlled phase-2a Clinical Trial. J. Gastroenterol. 2021, 56, 147–157. [CrossRef] 49. Kubo, K.; Tokashiki, M.; Kuwasako, K.; Tamura, M.; Tsuda, S.; Kubo, S.; Yoshizawa-Kumagaye, K.; Kato, J.; Kitamura, K. Biological Properties of Adrenomedullin Conjugated with Polyethylene Glycol. Peptides 2014, 57, 118–121. [CrossRef] 50. Nagata, S.; Yamasaki, M.; Kitamura, K. Anti-Inflammatory Effects of Pegylated Human Adrenomedullin in a Mouse DSS-Induced Colitis Model. Drug Dev. Res. 2017, 78, 129–134. [CrossRef][PubMed] Biomedicines 2021, 9, 1068 12 of 12

51. Nagata, S.; Yamasaki, M.; Kawano, A.; Kitamura, K. Developments of Human Adrenomedullin-IgG1 Fc Fusion Proteins. J. Biochem. 2019, 166, 157–162. [CrossRef][PubMed] 52. Kuroishi, N.; Nagata, S.; Akashi, E.; Ashizuka, S.; Kato, J.; Yamasaki, M.; Kitamura, K. Development of a Novel Human Adrenomedullin Derivative: Human Serum Albumin-Conjugated Adrenomedullin. J. Biochem. 2021.[CrossRef][PubMed]