USOO8314149B2 (12) United States Patent (10) Patent No.: US 8,314,149 B2 Zamora et al. (45) Date of Patent: *Nov. 20, 2012 (54) PROTECTIVE ROLE OF SEMAPMOD IN (60) Provisional application No. 60/685,875, filed on Jun. NECROTZNG ENTEROCOLTS 1, 2005. (75) Inventors: Ruben Zamora, Pittsburgh, PA (US); Henri R. Ford, La Canada, CA (US); (51) Int. Cl. Thais Sielecki-Dzurdz, Kennett Square, A613 L/65 (2006.01) PA (US); Vidal F. De La Cruz, A613 L/55 (2006.01) Phoenixville, PA (US) (52) U.S. Cl. ....................................................... S14/615 (58) Field of Classification Search ........................ None (73) Assignees: Feering B.V., Hoofddorp (NL); See application file for complete search history. University of Pittsburgh-Of The Commonwealth System Of Higher (56) References Cited Education, Pittsburgh, PA (US) (*) Notice: Subject to any disclaimer, the term of this PUBLICATIONS patent is extended or adjusted under 35 Caplanet al., Pediatric Pathology, (Nov.-Dec. 1994), 14(6), pp. 1017 U.S.C. 154(b) by 0 days. 1028 (Abstract).* This patent is Subject to a terminal dis claimer. * cited by examiner (21) Appl. No.: 12/879,144 Primary Examiner — Phyllis G. Spivack (74) Attorney, Agent, or Firm — Fish & Richardson P.C. (22) Filed: Sep. 10, 2010 (65) Prior Publication Data (57) ABSTRACT US 2011 FO224301A1 Sep. 15, 2011 A method is disclosed wherein semapimod, and/or the mesy late salt thereof, is administered for the treatment of necro Related U.S. Application Data tizing enterocolitis. (63) Continuation of application No. 1 1/421,666, filed on Jun. 1, 2006, now Pat. No. 7,795,314. 5 Claims, 12 Drawing Sheets U.S. Patent Nov. 20, 2012 Sheet 1 of 12 US 8,314,149 B2 Fig. 1. HMGB1 - . i : - ge f ... {- 30 kDa - 10 kDa 7kDa B-actin -> 30 kDa 10 kDa 7 kDa U.S. Patent Nov. 20, 2012 Sheet 2 of 12 US 8,314,149 B2 Fig. 2. 100 FFH CD FFH + Semapimod - 80 S. O L Z 60 an O (9 ss 40 U 20 O O.1 0.25 0.50 0.75 1 Semapimod mg/kg, IP U.S. Patent Nov. 20, 2012 Sheet 3 of 12 US 8,314,149 B2 Fig. 3a. BF FFH FFH Semapimod HMGB-1 u 2.5 2.0 - 1.5 - k, 10 - 0.5 OO BF FFH FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 4 of 12 US 8,314,149 B2 Arg F-Jaw: *::YN ...e. -- . ". 3. - A . Fig 3b. U.S. Patent Nov. 20, 2012 Sheet 5 of 12 US 8,314,149 B2 Fig. 3c. BF FFH FFH + Semapimod RAGE 45 kDa - inwn st c - s “P up ... m. edmousumoto B-actin "tural auhaumuruu 6 O BF FFH 5 O FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 6 of 12 US 8,314,149 B2 Fig. 4a. BF FFH FFH + Semapimod Bad -> sue on the q m an are ster par ( 23 kDa B-actin -> Nandanmummim 18 15 1.2 0.9 0.6 0.3 0.0 BF FFH FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 7 of 12 US 8,314,149 B2 Fig. 4b. BF FFH FFH + Semapimod Bax -> e is as as - 21 kDa B-actin -> OC O.30 S w 0.25 y O 020 k, NE E. 5 0.15 s 0.10 g O O O,05 OOO BF FFH FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 8 of 12 US 8,314,149 B2 Fig. 5a. BF FFH FFH + Semapimod iNOS --> xx * * * * * oneeasis. - - -- 130 kDa B-actin - -u 18 1.5 1.2 O.9 O.6 O.3 0.0 BF FFH FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 9 of 12 US 8,314,149 B2 Fig. 5b. BF FFH FFH + Semapimod COX-2-> . - but opoufaoens emale-72 kDa f-actin -> unupuupular untinuu 2.0 - BF FFH FFH + Semapimod U.S. Patent Nov. 20, 2012 Sheet 10 of 12 US 8,314,149 B2 Fig. 6a. Semapimod uM pp38 -) Ctrl O 0.1 0.5 1 5 10 SemapimodulM) U.S. Patent Nov. 20, 2012 Sheet 11 of 12 US 8,314,149 B2 Fig. 6b. Ctrl LPS ONOO Semapimod - H pp38 -> t dip to p38 -) in mo an elo - U.S. Patent Nov. 20, 2012 Sheet 12 of 12 US 8,314,149 B2 Fig. 7. Control NEC HMGB-1 -> m an amumumap (-30 kDa B-actin - bu. uru Control NEC US 8,314,149 B2 1. 2 PROTECTIVE ROLE OF SEMAPMOD IN to understand the pathogenesis of NEC, others have relied on NECROTZNG ENTEROCOLTS intravenous administration of pro-inflammatory cytokines or invasive Surgical procedures, such as occlusion of the mesen CROSS REFERENCE TO RELATED teric vessels, to reproduce the morphological and clinical APPLICATIONS changes seen in infants with NEC (16). However, these arti ficial insults do not parallel the human disease. To understand This application is based on, and claims priority to, U.S. the pathogenesis of this disease, the present inventors have Provisional Application Ser. No. 60/685,875, filed Jun. 1, developed a consistent and reproducible rat model that relies 2005, the entire contents of which are hereby incorporated by on formula feeding and hypoxia, two major risk factors for reference. 10 NEC (32). This work was supported in part by the RO1-AI-14032 The present inventors modified an experimental model grant from the National Institutes of Health (Bethesda, Md.). (32) so that newborn rats were fed with formula twice a day The government may have rights in this invention. and exposed to hypoxia for a longer period of time (10 min. 15 instead of 3 min.). Examining a larger number of animals the BACKGROUND present inventors observed a higher mortality rate in the FFH Field animals as compared to the BF group. As used herein, BF represents breast-fed animals left with their mothers (con The present invention relates to guanylhydrazone-moiety trols), and FFH are formula-fed pups exposed to hypoxia. containing compounds and their use in the prevention, treat Although malnutrition could be a contributing factor (the ment, and inhibition of conditions, such as inflammatory Volume of formula consumed by the neonatal rats is less than conditions. the volume of milk consumed by breast-fed pups), there was no apparent cause of death for most of the animals in the FFH DESCRIPTION OF THE FIGURES group. The significant difference in mortality could only be 25 attributed to aspiration during gavage. The longer exposure to FIG. 1 shows that HMGB1 is elevated in formula-fed ani hypoxia, however, exacerbated the inflammatory changes mals exposed to hypoxia. seen in the intestine of the FFH group and rendered the FIG. 2 shows that administration of semapimod in vivo is neonatal rats more Susceptible to gut barrier failure as shown protective in an experimental NEC model. by the increased incidence of NEC starting at day 3 after birth. FIG. 3 shows that administration of semapimod in vivo 30 The present inventors have shown that NEC is character decreases the expression of HMGB1 and its receptor RAGE ized by the presence of a number of pro-inflammatory cytok in ileal samples from formula-fed animals exposed to ines, both inexperimental and in human NEC (32:33). There, hypoxia. the present inventors have examined the expression of the FIG. 4 shows that administration of semapimod in vivo protein called high mobility group box 1 (HMGB1) in the decreases the expression of the Bcl-2 family members Bad 35 small intestine of newborn rats with NEC. Wang et al. have and Bax in an experimental NEC model in rats. identified and characterized the high mobility group box-1 FIG. 5 shows that administration of semapimod in vivo protein (HMGB-1, previously known as HMG-1 or amphot decreases the expression of iNOS and COX-2 in an experi erin) as a potential late mediator of lethality in a mouse model mental NEC model in rats. (44) and a regulator of human monocyte proinflammatory FIG. 6 shows that semapimod decreases the activation of 40 cytokine synthesis (3). This protein belongs to the HMGB p38 MAP kinase by LPS in vitro. group, one of the three superfamilies of the HMG nuclear FIG. 7 shows HMGB1 protein expression in human NEC. proteins Subdivided according to their characteristic func tional sequence motifs. The functional motif of this family is DESCRIPTION OF THE SEVERAL called the HMG-box and according to the revised nomencla EMBODIMENTS 45 ture of the HMG nuclear proteins, the HMG-1 has been renamed to HMGB1 (9). HMGB1 is 30 kDa nuclear and Necrotizing enterocolitis (NEC) is the most frequent and cytosolic protein member of the high-mobility group protein the most lethal disease that affects the gastrointestinal tract of Superfamily that has been widely studied as a transcription as the premature infant (16). The overall mortality rate for well as growth factor. HMGB1 is also defined as a cytokine patients with NEC ranges from 10 to 70% (22) and 50 because it stimulates proinflammatory responses in mono approaches 100% for patients with the most severe form of cytes/macrophages, is produced during systemic and local the disease, which is characterized by involvement of the inflammatory responses, has been identified as a late mediator entire bowel (pan-necrosis) (37). Survivors may develop of lethal systemic inflammation (e.g. endotoxaemia and sep short-bowel syndrome, recurrent bouts of central line associ sis), and is required for the full expression of inflammation in ated sepsis, malabsorption and malnutrition, liver failure as a 55 animal models of endotoxemia, sepsis and arthritis (2: 45).