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). result of longstanding administration of total parenteral nutri Until the present invention, however, the expression of tion, and eventually may require liver-Small bowel transplan HMGB1 in the injured intestine and its role in intestinal tation (27:37). Thus, NEC represents one of the most impor inflammation and in particular in NEC, was not known. tant diseases seen in the neonatal population. HMGB1 is produced by nearly all cell types, but cellular Several risk factors for NEC have been identified, includ 60 levels vary with development and age (45). As expected, the ing low birth weight, formula feeding, bacterial colonization present inventors found a high expression of HMGB1 in the of the gut, and hypoxia (1-3). Several experimental animal ileum of BF newborn rats. However, maximal expression of models have been reported (4; 10; 12; 14: 20) which have HMGB1 in the ileal mucosal scrapings of FFH newborn rats intestinal hypoxia as the common denominator. Other risk at day 4 coincided with the marked appearance of morpho factors including prematurity (47), hypoxia (26), formula 65 logical characteristics of NEC. Those animals that showed feeding (18), bacterial infection (40), and intestinal ischemia moderate to severeintestinal damage as assessed by histology (1) have been implicated in the pathogenesis of NEC. In order also showed an increased expression of a lower molecular US 8,314,149 B2 3 4 weight (e.g., below 30 kDa) protein detected with the same deactivator semapimod (formerly known as CNI-1493) on anti-HMGB1 polyclonal antibody (as a 7-10 kDa band). the expression of pro-apoptotic and pro-inflammatory pro Numerous inflammatory mediators have been implicated teins and on the severity of intestinal inflammation in their in the cascade leading to the hemodynamic instability asso experimental model of NEC, as well as its effects on rat ciated with sepsis. Elevated levels of IL-6 and TNF-C. have 5 intestinal epithelial cells challenged with bacterial LPS. been measured in infants with NEC, bacterial sepsis, or men Semapimod (formerly known as CNI-1493), is a tetravalent ingitis (21). The present inventors have shown that nitric guanylhydraZone known to inhibit mouse macrophage argi oxide (NO) modulates intestinal changes in septic shock (17; nine transport and NO production and to suppress the release 43). Furthermore, the end products of NO metabolism have of proinflammatory cytokines (7). The present inventors been shown to be elevated in newborn infants as well as in 10 show that this macrophage-deactivating drug inhibits the adult patients with clinical sepsis (34: 41). Several studies expression and accumulation of HMGB1 in the ileal mucosal Suggest these and other mediators of sepsis are also involved scrapings of FFH animals thereby limiting the degree of in the pathogenesis of NEC. In this context, the present inven intestinal injury caused by formula feeding and exposure to tors have previously reported the upregulation of iNOS hypoxia. The fact that semapimod did not completely prevent mRNA in the intestine of infants with acute NEC, and its 15 the occurrence of NEC-like morphological changes in the downregulation by the time of intestinal stoma closure, after experimental model merely confirms the complexity of this the infants have recovered from the acute inflammatory pro disease and implies that other mechanism(s) are also involved cess (17). A similar pattern was seen for IFN-Y, but not for in its pathogenesis. It is clear, however, that HMGB1 is an IL-1, IL-6, TGF-beta, or TNF-C. The pattern of cytokine important mediator of hypoxia-induced gut injury and that mRNA expression in our model of experimental NEC paral- 20 Suppression of pro-inflammatory cytokines with inhibitors lels that seen in patients with NEC (17). Such as semapimod partially protects against intestinal epi It has been shown that HMGB1 necrotic cells have a thelial cell death both in vitro and in vivo. greatly reduced ability to promote inflammation, which Accordingly, one embodiment is the use and/or adminis proves that the release of HMGB1 can signal the demise of a tration of semapimod and other guanylhydraZone containing cell to its neighbors (39). Apoptotic cells, however, do not 25 compounds for the inhibition, treatment, and/or prevention of release HMGB1 even after undergoing secondary necrosis NEC to a subject in need thereof. and partial autolysis, and thus fail to promote inflammation Another embodiment is the use and/or administration of even if not cleared promptly by phagocytic cells (39). semapimod and other guanylhydraZone containing com HMGB1 is either actively secreted by monocytes/macroph pounds for the inhibition, treatment, and/or prevention of ages or passively released from necrotic cells from any tissue 30 NEC to a human subject in need thereof. In one embodiment, (45). Since in experimental NEC enhanced epithelial apop the Subject is a human infant, for example one with one or tosis is an initial event underlying the gross tissue necrosis of more of low-birth weight, formula feeding, bacterial coloni the intestinal wall (11; 25), it is believed that the elevated Zation of the gut, hypoxia, and/or other conditions associated levels of HGMB 1 found in the ileal mucosal scrapings of FFH with the release of HMGB1. newborn rats suggest that there is more synthesis of HMGB1 35 Outside the cell HMGB1 binds with high affinity to the protein and/or more translocation from the nucleus to the receptor for advance glycation end-products (RAGE), a trans cytoplasm of the protein ready to get released into the extra membrane receptor of the immunoglobulin Superfamily cellular milieu, where it mediates downstream inflammatory which is in part responsible for the receptor signal transduc responses. Such HMGB1-related responses have been tion of HMGB1 (24; 44). RAGE is expressed on monocytes/ reported in endotoxemia, arthritis and sepsis (2). Like their 40 macrophages, endothelial cells, neurons and Smooth-muscle finding with rat NEC, the present inventors found that the cells (2). The accumulation of RAGE ligands leads to inflam intestinal segments of infants with acute NEC showed greater matory disorders and the biology of RAGE is driven by the expression of HMGB1 than the intestinal segments from settings in which these ligands accumulate. Such as diabetes, non-NEC patients. Thus, without wishing to be bound by inflammation, neurodegenerative disorders and tumors (8). theory, it is believed that an overexpression of HMGB1 may 45 Although one study has shown that a human colon adenocar be related to the intestinal damage induced by hypoxia and cinoma cell line expresses RAGE (51), until the present formula feeding in experimental NEC as well as in human invention, the expression of this protein in nontransformed NEC. intestinal epithelial cells or normal intestinal tissue has not Little is known about how the HMGB1 mRNA is transcrip been reported. In their rat model, the present inventors could tionally regulated. The present inventors previously found 50 hardly detect any RAGE in the ileal mucosal scrapings of BF that among the several DNA repair genes that were affected newborn rats. However, there was a higher expression of by iNOS in murine hepatocytes, there was an increased RAGE in the intestinal samples from FFH animals analyzed expression of the gene coding for the high mobility group using an anti-RAGE antibody that detects two bands in the 45 protein HMG-2 as determined by microarray DNA analysis kDa range (the RAGE protein pre and post-glycosylation in (49). However, the exact relationship and whether iNOS- 55 mouse lung extract) and a 25 kDa protein believed to be a derived NO alters the expression or activity of HMG proteins proteolytic degradation product. The intensity of the 25 kDa and specifically of HMGB1 remain to be investigated. band was significantly higher than the 45 kDa band in the Although HMGB1 has not been associated with gene tran FFH animals. Without wishing to be bound by theory, it is Scription in Vivo, it can stimulate transcription in vitro (45). believed that this finding is not due to total protein degrada Thus, it is also possible that elevated levels of HMGB1 affect 60 tion as shown by the normal B-actin levels. The present inven the expression of other cell-death related genes in the experi tors have found that not only the expression of HMGB1 was mental model of NEC. significantly reduced by Semapimod but also that of its recep The present inventors have found that the cell death related tor RAGE. Accordingly, it is believed that upregulation of protein HMGB1 is differentially expressed in the injured RAGE is involved in the intestinal injury caused by formula small intestine of newborn rats in experimental NEC and in 65 feeding and hypoxia. In this respect, a recent study using the inflamed intestine of human infants with acute NEC. The novel animal models with defective or tissue-specific RAGE present inventors evaluated the effects of the macrophage expression showed that deletion of RAGE provides protec US 8,314,149 B2 5 6 tion from the lethal effects of septic shock caused by cecal tissue specific and confer protection against the hemody ligation and puncture (29). It should be noted, however, that namic and inflammatory responses to LPS (31). Also, although RAGE has been shown to interact with HMGB1, semapimod has previously been shown to inhibit the expres other putative HMGB1 receptors are known to exist but have sion of the pro-inflammatory cytokines through a pathway not been characterized yet. A recent study showed that while involving the MAP kinases, specifically to inhibit the phos RAGE played only a minor role in macrophage activation by phorylation and activation of p38 MAP kinase in both human HMGB1, the interactions of HMGB1 with TLR 2 and TLR 4 monocytes and the murine macrophage cell line RAW264.7 could explain the ability of HMGB1 to generate inflamma (13:30). Inflammatory MAP kinases, in particular p38 and tory responses that are similar to those initiated by LPS (35). JNK, are critically involved in the pathogenesis of Crohn's Accordingly, another embodiment relates to the role of 10 disease (23), and their involvement in the regulation of RAGE and other receptors in mediating inflammatory inflammatory responses in the gut is within the scope of the responses to HMGB1, and it is believed that RAGE accumu present invention. lation and degradation accompanies the upregulation of its A neural pathway, termed the “cholinergic anti-inflamma ligand HMGB1 associated with formula feeding and tory pathway', involves vagus nerve stimulation that inhibits hypoxia-induced intestinal injury in experimental NEC. As 15 the release of TNF, HMGB1, and other cytokines, and pro Such, another embodiment is the inhibition and/or downregu tects against endotoxemia and ischemia-reperfusion injury lation of the HMGB1 receptor, RAGE, by the administration (6). Examining the effects of pharmacological and electrical with semapimod and/or guanylhydrazone containing com stimulation of the intact vagus nerve in adult male Lewis rats pounds. Subjected to endotoxin-induced shock, it was found that intact As in many other cell types and tissues, intestinal apoptosis vagus nerve signaling was required for the anti-inflammatory and cell death are the result of different pathways that involve action of semapimod (6). In that study, intracerebroVentricu a number of pro-inflammatory cytokines and mediators other lar (icv.) administration of semapimod was 100,000-fold than HMGB1. The present inventors found that expression of more effective in suppressing endotoxin-induced TNF Bax and Bad, two pro-apoptotic members of the Bcl-2 family release and shock as compared with intravenous route. of proteins, were significantly upregulated by formula feed 25 Although icv. administration of the drug is extremely difficult ing and hypoxia in the mucosal Scrapings of FFH animals as due to the Small size and sensitivity of newborn pups, such a compared to BF controls. Expression of both proteins in FFH finding would not preclude this administrative route to human pups was also significantly decreased when the animals subjects, such as neonates. Without wishing to be bound by where administered the drug semapimod. Similarly, other theory, one possible mechanism through which semapimod proteins like iNOS and COX-2 whose altered expression has 30 may be protective is the “cholinergic anti-inflammatory path been associated with inflammation in a number of experimen way’ in the development of both experimental and human taland clinical conditions (see (28:46:50) for reviews), were NEC. elevated in the terminal ileum of FFH newborn rats compared In Summary, the present inventors have found that the to BF controls and downregulated when the animals were pro-inflammatory protein HMGB1 and its receptor RAGE are administered the drug semapimod. Although the present 35 elevated in the ileal mucosa of formula-fed newborn rats inventors did not assess the integrity of the mucosal barrier, it exposed to hypoxia. The present inventors show that the has previously been shown that HMGB1 is capable of causing synthetic guanylhydraZone semapimod, a cytokine inhibitor derangements in intestinal barrier function in cultured Caco-2 and MAP kinase blocker, inhibits the upregulation of both human enterocytic monolayers and that this effect depends on HMGB1 and RAGE as well as of other proinflammatory the formation of NO and peroxynitrite (38). Thus, without 40 mediators, thereby limiting the intestinal injury caused by wishing to be bound by theory, it is believed that the protec formula feeding and hypoxia in experimental NEC. In addi tion conferred by semapimod may also be related to its ability tion, the present inventors have found that semapimod selec to inhibit the formula feeding plus hypoxia-induced increase tively affects the activation of p38 MAP kinase by LPS in in the intestinal permeability by decreasing both HMGB1 and IEC-6 intestinal epithelial cells supporting the involvement of iNOS protein expression. 45 the MAP kinases in the protective effect of semapimod Accordingly, another embodiment relates to the adminis against bacterial LPS-induced intestinal injury. The present tration of semapimod and/or guanylhydrazone containing inventors have identified HMGB1 and RAGE as important compounds to decrease one or both of HMGB1 and iNOS mediators of enterocyte apoptosis/cell death and hypoxia protein expression. induced gut barrier failure associated with NEC. Semapimod Since bacterial colonization is an important factor in the 50 is currently being developed as a potential treatment for pathogenesis of NEC, it is necessary to understand the Crohn's disease and other inflammatory pathologies (42). response of intestinal epithelial cells to LPS. In the model The present inventors have found a method to suppress the described herein, this becomes even more important because release of pro-inflammatory cytokines and inhibit the expres the animals are not kept in a pathogen free environment. The sion and activity of cytotoxic molecules such as HMGB1 with present inventors have found that LPS activates the mitogen 55 inhibitors like semapimod, and the present compounds are activated protein kinase (MAP kinase) p38 both in IEC-6 rat also useful in a therapeutic modality to combat inflammatory intestinal epithelial cells and in ileal mucosal scrapings cul conditions such as NEC. Other guanylhydrazone-containing tured ex vivo (19). This increase in p38 activation could compounds described herein are expected to have the same significantly be inhibited upon pre-incubation of IEC-6 cells effect. with Semapimod in a concentration-dependent fashion. More 60 Suitable compounds include guanylhydrazone-containing importantly, the inhibitory effect of semapimod rather than a compounds described herein, such as semapimod. The gua non-specific inhibitory effect seems to be a selective effect on nylhydrazone-containing compound may be used in pharma the LPS signaling pathway. That is illustrated by the fact that ceutical, medicinal, veterinary, agricultural, and analytical semapimod inhibited p38 activation only in LPS treated cells applications and/or for other commercial purposes in accor but not in cells exposed to ONOO, the toxic NO-related 65 dance with the present methods. species that also activates p38. Without wishing to be bound Accordingly, one embodiment is the use and/or adminis by theory, it is believed that diverse effects of semapimod are tration of semapimod and other guanylhydrazone-containing US 8,314,149 B2 7 8 compounds for the inhibition, treatment, and/or prevention of wherein Z is one or more selected from the group consist NEC to a subject in need thereof. ing of Another embodiment is the use and/or administration of semapimod and other guanylhydrazone-containing com pounds for the inhibition, treatment, and/or prevention of any of NEC, a condition associated with the release of HMGB1, a condition associated with the release of iNOS protein, or a condition associated with the release of RAGE, or a combi nation thereof to a human Subject in need thereof. In one and combinations thereof; embodiment, the Subject is a human infant, for example one 10 whereina is selected from the group consisting of 0, 1,2,3, with one or more of NEC, low-birth weight, formula feeding, 4, 5, 6, 7, 8, and 9: bacterial colonization of the gut, hypoxia, a condition asso wherein b is selected from the group consisting of 0,1,2,3, ciated with the release of HMGB1, a condition associated 4, 5, 6, 7, 8, and 9: with the release of iNOS protein, or a condition associated wherein X is selected from the group consisting of 0,1,2,3, with the release of RAGE, or a combination thereof. 15 4, 5, 6, 7, 8, and 9: Another embodiment relates to the administration of whereiny is selected from the group consisting of 0,1,2,3, semapimod and/or guanylhydrazone containing compounds 4, 5, 6, 7, 8, and 9: to decrease one or both of HMGB1 and iNOS protein expres wherein Z is selected from the group consisting of 0, 1,2,3, sion to a subject in need thereof. 4, 5, 6, 7, 8, and 9: Another embodiment relates to the administration of 20 wherein m is selected from the group consisting of 0, 1, 2, semapimod and/or guanylhydrazone containing compounds 3, 4, 5, 6, 7, 8, and 9: to decrease RAGE to a subject in need thereof. whereinn is selected from the group consisting of 0,1,2,3, Semapimod has the following formula: 4, 5, 6, 7, 8, and 9:

CH NH H H HN N-2 N r N s NH O H3C 2 N

r CH O NH HN NH N l r NH 21 SN NH2 NH H3C

Other embodiments relate to methods and compositions 40 wherein A' and A are each independently selected from that prevent or treat the conditions described herein. One the group consisting of NR(CO)NR , —(CO)NR , embodiment relates to the use of guanylhydrazone (“Ghy', NR(CO) , NR, O—, S—, S(=O) , termed as an amidinohydrazone, i.e., (—N NH C (—NH)—NH) containing compounds and compositions. SO. , SONR , NRSO. , and salts thereof; One embodiment relates to the use of aromatic guanylhydra wherein Q and T are each independently selected from the Zone-containing compounds and compositions. group consisting of NR'(CO)NR''-, -(CO)NR' , One class of compounds useful for the purposes of the - NR'(CO) , – NR' -O-, -S , S(=O) , invention includes but is not limited to aromatics substituted —SO-, -SONR' , NR'SO. , salts thereof, with multiple guanylhydrazone (Ghy) moieties (i.e., amidi branched or unbranched, Saturated or unsaturated, Substituted nohydrazones). The synthesis and use of Such compounds is or unsubstituted C-C alkylene, saturated or unsaturated, described in U.S. Pat. No. 5,599.984 (the entire contents of 50 Substituted or unsubstituted C-C cycloalkylene, Substi which being hereby incorporated by reference) with respect tuted or unsubstituted Cs-Cs arylene, and combinations to treating inflammatory conditions. The compounds can also thereof; be used in Screening assays to test additional compounds for activity. wherein one or more carbon atoms in any of said alkylene, 55 cycloalkylene or arylene in said Q and/or T may each be Other Suitable guanylhydrazone compounds include those independently replaced with one or more heteroatoms having the following formula: selected from the group consisting of nitrogen, oxygen, Sul fur, and a combination thereof. and wherein when substituted, each of said alkylene, XC - Z oy 60 cycloalkylene or arylene in said Q and/or T may be indepen dently Substituted with one or more Substituent groups x / \ Žx. defined herein, and the Substituent groups themselves may be optionally and independently substituted as defined herein. and/or salts thereof; The R. R. R. R. R. R. R. R. R', and R'' are defined wherein X', X, X, and X each independently represent 65 herein, and may be optionally and independently substituted H. GhyCH , GhyCCH , or CHCO , with the provisos with one or more substituents defined herein. If desired, two that X', X, X and X’ are not simultaneously H: independently chosen R. R. R. R. R. R. R. R. R', and US 8,314,149 B2 10 R'' alkyl-containing groups may be taken together with any In another embodiment, Z is a -A-(CH2)-A- group, atom to which they are attached to form a three to forty wherein p is 1, 2, 3, 4, 5, 6,7,8,9, or 10, and wherein the As membered cyclic, heterocyclic or heteroaryl ring. are each independently —NH(CO)— —(CO)NH , or In the present application, GhyCH is NH CO NH)— - NH(CO)NH groups. NH-N=CH-; and GhyCH is NH C(=NH)– In another embodiment, Z is a -A-(CH)-A- wherein the NH N=CCH . As are each independently —CO— —NH(CO)— —(CO) In one embodiment, Z is a C-C alkylene, which may be NH-, or - NH(CO)NH groups. branched or unbranched, Saturated or unsaturated, Substituted In another embodiment, Z is —O—(CH)—O—, or unsubstituted, and which may have one or more carbon wherein the two"—O groups are para to each other about atoms replaced by one or more heteroatoms selected from the 10 the phenylene ring. group consisting of oxygen, nitrogen, Sulfur, and a combina In another embodiment, Z is —O—(CH)—O—, tion thereof. This includes alkylenes having 1, 2, 3, 4, 5, 6, 7, wherein the two" O groups are meta to each other about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. the phenylene ring. In another embodiment, Z is a branched C-Co alkylene. In another embodiment, Z is —O CH, CH, O In another embodiment, Z is an unbranched C-C alky 15 CH2—CH2—O-. lene. In another embodiment, Z is a group having the formula: In another embodiment, Z is a saturated C-C alkylene. In another embodiment, Z is an unsaturated C-C alky lene. In another embodiment, Z is an unsubstituted C-C alky lene. In another embodiment, Z is a Substituted C-C alkylene. In another embodiment, Z is a C-C alkylene in which one or more carbons is replaced with one or more heteroatoms selected from the group including oxygen, nitrogen, Sulfur 25 and a combination thereof. In one embodiment, Z is a Saturated or unsaturated, Sub In another embodiment, Z is a group having the formula: stituted or unsubstituted C-C cycloalkylene, and which may have one or more carbonatoms replaced by one or more heteroatoms selected from the group consisting of oxygen, 30 nitrogen, Sulfur, and a combination thereof. This includes cycloalkylenes having 3, 4, 5, 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20 carbons. In another embodiment, Z is a Saturated C-C cycloalky lene. 35 In another embodiment, Z is an unsaturated C-Co In another embodiment, Z is a group having the formula: cycloalkylene. In another embodiment, Z is an unsubstituted C-Co cycloalkylene. In another embodiment, Z is a substituted C-Co 40 cycloalkylene. In another embodiment, Z is a C-C cycloalkylene in which one or more carbons is replaced with one or more heteroatoms selected from the group including oxygen, nitro puch gen, Sulfur and a combination thereof. 45 In one embodiment, Z is a substituted or unsubstituted Cs-Cs arylene, wherein one or more carbon atoms in the In another embodiment, Z is a group having the formula: cycloalkylene and arylene may be replaced with one or more heteroatoms selected from the group consisting of nitrogen, oxygen, Sulfur, and a combination thereof. This includes 50 O arylenes having 6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19. 20, 21, 22, 23, 24, and 25 carbons. N In another embodiment, Z is a substituted Cs-Cs arylene. In another embodiment, Z is an unsubstituted Cs-Cs H H arylene. In another embodiment, Z is a Cs-Cs arylene in which one 55 or more carbons is replaced with one or more heteroatoms '--O selected from the group including oxygen, nitrogen, Sulfur and a combination thereof. In one embodiment, Z is an NR(CO)NR' group, In another embodiment, Z is a group having the formula: optionally in the salt form, wherein the R groups are both 60 hydrogen. In another embodiment, Z is a —(CH)—group. In another embodiment, Z is a -(CH2) group, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In another embodiment, Z is a —(CHN)—group. 65 In another embodiment, Z is a -O-(CH2). O—group, wherein p is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. US 8,314,149 B2 11 12 In another embodiment, Z is a group having the formula: -continued O . HN N 2 In another embodiment, Z is a group having the formula: 10 N NH In one embodiment, the compound includes the structure: In one embodiment, the compound includes the structure: '-- 15 In another embodiment, Z is a group having the formula: . O HN 2 H ls 2 r NN 25 NH

'sO N In one embodiment, the compound includes the structure: In one embodiment, the compound includes the structure: 30 O

HN H 2 Z s 35 NH g . O HN 2 NN 2 In one embodiment, the compound includes the structure: 40 NH O In one embodiment, the compound includes the structure:

45 H HN N Na Z or . NH In one embodiment, the compound includes the structure: 50

HN2 2

In one embodiment, the compound includes the structure: 55 Cri "N'sH 21 . In one embodiment, the compound includes the structure: NH

HN2 2 Z HN N 2 r NN s 65 NH NH US 8,314,149 B2 13 14 In one embodiment, the compound includes the structure: with the proviso that if Q is NR' or O-andy is 1 then T is not NR' or —O—. In one embodiment, Z has the formula: H HN N Z rise wherein each of the variables a, m, n, and b are equal to 1; NH and the sum of the variables X, y and Z does not exceed 12; wherein Q and T are each independently selected from the group consisting R'(CO)NR'' ,-(CO)NR' , – NR' In the guanylhydrazone containing compound, X', X, X. 10 (CO)— —NR' , salts thereof. -O-, optionally substi and X may each individually adopt the ortho, meta or para tuted alkylene, optionally Substituted arylene, optionally Sub position on the phenylene ring relative to the Z group. In stituted heteroarylene, and combinations thereof; another embodiment, the X', X, X, and X’ are meta or para and wherein A", R,R,R,R,R,R, A, R', and R'' are to the Zgroup. In another embodiment, the non-HX', X, X, defined herein; and X groups are meta to both the Z group and to each other. 15 with the proviso that if Q is NR' or O-andy is 1 As used herein, the formula " NH(CO)— includes the then T is not NR' or —O : “ (CO)NH isomer. and with the proviso that if Q is —(CO)NR' , NR' In one embodiment, at least one of X, X, X and X* is (CO)—, NR' , or O-andy is 1 then T is not —(CO) GhyCH or GhyCCH. , X' and X’ are not simultaneously NR' NR' (CO) NR' , or - O -. H, and X and X’ are not simultaneously H. In another embodiment, Z has the formula: In another embodiment, X', X, X, and X’ are selected from the group including GhyCH or GhyCCH . In another embodiment, X', X, X, and X’ are selected wherein Q and T are each independently selected from the from the group including GhyCH , GhyCCH , or group consisting R'(CO)NR'-, -NR' (CO) , CHCO-. 25 —NR' , salts thereof, —O , optionally substituted alky In another embodiment, X', X, X, and X’ are each lene, optionally substituted arylene, optionally Substituted GhyCH-. heteroarylene, and combinations thereof. In another embodiment, the X', X, X, and X’ are each wherein A. R. R. R. R. R. R7, A, R', and R'' are GhyCCH . defined herein; In another embodiment, the X', X, X, and X’ are each 30 wherein if substituted, the alkylene, arylene, and/or het CHCO eroarylene are each independently substituted with 0 to 4 In another embodiment, at least one of X, X, X, and X* groups selected from the group consisting of H, halogen, OR, is CHCO-. NR'R'', NRCO, CONR, COR', SR', SOR', SONR', In one embodiment, the compound is in the salt form. SOR', alkyl, aryl, heteroalkyl, and heteroaryl, salts thereof, In another embodiment, the compound is in the salt form 35 and combinations thereof; having a compound:Salt ratio of 1:1, 1:2, 1:3, 1:4 or 2:1. R" and R' being each independently selected from the In one embodiment, Z has the formula: group including hydrogen, hydroxy, halo, bromo, chloro, iodo, fluoro, N, CN, NC. —SH, NO, NH, (C-Co.)alkyl, phenyl, (C-Co)cycloalkyl, (C-Co.)alkoxy, wherein each of the variables a, m, n, and b are equal to 1; 40 (C-Cs)heteroaryl, (C-Cs)heterocyclic, (C-C)alkenyl, and the Sum of the variables X, y and Z does not exceed 12; (Cs-Co) cycloalkenyl, (C-Co.)alkynyl, (Cs-Co)cycloalky and wherein Q, T., A', R. R. R. R. R. R7, Al, R', and nyl, (C-Cs)aryl, perhalo (C-C)alkyl, (C-C)alkyl-O-, R'' are defined herein. phenyl-O-, (C-C)cycloalkyl-O-, (C-Cs)heteroaryl In one embodiment, Z has the formula: O—, (C-Cs)heterocyclic-O-, (C-Co.)alkenyl-O-, (C- 45 Co) cycloalkenyl-O-, (C-C)alkynyl-O-, (C-C)cy cloalkynyl-O-, (Cs-Cs)aryl-O-, perhalo(C-C)alkyl wherein each of the variables a, m, n, and b are equal to 1; O—, (C-C)alkyl-S—, phenyl-S , (C-C)cycloalkyl and the Sum of the variables X, y and Z does not exceed 12; S—, (C-Cs)heteroaryl-S—, (C-Cs)heterocyclic-S , wherein Q and T are each independently selected from the (C-C)alkenyl-S , (C-C)cycloalkenyl-S-, (C-C) group consisting R'(CO)NR'-, -(CO)NR' , NR' 50 alkynyl-S , (Cs-Co)cycloalkynyl-S—, (Cs-Cs)aryl-S , (CO) , NR' , salts thereof, —O , optionally substi perhalo(C-C)alkyl-S—, (C-Co.)alkyl-SO , phenyl tuted alkylene, optionally Substituted arylene, optionally Sub SO. , (C-C)cycloalkyl-SO , (C-C)alkoxy-SO , stituted heteroarylene, and combinations thereof; (C-Cs)heteroaryl-SO , (C-Cs)heterocyclic-SO , and wherein A', R,R,R,R,R,R7, A, R', and R'' are (C-C)alkenyl-SO. , (C-C) cycloalkenyl-SO , (C- defined herein. 55 Co)alkynyl-SO , (C-C)cycloalkynyl-SO (Cs-Cs) In one embodiment, Z has the formula: aryl-SO. , perhalo(C-C)alkyl-SO. , HN SO , (C-Co.)alkyl-NH SO , phenyl-NH-SO , (C-C) cycloalkyl-NH-SO , (C-Co.)alkoxy-NH SO , (C- wherein each of the variables a, m, n, and b are equal to 1; Cs)heteroaryl-NH SO (C-Cs)heterocyclic-NH and the Sum of the variables X, y and Z does not exceed 12; 60 SO , (C-C)alkenyl-NH-SO , (C-C20) wherein Q and T are each independently selected from the cycloalkenyl-NH-SO. , (C-C)alkynyl-NH-SO , group consisting R'(CO)NR'-, -(CO)NR' , NR' (Cs-Co)cycloalkynyl-NH-SO , (Cs-Cs)aryl-NH (CO) , NR' , salts thereof, —O , optionally substi SO. , perhalo(C-Co.)alkyl-NH-SO. , {(C-C) tuted alkylene, optionally Substituted arylene, optionally Sub alkyl-N-SO , {phenyl)-N-SO , {(C-C20) stituted heteroarylene, and combinations thereof; 65 cycloalkyl-N-SO. , {(C-Co)alkoxy},N-SO , and wherein A", R. R. R. R. R. R7, A, R', R'' are {(C-C2s)heteroary1}N SO , {(C-C2s) defined herein; heterocyclic N–SO. , {(C-Co)alkenyl-2N SO ,

US 8,314,149 B2 21 22 (C-Co)alkyl-N-(C=O)—, {(C, -Co)alkoxy}{(C-C) alkoxycarbonyl groups, alkylaminocarbonyl groups, dialky alkyl-N-(C=O) , {(C-Cs)heteroaryl{(C-C) lamino carbonyl groups, arylcarbonyl groups, aryloxycarbo alkyl-N-(C=O) , {(C-C2s)heterocyclic}{(C-Co) nyl groups, alkylsulfonyl groups, arylsulfonyl groups and the alkyl-N-(C=O) , {(C-C20)alkenyl}{(C-Co) like. alkyl-N-(C=O) , {(Cs-Co)cycloalkenyl}{(C-C) As used herein, the term, “alkylene' refers to a diradical alkyl-N-(C=O) , {(C-C20)alkynyl) {(C-Co) alkane species that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, alkyl-N-(C=O) , ((Cs-Co)cycloalkynyl) {(C-C) 13, 14, 15, 16, 17, 18, 19 and 20 carbons or any subrange of alkyl-N-(C=O) , (C-C)ary1}{(C-C)alkyl-N- carbons therebetween. The alkylene may be branched or (C=O) , (perhalo(C-Co)alkyl{(C-Co)alkyl)-N- unbranched, saturated or unsaturated, and substituted or 10 unsubstituted. In addition, any carbon atom therein may be (C=O)—, salts thereof, and a combination thereof. optionally replaced with one or more heteroatoms such as In another embodiment, each of said alkylene, cycloalky nitrogen, oxygen or sulfur or any combination thereof. lene or arylene in said Q and/or T may be independently As used herein, the term, "cycloalkylene' refers to a diradi substituted with one or more substituent groups selected from cal cycloalkane species that contains 3, 4, 5, 6,7,8,9, 10, 11, the group consisting of phenyl-N-(C=O) , (C-C) 12, 13, 14, 15, 16, 17, 18, 19 and 20 ring carbons or any 15 subrange of carbons therebetween. The cycloalkylene may be branched or unbranched, saturated or unsaturated, and sub stituted or unsubstituted. In addition, any carbonatom therein may be optionally replaced with one or more heteroatom such alkenyl phenyl-N-(C=O) , (C-C20) as nitrogen, oxygen or sulfur or any combination thereof. cycloalkenyl phenyl-N-(C=O) , (C-C20) As used herein, the term “arylene' means an aromatic alkynyl) phenyl-N-(C=O) , (Cs-Co) diradical species having 5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, cycloalkynyl)phenyl-N-(C=O) , (Cs-C2s) 17, 18, 19, 20, 21, 22,23, 24 and 25 carbons and any subrange aryl phenyl-N-(C=O) , {perhalo(C-Co) of carbons thereof. These may be unsubstituted or substituted alkyl phenyl-N-(C=O) , salts thereof, and a as indicated herein. In addition, any carbon atom therein may combination thereof. 25 be optionally replaced with one or more heteroatom such as In another embodiment, each of said alkylene, cycloalky nitrogen, oxygen or sulfur or any combination thereof to form lene or arylene in said Q and/or T may be independently a heteroarylene. substituted with one or more substituent groups selected from As used herein, the term “alkyl” as well as the alkyl moi the group consisting of HO—(C=O) , (C-C)alkyl eties of or within other groups referred to herein (e.g., (C- (C=O) , (C-Cs)heteroaryl-(C=O) , (C-Cs)hetero 30 Co)alkyl, (C-Co)alkoxy, (C-Co.)alkenyl, (C-Co.)alky cyclic-(C=O) , (C-Co.)alkenyl-(C=O) , (C-C) nyl, and perhalo(C-Co.)alkyl) include alkyl moieties having cycloalkenyl-(C=O) , (C-C)alkynyl-(C=O) , (C- 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and C2s)aryl-(C=O)—, perhalo (C-Co)alkyl-(C=O) , phe 20 carbons or any subrange of carbons therebetween. They nyl-(C=O) , and a combination thereof. may be linear or branched (such as methyl ethyl, n-propyl. In another embodiment, each of said alkylene, cycloalky isopropyl. n-butyl, iso-butyl, secondary-butyl, tertiary-butyl, lene or arylene in said Q and/or T may be independently 35 etc.). They may be saturated or unsaturated as indicated by the substituted with one or more substituent groups selected from “alkenyl' or "alkynyl' terminology. Other than the perha the group consisting of (C-C)alkyl-O-(C=O) , (C- loalkyl, which are completely substituted by one or more of C2s)heteroaryl-O-(C=O)—, (C-Cs)heterocyclic-O- the same or different halogens, the alkyl groups may be (C=O) , (C-Co.)alkenyl-O-(C=O) , (C-C) unsubstituted or substituted as indicated herein. cycloalkenyl-O-(C=O) , (C-Co.)alkynyl-O- 40 As used herein, the term "cycloalkyl” as well as the other (C=O) , (Cs-Cs)aryl-O-(C=O) , perhalo(C-C) moieties having cyclic groups referred to herein (for example alkyl-O-(C=O) , phenyl-O (C=O)—, and a combina (C-C20)cycloalkyl, (C-C20) cycloalkenyl and (Cs-Co)cy tion thereof. cloalkynyl) refers to mono carbocyclic moieties having 3, 4, In another embodiment, each of said alkylene, cycloalky 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 ring lene or arylene in said Q and/or T may be independently 45 carbons or any Subrange of carbons therebetween. They may substituted with one or more substituent groups selected from be unsubstituted or substituted as indicated herein. the group consisting of (C-C)alkyl-(C=O)-O-, (C- As used herein, the terms, “alkenyl,” “alkynyl.” Cs)heteroaryl-(C=O) O , (C-C2s)heterocyclic “cycloalkynyl.” and “cycloalkenyl refer to unsaturated radi (C=O)-O-, (C-C)alkenyl-(C=O)-O-, (C-C) cal species having 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, cycloalkenyl-(C=O) O , (C-C)alkynyl-(C=O)– 50 16, 17, 18, 19 and 20 carbons (or, for the cyclic species 3, 4, O—, (C-Cs)aryl-(C=O)-O-, phenyl-(C=O) O-, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20 ring perhalo(C-Co.)alkyl-(C=O)—O , and a combination carbons) or any subrange of carbons or ring carbons therebe thereof. tween. They may be branched or unbranched, and they may When the Z group or any of its constituent A, Q, T, or CRR be unsubstituted or substituted as indicated herein. These groups are substituted, the substituent is preferably a phar groups have one or more than one site of unsaturation, i.e., maceutically acceptable or suitable substituent. This type of 55 one or more double or triple bonds. For example, these moi Substituent is intended to mean a chemically and pharmaceu eties may have one, two, three, four or more sites of unsat tically acceptable functional group (e.g., a moiety that does uration. Some nonlimiting examples of these include ethenyl, not negate the desired activity of the active compound.) 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1- In one embodiment, suitable pharmaceutically acceptable propenyl, 1-butenyl, 2-butenyl, ethynyl, 1-propynyl 2-pro Substituents include, but are not limited to halo groups, per 60 pynyl, 1-butynyl, and 2-butynyl. fluoroalkyl groups, perfluoroalkoxy groups, alkyl groups, As used herein, the term, “alkoxy” refers to alkylO radical alkenyl groups, alkynyl groups, hydroxy groups, oxo groups, species having 1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, mercapto groups, alkylthio groups, alkoxy groups, aryl or 17, 18, 19 and 20 carbons or any subrange of carbons ther heteroaryl groups, aryloxy or heteroaryloxy groups, aralkyl ebetween. They may be unsubstituted or substituted as indi or heteroaralkyl groups, aralkoxy or heteroaralkoxy groups, 65 cated herein. HO-(C—O)— groups, amino groups, alkyl- and dialky As used herein, the term “halogen' or “halo' includes lamino groups, carbamoyl groups, alkylcarbonyl groups, fluoro, chloro, bromo or iodo, and any combination thereof. US 8,314,149 B2 23 24 As used herein, the term “aryl means aromatic radicals -continued having 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, II O R O 21, 22, 23, 24 and 25 carbons and any Subrange of carbons | thereof. These may be unsubstituted or substituted as indi HO-C-C-C*-no-C-OH cated herein. Nonlimiting examples include phenyl, naph thyl, tetrahydronaphthyl, indanyl and the like. R As used herein, the term "heteroaryl” refers to an aromatic heterocyclic group with at least one heteroatom selected from In the carboxylic acid formulas I and II above, C* repre O, S and N in the ring and having 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, sents a potentially chiral carbon that can be in either the D or 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 and 25 ring 10 L enantiomeric configuration, and R represents a Suitable carbons and any Subrange of carbons thereof. The heteroat Substituent such as, but not limited to, hydrogen (H), or oms may be present either alone or in any combination. The methyl (CH) or other alkyl. The “0-20' range includes all heteroaryl groups may be unsubstituted or Substituted as indi values and Subranges therebetween, including 0, 1, 2, 3, 4, 5, cated herein. One, two, three, four or more heteroatoms may be present. In addition to said heteroatom, the aromatic group 15 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20. may optionally have up to four Natoms in the ring. Nonlim In the carboxylic acid formulas I and II above, a “suitable iting examples of heteroaryl groups include pyridyl, pyrazi Substituent is intended to mean a functional group that does nyl, pyrimidinyl, pyridaZinyl, thienyl, furyl, imidazolyl pyr not negate the intended activity of the active guanylhydrazone rolyl, oxazolyl (e.g., 1.3-oxazolyl, 1.2-oxazolyl), thiazolyl compound in the salt. Nonlimiting examples of Suitable Sub (e.g., 1,2-thiazolyl, 1.3-thiazolyl), pyrazolyl, tetrazolyl, tria stituents include halo groups, perfluoroalkyl groups, perfluo Zolyl (e.g., 1,2,3-triazolyl, 1,2,4-triazolyl), oxadiazolyl (e.g., roalkoxy groups, alkyl groups, alkenyl groups, alkynyl 1.2.3-oxadiazolyl), thiadiazolyl (e.g., 1,3,4-thiadiazolyl), groups, hydroxy groups, oxo groups, mercapto groups, alky quinolyl, isoquinolyl, benzothienyl, benzofuryl, indolyl, and lthio groups, alkoxy groups, aryl or heteroaryl groups, ary the like; which are optionally unsubstituted or substituted loxy or heteroaryloxy groups, aralkyl or heteroaralkyl with one or more substituent groups as indicated herein. 25 groups, aralkoxy or heteroaralkoxy groups, HO—(C O)— The term "heterocyclic” as used herein refers to a cyclic groups, amino groups, alkyl- and dialkylamino groups, car group containing 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, bamoyl groups, alkylcarbonyl groups, alkoxycarbonyl 17, 18, 19, 20, 21, 22, 23, 24 and 25 ring carbons and any groups, alkylaminocarbonyl groups, dialkylamino carbonyl Subrange of carbons thereof carbon atoms and hetero atoms groups, arylcarbonyl groups, aryloxycarbonyl groups, alkyl selected from N, O, S or NR'. Nonlimiting examples include 30 Sulfonyl groups, arylsulfonyl groups and the like. Mixtures aZetidinyl, tetrahydrofuranyl, imidazolidinyl, pyrrolidinyl, are possible. piperidinyl, piperazinyl, oxazolidinyl, thiazolidinyl, pyrazo lidinyl, thiomorpholinyl, tetrahydrothiazinyl, tetrahydrothia More particularly, in the carboxylic acid formulas I and II diazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl, oxazi above, each R may be independently selected from Y— nyl, Oxathiazinyl, indolinyl, isoindolinyl, quinuclidinyl, 35 Y O— Y S Y SO (Y). N SO , chromanyl, isochromanyl, benzoxazinyl and the like. Y (C=O) , Y-(C=O)O. , Y O (C=O) , (Y)- Examples of such monocyclic Saturated or partially saturated N Y (C=O)–(Y N) , (Y (C=O)) N-, ring systems are tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, Y—(SO)(Y N)—, or (Y—(SO)) N. ; wherein two imidazolidin-1-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyr independently chosen Y alkyl-containing groups may be rolidin-1-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-1-yl, 40 taken together with any nitrogen atom to which they are piperidin-2-yl, piperidin-3-yl, piperazin-1-yl, piperazin-2-yl, attached to form a three to twelve membered cyclic, hetero piperazin-3-yl, 1.3-oxazolidin-3-yl, isothiazolidine, 1,3-thia cyclic or heteroaryl ring, and each Y is independently selected Zolidin-3-yl, 1.2-pyrazolidin-2-yl, 1.3-pyrazolidin-1-yl, thio from hydrogen, carboxyl, halo, hydroxyl, thiol, nitro, amine, morpholinyl, 1,2-tetrahydrothiazin-2-yl, 1,3-tetrahydrothi NC , (C-C)alkyl, (C-C)cycloalkyl, (C-C)alkenyl, azin-3-yl, tetrahydrothiadiazinyl, morpholinyl, 1.2- 45 (C-C) cycloalkenyl, (C-C)alkynyl, (C-C)cycloalkynyl, tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, 1,4-oxazin (C-C)alkoxy, (C-C)aryl, (C-Cs)heteroaryl, and (C-C) 2-yl, 1,2,5-oxathiazin-4-yl and the like; which may be heterocyclic; wherein each of the aforesaid (C-C)alkyl, unsubstituted or optionally substituted with one or more sub (C-C)cycloalkyl, (C-C)alkenyl, (C-C) cycloalkenyl, stituents as indicated herein. (C-C)alkynyl, (C-C)cycloalkynyl, (C-C)alkoxy, (Cs In another embodiment, any of the guanylhydrazone-con 50 C.)aryl, (C-Cs)heteroaryl, and (C-Cs)heterocyclic Sub taining compounds of this invention, with or without addi stituents may be optionally and independently Substituted, as tional active agents, may beformulated into therapeutic com in for instance a halo-substituted alkyl, by one to ten moieties positions as natural or salt forms. independently selected from the group consisting of car According to one embodiment, a guanylhydraZone salt boxyl, halo, hydroxyl, thiol, nitro, amine, NC , (C-C) according to the invention includes one or more guanylhy 55 alkyl, (C-C)alkoxy, (C-C)alkenyl, and (C-C)alkynyl. draZone compounds combined with a carboxylic acid. One According to another embodiment, the carboxylic acid in suitable carboxylic acid for the salt combination includes a the guanylhydrazone salt is described by the following gen chemical structure having one of the following formulas (I eral formula (III): and II): 60 III O RC-C-OH 65 wherein C* and each R, independently, are defined as described for the carboxylic acid formulas I and II above. In US 8,314,149 B2 25 26 one aspect of this embodiment the carboxylic acid is acetic wherein C* and Rare defined as described for the carboxylic acid: acid formulas I and II above. In one aspect of this embodiment the carboxylic acid is L-glutamic acid:

O 5 O H H H O (H)3C-C-OH | | | | || Acetic Acid HO- ----e-oil H H NH2 According to another embodiment, the carboxylic acid in 10 L-Glutamic Acid the guanylhydrazone salt according to the invention is described by the following general formula (IV): According to another embodiment, the guanylhydra Zone—carboxylic acid salts according to the invention IV 15 include a guanylhydrazone compound combined with a car H. O boxylic acid wherein a suitable carboxylic acid for the salt | s: || combination, according to this embodiment, is a chemical (R)-C-C-C-OH structure described by the following general formula (VII): OH 2O VII wherein C* and Rare defined as described for the carboxylic O O acid formulas I and II above. In one aspect of this embodiment the carboxylic acid is L-lactic acid: HO-C-L-C-OH wherein Lisa diradical moiety selected from a (C-C)alkyl, H. O (Cs-Co)cycloalkyl, (C-Co)alkenyl, (Cs-Co) cycloalkenyl, | || (C-Co.)alkynyl, (C-C)cycloalkynyl, (C-Co.)alkoxy/ ac--c-oil thiol, (C-C)aryl, (C-Cs)heteroaryl, (C-Cs)heterocy OH clic and (C-C)cycloalkyl, wherein each of the aforesaid 3 (C-C-o)alkyl, (C-Co)cycloalkyl, (C-Co)alkenyl, (C- L-Lactic Acid Co) cycloalkenyl, (C-Co.)alkynyl, (C-Co)cycloalkynyl, (C-Co.)alkoxy/thiol, (C-C)aryl, (C-Cs)heteroaryl, (C- According to another embodiment, the carboxylic acid in Cs)heterocyclic and (C-C)cycloalkyl diradical moieties may optionally be substituted, for example, with a halo-sub the guanylhydrazone salt according to the invention is 35 stituted alkyl, by one to twenty moieties independently described by the following general formula (V): selected from the group including carboxyl, halo, hydroxyl, thiol, nitro, amine, Y or R wherein these terms are defined V herein. O R. R. O According to another embodiment, the anion oracid for the | | | || 40 guanylhydrazone salt is described by the following general to-----oil formulas: R. R. wherein C* and Rare defined as described for the carboxylic f s: s: acid formulas I and II above. In one aspect of this embodiment 45 R -- C--S-O| 020 || O-S--(-)-S-O| 020 || the carboxylic acid is L-Aspartic Acid: R O O R. O

O H H O O-S--C - C-OH | | | | || 50 | HO- ---c-ol O R. H NH2 wherein C and R represent suitable substituents as defined in L-Aspartic Acid the formulas I and II above. 55 According to another embodiment, the anion or acid in the - 0 guanylhydraZone salt according to the invention is described According to another embodiment, the carboxylic acid in by the following general formula: the guanylhydrazone salt according to the invention is described by the following general formula (VI): O 60 VI RC'-S-O O R. R. R. O | | | | || O HO-C-C-C-C-C-OH 65. In one aspect of this embodiment, the anion is mesylate. One embodiment includes a salt wherein a guanylhydra Zone compound is combined with L-lactic acid. In another US 8,314,149 B2 27 28 embodiment, the salt is guanylhydraZone compound contain expression of Bax protein, to reduce or downregulate the ing multiple guanylhydrazone moieties and combined with release or expression of Bad protein, to reduce or down L-lactic acid. In another embodiment, the invention relates to regulate the release or expression of COX-2 protein, or to a salt combining the semapimod compound with L-lactic reduce or downregulate the release or expression of RAGE, or acid. a combination thereof, for example. The term “therapeuti Other Suitable salts include acid addition salts of a guanyl cally effective amount also refers to an amount of a guanyl hydrazone compound, and in particular semapimod. Suitable hydrazone compound which is effective in providing inhibi acids which are used to prepare the acid addition salts of the tor or preventive activity in a patient in need thereof or guanylhydraZone compounds invention are those which form reasonably expected to be in need thereof. As used herein the non-toxic acid addition salts, i.e., salts containing pharmaco 10 term “inhibiting does not necessarily refer to a total elimi logically acceptable anions, such as the bromide, iodide, nation of the malady or condition. nitrate, Sulfate, bisulfate, phosphate, acid phosphate, citrate, A therapeutically effective amount can be readily deter acid citrate, tartrate, bitartrate. Succinate, fumarate, tosylate, mined by the attending diagnostician, as one skilled in the art, mesylate, gluconate, saccharate, benzoate, methane by the use of known techniques and by observing results Sulfonate, ethanesulfonate, benzenesulfonate, p-toluene 15 obtained under analogous circumstances. In determining the Sulfonate and pamoate (i.e., 1,1'-methylene-bis-(2-hydroxy therapeutically effective amount or dose, a number of factors 3-naphthoate)) salts. are considered by the attending diagnostician, including, but Other salts can also be made using a guanylhydraZone, not limited to: the species of mammal; its size, age, and Such as semapimod and benzensulfonate, benzoate, bicarbon general health; the specific disease involved; the degree of or ate, bitartrate, edetate, camsylate, carbonate, citrate, dihydro involvement or the severity of the disease; the response of the chloride, edentate, edisylate, estolate, esylate, fumarate, glu individual mammal; the particular compound administered; ceptate, glucoheptonate, gluconate, glutamate, the bioavailability characteristics of the preparation adminis glycolylarsanilate, hexylresorcinate, hydrabamine, hydro tered; the dose regimen selected; the use of concomitant bromide, hydrochloride, hydroxynaphthoate, chloride, bro medication; and other relevant circumstances. mide, iodide, isethionate, lactate, lactobionate, malate, man 25 A therapeutically effective amount of the guanylhydra delate, mesylate, methylbromide, methylnitrate, Zone-containing compound may range from about 0.0001 methylsulfate, mucate, napsylate, nitrate, pamoate, pantoth milligram per kilogram of body weight per day (mg/kg/day) enate, phosphate or diphosphate, polygalacturonate, salicy to about 10,000 mg/kg/day. Preferred amounts may range late, Stearate, Subacetate. Succinate, Sulfate, tannate, teoclate, from about 0.001 to about 100 mg/kg/day. These ranges and triethiodide salts. 30 include all values and Subranges therebetween, including Other Suitable salts include Sodium, potassium, ammo 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, nium, calcium, or ferric hydroxide salts, and isopropylamine, 6, 7, 8, 9, 10, 100, 1,000, 5,000, and 10,000 mg/kg/day, and trimethylamine, 2-ethylamino ethanol, histidine, procaine, any combination thereof. hydrochloric or phosphoric acids, organic acids such as ace The guanylhydrazone compound according to the inven tic, oxalic, tartaric, mandelic, and the like. 35 tion can be administered to the mammal in any form or mode Any ratio of guanylhydrazone:counterion in the salt form, which makes the compound bioavailable in effective for example, guanylhydraZone:counterion ratios of 10,9,8,7, amounts, including oral and parenteral routes. For example, 6, 5, 4, 3, 2, 1:1, 2, 3, 4, 5, 6, 7, 8, 9, 10 is suitable. The ratio semapimod can be administered orally, intracerebroVentricu can be expressed as the number of "Ghy' groups:counterions larly, Subcutaneously, intramuscularly, intravenously, trans or as the number of ionic guanylhydrazone molecules:coun 40 dermally, intranasally, rectally, topically, intramucosaly, terions as appropriate. In one embodiment, either the guanyl intravaginally, parenterally, and the like. Oral, topical, intra hydrazone or the counterion or both may be multivalent, and venous or intramuscular administration is generally pre the ratio is adjusted accordingly such that the salt may adopt ferred. One skilled in the art of preparing formulations can a Zero or non-Zero charge. Mixed salts are possible. readily select the proper form and mode of administration The term “treating as used herein includes inhibition or 45 depending upon the particular characteristics of the com prophylaxis of the named condition or amelioration or elimi pound selected, the disease state to be treated, the stage of the nation of the condition once it has been established. As noted disease, and other relevant circumstances. above, the guanylhydraZone compounds described herein are The compounds can be administered alone or in the form of useful for agricultural or veterinary health purposes as well as a pharmaceutical composition in combination with pharma for the treatment of a human patient. 50 ceutically acceptable carriers or excipients, the proportion As used herein, the term “therapeutically effective and nature of which are determined by the solubility and amount refers to an amount of the compound or salt or a chemical properties of the compound selected, the chosen combination of compounds or salts which is effective, upon route of administration, and standard pharmaceutical prac single or multiple dose administration or continuous admin tice. The compounds of the invention, while effective them istration, infusion or application to the patient, for the inhibi 55 selves, may beformulated and administered, where appropri tion, treatment, and/or prevention of NEC, a condition asso ate, in the form of their pharmaceutically acceptable acid ciated with the release of HMGB1, a condition associated addition salts for purposes of stability, convenience of crys with the release of iNOS protein, a condition associated with tallization, increased solubility and the like. the release of Bax protein, a condition associated with the These compounds and compositions can be administered release of Bad protein, a condition associated with the release 60 to mammals for veterinary use. For example, domestic ani of COX-2 protein, or a condition associated with the release mals can be treated in much the same way as a human clinical of RAGE, or a combination thereof, and/or maladies associ patient. In general, the dosage required for therapeutic effect ated with low-birth weight, formula feeding, bacterial colo will vary according to the type of use, mode of administration, nization of the gut, or hypoxia, or a combination thereof, as well as the particularized requirements of the individual and/or to reduce or downregulate the release or expression of 65 hosts. Typically, dosages will range from about 0.0001 to HMGB1, to reduce or downregulate the release or expression 10,000 mg/kg, and more usually 0.001 to 100 mg/kg of the of iNOS protein, to reduce or downregulate the release or host body weight. These ranges include all values and Sub US 8,314,149 B2 29 30 ranges therebetween, including 0.0001, 0.0005, 0.001, 0.005, Biotechnology (Lake Placid, N.Y.), the rabbit polyclonal 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 100, 1,000, anti-iNOS antibody was from BD Transduction Laboratories 5,000, and 10,000 mg/kg, and any combination thereof. (Lexington, Ky.), the rabbit polyclonal anti-RAGE was from Alternatively, dosages within these ranges can be admin Affinity BioReagents (Golden, Colo.), and the anti-p38 and istered by constant infusion over an extended period of time, 5 anti-phospho-p38 were from Cell Signaling Technology for example, exceeding 24 hours, until the desired therapeu (Beverly, Mass.). tic, preventive, and/or inhibiting benefits are obtained. Animal Model of NEC Indeed, drug dosage, as well as route of administration, All the experiments were carried out according to an ani should be selected on the basis of relative effectiveness, rela mal protocol approved by the Animal Research and Care tive toxicity, microbial infection size, presence of other infec 10 Committee (ARCC) of the Children’s Hospital of Pittsburgh. tions, and effect of the guanylhydrazone compound on cell Pregnant time-dated Sprague-Dawley rats (Charles River cycle, drug pharmacokinetics, age, sex, physical condition of Labs, Wilmington, Mass.) were induced at term using a Sub the patient and prior treatment. cutaneous injection (1 to 2 Uper animal) of Pitocin R (Mon The suitability of particular carriers for inclusion in a given arch Pharmaceuticals, Bristol, Tenn.). Immediately after therapeutic composition depends on the preferred route of 15 birth, the neonates were weighed and randomized into one of administration. For example, compositions may be formu the different treatment groups. Group 1 consisted of neonatal lated for oral administration. Such compositions are typically rats left with their mother, and thus they were breast-fed. prepared as liquid solution or Suspensions or in Solid forms. Group 2 consisted of neonates separated from their mothers, Oral formulations usually include such additives as binders, housed in a temperature and humidity controlled incubator fillers, carriers, preservatives, stabilizing agents, emulsifiers, (Ohio Medical Products, Madison, Wis.) and gavaged with a buffers, mannitol, lactose, starch, magnesium Stearate, special rodent formula (0.2 ml, see below) two times per day Sodium saccharin, cellulose, magnesium carbonate, and the and subjected to 10 min. of hypoxia (5% O2, 95% N) (Prax like. These compositions may take the form of Solutions, Air, Pittsburgh, Pa.) thrice daily in a modular Chamber (Bil Suspensions, tablets, pills, capsules, Sustained release formu lups-Rothenberg Inc, Del Mar, Calif.) as follows: pups were lations, or powders, and typically contain 1% to 95% by 25 fed in the morning post-hypoxia, exposed to a 2" hypoxic weight of active ingredient. More preferably, the composition insult after 4 hrs, and then subjected to the final hypoxic insult contains from about 2% to about 70% active ingredient. followed by the final feed. Rats in Group 3 were treated in a These ranges include all values and Subranges therebetween, similar fashion to those in Group 2; however the animals including 1,2,3,4,5,6,7,8,9, 10, 20, 30, 40, 50, 60, 70, 80, received the experimental drug semapimod (0.1-1 mg/kg, 90, and 95%, and any combination thereof. 30 i.p., once daily, vehicle: 5% dextrose) right before hypoxia. Suitable compositions may be prepared as injectables, The formula composition consists of 15 g. Similac 60/40 either as liquid solutions, suspensions, or emulsions; solid (Ross Pediatrics, Columbus, Ohio) in 75 ml of Esbilac canine forms suitable for Solution in or Suspension in liquid prior to milk replacer (Pet-Ag Inc., Hampshire, Ill.) as described by injection. Such injectables may be administered Subcutane Barlow etal. (5), and was designed to approximate the protein ously, intravenously, intraperitoneally, intramuscularly, 35 and caloric content of rat breast milk. The rats were sacrificed intrathecally, or intrapleurally. The active ingredient or ingre on different days as indicated and the intestinal samples (seg dients are often mixed with diluents, carriers, or excipients ments ofterminal ileum for the present study) were harvested which are physiologically tolerable and compatible with the for morphological studies and Western blotting as described active ingredient(s). Suitable diluents and excipients are for below. example, water, Saline, dextrose, glycerol, or the like and 40 Morphologic Evaluation of Intestinal Samples combinations thereof. In addition, if desired, the composi Rats were sacrificed on different days as indicated and the tions may contain minor amounts of auxiliary Substances distal ileum was harvested for morphological studies. For Such as wetting or emulsifying agents, stabilizing or pH buff light microscopy, hematoxylin and eosin slides were prepared ering agents. per standard protocol (15). A pathologist from Children's Semapimod is a known compound. As for the other gua 45 Hospital of Pittsburgh (Dr. R. Jaffe) blinded to the experimen nylhydrazone compounds and salt molecules described tal groups graded the morphological changes in the intestinal herein, suitable methods for obtaining them may be found in epithelium. The criteria for each histological grade (0-3) were U.S. Provisional Application Ser. Nos. 60/601,992, filed Aug. as follows: (0) normal: no pathologic change, different epi 17, 2004, and 60/582,532, filed Jun. 25, 2004, incorporated thelial patterns are noted: clear, vacuolar and inclusion-type; herein by reference. 50 (1) mild: occasional neutrophils, separation of villus core and mild damage to enterocytes; (2) moderate: Submucosal EXAMPLES edema, epithelial sloughing and marked presence of neutro phils; and (3) severe: denudation of epithelium with loss of Materials and Methods villi and transmural necrosis or perforation (32). 55 Western Blotting Reagents Newborn rats were sacrificed as indicated and segments of LPS from Escherichia coli 0127:B8 was from Sigma (St. the terminal ileum were isolated. The mucosa was gently Louis, Mo.). Peroxynitrite was from Alexis Biochemicals, scraped from each segment and immediately placed in cold San Diego, Calif. The drug semapimod was kindly provided lysis buffer containing 62.5 mM Tris (pH 6.6), 10% glycerol, by Cytokine PharmaSciences, Inc., King of Prussia, Pa. The 60 1% sodium dodecyl sulfate (SDS), and protease inhibitors (10 different antibodies used for Western blotting were purchased ug/ml leupeptin, 5 g/ml pepstatin, 2 Lig/ml aprotinin, and 0.5 from the following sources: the rabbit polyclonal anti-Bad mM phenylmethylsulfonyl fluoride (PMSF), all from Sigma, (sc-943) and anti-Bax (P-19) were from Santa Cruz, Biotech St Louis, Mo.). The samples were then homogenized and nology, Inc. (Santa Cruz, Calif.), the COX-2 (murine) poly boiled for 1 min. followed by centrifugation at 10,000xg for clonal antibody was from Cayman Chemical (Ann Arbor, 65 30 min. in order to remove the cell debris. Protein concentra Mich.), the anti-HMGB1 was a polyclonal rabbit antisera tion in the Supernatant was determined using the bicincho against native calf thymus HMG1/HMGB1 from Upstate ninic acid (BCA) Protein Assay kit from Sigma (St Louis, US 8,314,149 B2 31 32 Mo.) with bovine serum albuminas standard. Protein samples Results in graph bars are the meantS.E.M. (n=13 animals for (equivalent to 50 lug) were resolved on 12% SDS-polyacry each group, *P<0.05 vs. BF control, analyzed by one-way lamide gels using a BioRad mini-gel system (Hercules, ANOVA followed by Fisher's LSD Test). Calif.) and then electroblotted onto PVDF membranes (Mil As shown in FIG. 2, the administration of semapimod in lipore. Beford, Mass.). After blocking for 1 hr with milk (5% vivo is protective in an experimental NEC model. Newborn in PBS with 0.1% Tween-20) at room temperature, the mem rats separated from their mothers were fed a conventional branes were probed for 1 hr at room temperature with the formula and exposed to hypoxia after administration of the primary antibodies dissolved in 1% milk PBS/Tween at the drug semapimod (0.1-1 mg/kg, i.p.) as described in Methods. following dilutions: HMGB1 (1:1000), Bad (1:1000), Bax A segment of the terminal ileum of each rat was harvested on (1:500), iNOS (1:750), COX-2 (1:500) and RAGE (1:1000). 10 day 4 for morphological analysis as described. Incidence of The membranes were then thoroughly washed and incubated NEC was calculated as the percentage of animals displaying with the secondary antibody (horseradish peroxidase-conju pathology scores higher than 0 as assessed by histology. gated goat anti-rabbit or goat anti-mouse IgG, Pierce, Rock Results in graph bars are the mean+S.E.M. (n=15-45 animals ford, Ill.) at 1:15,000 dilution (in PBS/Tween with 1% milk) for each group, *P<0.05 vs. FFH without semapimod, ana for 1 hr prior to detection. Protein bands were visualized 15 lyzed by one-way ANOVA followed by Fisher's LSD Test). using a SuperSignalTM chemiluminescence substrate (Pierce, As shown in FIG. 3, the administration of semapimod in Rockford, Ill.) according to the manufacturers instructions. vivo decreases the expression of HMGB1 and its receptor Cell Culture and Western Blotting RAGE in ileal samples from formula-fed animals exposed to The rat intestinal crypt cell line IEC-6 was purchased from hypoxia. Newborn rats were randomized into two groups: the American Type Culture Collection (ATCC, Manassas, breast-fed (BF) animals were left with their mothers, for Va.). Cells were grown in tissue culture medium consisting of mula-fed pups were exposed to hypoxia without (FFH) or Dulbeco's modified Eagle's medium with 4.5gm/L glucose with semapimod (FFH+semapimod) as described in Meth (Bio-Whittaker, Walkersville, Md.) supplemented with 5% ods. The terminal ileum of each rat was harvested on day 4 fetal bovine serum (Bio-Whittaker), 0.02 mM glutamine and the mucosal scrapings were processed for protein isola (GIBCO; Grand Island, N.Y.), 0.1 U/ml insulin, 100 U/ml 25 tion followed by Western blotting. (a) A representative blot penicillin, and 100 ug/ml streptomycin (GIBCO) at 37° C. with 3 animals/group and densitometric analysis for HMGB1 and 10% CO. Cells were pretreated with semapimod (0.1-10 protein (30 kDa band). Results in graph bars are the uM solutions prepared in 5% dextrose) followed by short meantS.E.M. (n=6-13 animals for each group, *P<0.001 vs. exposure to LPS (5ug/ml), peroxynitrite (ONOO, 50 uM) or BF control, "P<0.05 vs. FFH group, analyzed by one-way cytomix (a combination of tumor necrosis factor (TNFC., 10 30 ANOVA followed by Fisher's LSD Test). (b) Morphological ng/ml), interleukin-1B (IL-3, 1 ng/ml), and interferon analysis of rat intestinal samples in experimental NEC at day gamma (IFNY, 1000 U/ml) for 10 min. Preparation of the 4. Panel A represents an ileal segment from a BF animal ONOO solution and cell treatment was as previously showing normal histology (the villi are tall and healthy). reported (36). The lysates were collected for Western blotting Panel B represents an ileal segment from a FFH animal show analysis as follows: proteins were extracted for 10 min at 4 35 ing fewer Goblet cells, Villus core separation and presence of C. with lysis buffer (20 mM Tris pH 8.0, 150 mM. NaCl, 1% inflammatory cells (black arrows). Panel C represents an ileal NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl segment from a FFH animal that received semapimod (0.75 Sulfate) Supplemented with protease and phosphatase inhibi mg/kg, i.p.) and displays a similar morphology as BF control. tors: 1 mM PMSF, 1 mM benzamidine, 50 g/ml aprotinin, (c) A representative blot with 3 animals/group and densito 0.5 mM NaVO 20 mM NaF, and 0.5 mM phenylarsine 40 metric analysis for RAGE protein (45 and 25 kDa bands) oxide. Aliquots containing 50 ug total protein were resolved performed as described. Results in graph bars are the by SDS-polyacrylamide gel electrophoresis. Gels were elec meantS.E.M. (n=9-24 animals for each group, *P=0.001 vs. troblotted onto nitrocellulose membranes. Following 1 hr BF control, "P=0.009 vs. FFH, **P=0.016 vs. BF control, incubation in blocking Solution (phosphate-buffered saline, analyzed by one-way ANOVA followed by Tukey Test). 0.1% Tween-20, 2% fish gelatin, pH 7.5), membranes were 45 As shown in FIG. 4, the administration of semapimod in incubated with primary (phospho-p38 and p38) and second vivo decreases the expression of the Bcl-2 family members ary horseradish peroxidase-conjugated antibodies as recom Bad and Bax in an experimental NEC model in rats. Newborn mended by manufacturers. Membranes were then impreg rats were randomized into three groups: BF represents breast nated with luminol reagent and exposed to X-ray film. fed animals left with their mothers (controls), FFH are for Statistical Analysis 50 mula-fed pups exposed to hypoxia, and FFH-i-Semapimod Results are expressed as meant-SEM or SD as indicated. represents animals exposed to hypoxia after administration of Differences among groups were analyzed by the Students the drug semapimod as described in Methods. The terminal t-test, Chi-square test or one-way analysis of variance ileum of each rat was harvested on day 4 and the mucosal (ANOVA) followed by Tukey Test or Fisher's Least Signifi scrapings were processed for protein isolation followed by cance Difference (LSD) Test where appropriate (SigmaS 55 Western blotting. (a) A representative blot with 3 animals/ tattM 2.03: SPSS, Chicago, Ill.). group and densitometric analysis for Bad protein. Results in Results graph bars are the mean+S.E.M. (n=17-22 animals for each As shown in FIG. 1, HMGB1 is elevated in formula-fed group, *P-0.001 vs. BF control, "P-0.001 vs. FFH group, animals exposed to hypoxia. Newborn rats were randomized analyzed by one-way ANOVA followed by Tukey Test). (b) A into two groups: breast-fed (BF) animals were left with their 60 representative blot with 3 animals/group and densitometric mothers and formula-fed pups were exposed to hypoxia analysis for Bax protein. Results in graph bars are the (FFH) as described in Methods. The terminal ileum of each meani.S.E.M. (n=4 animals for each group, *P<0.005 vs. BF rat was harvested on day 4 and the mucosal scrapings were control, "PK0.005 vs. FFH group, analyzed by one-way processed for protein isolation followed by Western blotting. ANOVA followed by Fisher's LSD Test). A representative blot with 3 animals/group and densitometric 65 As shown in FIG. 5, the administration of semapimod in analysis for HMGB1 protein (30 kDa band) and low molecu vivo decreases the expression of iNOS and COX-2 in an lar weight products (7-10kDa bands) performed as described. experimental NEC model in rats. Newborn rats were random US 8,314,149 B2 33 34 ized into three groups: BF represents breast-fed animals left Newborn rats were randomized into two groups: breast-fed with their mothers (controls), FFH are formula-fed pups (BF) animals were left with their mothers, and formula-fed exposed to hypoxia, and FFH-i-Semapimod represents ani pups were exposed to hypoxia (FFH) as described in Meth mals exposed to hypoxia after administration of the drug ods. The terminal ileum of each rat was harvested on the day semapimod as described in Methods. The terminal ileum of 5 indicated, fixed and stained for morphological analysis as each rat was harvested on day 4 and the mucosal scrapings described. The data presented in Table 1 is the percentage of were processed for protein isolation followed by Western rats in each group with incidence of intestinal damage graded blotting. (a) A representative blot with 3 animals/group and between 1 and 3. The numbers in parentheses are the total densitometric analysis for iNOS protein. Results in graph number of animals/group. 10 Morphological Changes of Intestinal Samples in Experimen bars are the meant S.E.M. (n=29 animals for each group, tal NEC *P<0.001 vs. BF control, "P=0.006 vs. FFH group, analyzed The present inventors have previously shown that morpho by one-way ANOVA followed by Tukey Test). (b) A repre logic analysis of ilea from 4 day-old formula-fed rats revealed sentative blot with 3 animals/group and densitometric analy various degrees of intestinal inflammation compared to ileal sis for COX-2 protein. Results in graph bars are the 15 segments from breast-fed controls. These inflammatory meantS.E.M. (n=16-27 animals for each group, *P<0.001 vs. changes closely resembled the histologicalterations observed BF control, "P-0.05 vs. FFH group, analyzed by one-way in human NEC samples (32). In order to determine the kinet ANOVA followed by Tukey Test). ics of the development of intestinal inflammation in this As shown in FIG. 6, semapimod decreases the activation of model, both breast-fed and formula-fed animals were sacri p38 MAP kinase by LPS in vitro. (a) Rat intestinal epithelial ficed on each consecutive day after birth and the last 2 cm of cells (IEC-6) were pretreated with different doses of the drug their distal ilea were harvested for morphological analysis. (0.1-10M) for 1 hr. prior to stimulation with 5 g/ml LPS for The specimens for day 0 were from animals sacrificed within 10 minor (b) were pretreated with semapimod (5uM) for 1 hr. 3 hr after birth and showed minimal morphological changes prior to stimulation with 5ug/ml LPS or ONOO (50 uM) for (18.8% had a histological score of <1, n=48 animals). The 10 min. The protein lysates were then collected and Western 25 overall incidence of morphological and pathological changes blotting analysis for phospho-p38 and p38 (loading control) characteristic of NEC is shown in Table 1. Intestines of was performed as described in Methods. (a) A representative breast-fed pups were normal and rarely showed abnormal blot for pp38/p38 and densitometric analysis showing the morphology. In contrast, in the FFH group morphological ratio of pp38/p38 band intensity. Ctrl are non-stimulated evidence of NEC was detected as early as day 1 (mainly cells. Results in graph bars are the meant S.E.M. (n=3 inde 30 villous core separation and presence of occasional neutro pendent experiments, *P<0.05 vs. LPS treated cells without phils) with moderate to severe damage at day 4 (histological pretreatment with semapimod, analyzed by one-way ANOVA scores higher than or equal to 1). Analysis of ileal specimens followed by Fisher's LSD Test). from FFH newborn rats sacrificed at day 5 or day 7 showed As shown in FIG. 7, HMGB1 protein is expressed in severe intestinal damage but not significantly different from human NEC. Representative ileal segments from four neo FFH animals on day 4 (not shown). Based on this observation, nates undergoing bowel resection for NEC and ileal speci 35 Subsequent studies were performed utilizing the intestinal mens from four neonates undergoing intestinal resection for samples harvested from BF animals and FFH animals inflammatory conditions other than NEC (control) were ana exposed to hypoxia sacrificed on day 4 after birth. lyzed for the presence of HMGB1. The frozen intestinal Expression of High Mobility Group-1 (HMGB1) Protein and samples were processed and Western blotting was performed its Receptor Rage in the Intestine. as described under Materials and Methods with 50g protein 40 In order to determine whether the high mobility group-1 per lane. Results in graph bars are the mean+S.D. (n=7 (HMGB1) is expressed in the ileum of newborn rats and patients/group in two independent experiments, *P<0.05 vs. whether its expression is related to intestinal damage, the control, analyzed by Student's t-test). expression of this protein in ileal mucosa from both BF and Overall Mortality in Animal Model of NEC FFH animals sacrificed on different days was analyzed as Newborn rats were fed either breast milk (BF) or a com 45 described in Methods. Although, the mature HMGB1 protein mercial formula (FF) up to four days, with or without a generally migrates as a 30-kDa band on SDS-PAGE (48), it 10-min hypoxic insult thrice as indicated in the Methods can also appear as a doublet or two very close bands around 30 section. While breast fed animals gained weight in a normal kDa, where the lower band is usually more intense (K. Tracey, fashion, formula-fed animals gained significantly less weight unpublished observations). It was found that intestines of BF than their breast milk-fed counterparts (32). Moreover, the pups showed a basal expression of HMGB1, however, larger present inventors observed a higher mortality rate (days 0-3) 50 protein amounts were present in the ileal mucosa of FFH in the formula-fed animals as compared to the breast-fed animals at day 4 (FIG. 1). In some cases those animals that group: 34.33% for FFH rats (484/1410) vs. 1.13% for BF showed histologic evidence of moderate to severe intestinal controls (8/707) (P<0.001, analyzed by Chi-square test.) The damage also showed increased expression of a lower molecu total numbers of animals are cumulative. lar weight protein (below the 14 kDa molecular weight 55 marker and referred herein as 7-10 kDa bands) detected with the same anti-HMGB1 polyclonal antibody (FIG. 1). TABLE 1. Semapimod Protects Against Formula-Feeding/Hypoxia Time course effect of formula feeding and hypoxia on incidence of NEC. Mediated Intestinal Injury and Reduces Ileal HMGB1 and Rage Protein Expression. Incidence of NEC (% 60 The present inventors hypothesized that the macrophage deactivator semapimod will exert a protective effect against BF FFH formula-feeding/hypoxia-mediated intestinal injury and that day 1 6.6 (91) 22 (109) it will affect the expression of the HMGB1 protein. The day 2 () (55) 33.3 (84) day 3 2.1 (94) 40 (127) present inventors found that intraperitoneal administration of day 4 2.3 (390) 48.3 (487) 65 semapimod (0.1-1 mg/kg, daily) prevented intestinal damage at doses higher than 0.5 mg/kg, as represented in FIG. 2 as reduction in the incidence of NEC. The present inventors also US 8,314,149 B2 35 36 examined the effect of semapimod on HGMB1 protein ated. As shown in FIG. 6b, a similar exposure of IEC-6 cells expression. While the present inventors found a basal expres to ONOO resulted in a rapid phosphorylation of p38, which sion of HMGB1 protein in the ileal mucosa of BF animals, a in contrast to the LPS-induced effect, was not inhibited by 1 significant increase was observed in the ileal specimens of hr pretreatment with 5 uM semapimod. Pretreatment with FFH animals that was reduced after administration of semapi semapimod also failed to inhibit the stimulatory effect on p38 mod (FIG.3a). Administration of vehicle alone had no effect activation by a mixture of TNFO, IL-1B and IFN-Y (not and was not different from FFH group (not shown). This shown). inhibition of protein expression correlated with a decrease in HMGB1 is Increased in Human Intestinal Specimens from the severity of injury as assessed by histology (FIG. 3b). Patients with Acute NEC Receptor signal transduction of HMGB1 occurs in part 10 Intestinal specimens from seven infants with acute NEC through the receptor for advanced glycation endproducts and control intestinal specimens from seven patients who (RAGE) in different cell types (2). The present inventors also underwent intestinal resection for inflammatory conditions examined the expression of RAGE in the ileal mucosal scrap other than NEC were analyzed by Western blot for the pres ings in their NEC model. The anti-RAGE antibody used ence of HMGB1 protein in two independent experiments. detects two bands in the 45 kDa range representing the RAGE 15 The frozen samples (segments from whole distal ileum) were protein pre and post-glycosylation in mouse lung extract. In processed as described above, and the results are shown in addition, this antibody detects a 25kDa protein believed to be FIG. 7. In contrast to the intestinal specimens from the control a proteolytic degradation product. The present inventors patients, ileal specimens from NEC patients demonstrated a found that the mucosal scrapings of the FFH rats had a higher two-fold increase in the levels HMGB1 protein. This finding expression of RAGE (both the 45 and 25 kDa bands) than the correlates with upregulation of iNOS protein (Zuckerbraunet BF controls. Similar to the effect on HMGB1 in the FFH al., Am. J. Physiol. Gastrointest. Liver Physiol. in press) and Semapimod group, expression of RAGE was reduced after higher levels of nitroso species (Zamora et al., Free Rad. Biol. administration of semapimod (FIG. 3c). Med., in press) in resected ileal segments from infants with Ileal Expression of Inflammatory Proteins in Experimental acute NEC. NEC: Effect of Semapimod 25 The relevant portion of each of the following references is The present inventors examined the expression of three hereby incorporated by reference, the same as if set forth at members of the Bcl-2 family known to either promote or length: inhibit apoptosis, namely Bax, Bad and Bcl-2, in the ileal 1. Albanese CT and Rothe present inventors MI. Necrotizing mucosa of newborn rats. The present inventors found that enterocolitis. Semin Pediatr Surg 4: 200-206, 1995. formula feeding and hypoxia significantly upregulate the 30 2. Andersson U and Tracey K J. HMGB1 in sepsis. Scand J expression of the apoptotic proteins Bad and Bax in the Infect Dis 35: 577-584, 2003. mucosal scrapings of FFH animals as compared to BF con 3. Andersson U. Wang H, Palmblad K, Aveberger AC, Bloom trols (FIG. 4). Expression of both proteins in FFH pups was O, Erlandsson-Harris H. Janson A, Kokkola R. Zhang M. significantly decreased when the animals were administered Yang H and Tracey K J. High mobility group 1 protein the drug semapimod (0.75 mg/kg, IP, daily). There was no 35 (HMG-1) stimulates proinflammatory cytokine synthesis significant difference in the expression of Bcl-2 in BF and in human monocytes.J Exp Med 192: 565-570, 2000. FFH samples (not shown). 4. Barlow Band Santulli TV. Importance of multiple episodes Other proteins whose altered expression has been associ of hypoxia or cold stress on the development of enterocoli ated with inflammation in a number of experimental and tis in an animal model. Surgery 77: 687-690, 1975. clinical conditions are iNOS and COX-2. In the NEC model, 40 5. Barlow B, Santulli TV, Heird W. C., Pitt J, Blanc WA and intestinal samples from BF animals had very little or no iNOS Schullinger J. N. An experimental study of acute neonatal protein as shown by Western blotting (FIG.5a). Expression of enterocolitis—the importance of breast milk. J Pediatr COX-2, however, was present in those samples (FIG. 5b). Surg9:587-595, 1974. Elevated levels of iNOS and COX-2 were found in the termi 6. Bernik TR, Friedman SG, OchaniM, DiRaimo R, Ulloa L, nal ileum from FFH newborn rats as compared to BF controls. 45 Yang H. Sudan S, Czura C J, Ivanova S M and Tracey K.J. Expression of both proteins was significantly decreased when Pharmacological stimulation of the cholinergic antiinflam FFH animals where administered the drug semapimod (0.75 matory pathway.J Exp Med 195: 781-788, 2002. mg/kg, IP, daily) (FIG. 5). 7. Bianchi M, Bloom O, Raabe T. Cohen PS, Chesney J, Semapimod Inhibits Activation of p38 Mitogen-Activated Sherry B, Schmidtmayerova H, Calandra T. Zhang X, Protein Kinase (Map Kinase) by Bacterial LPS in Rat Intes 50 Bukrinsky M. Ulrich P. Cerami A and Tracey K.J. Suppres tinal Epithelial Cells sion of proinflammatory cytokines in monocytes by a tet The present inventors have previously shown that LPS ravalent guanylhydrazone. J Exp Med 183: 927-936, 1996. causes rapid but transient activation of p38 MAP kinase as 8. Bucciarelli L. G. Wendt T. Rong L, Lalla E. Hofmann MA, determined by increased phosphorylation of p38. In IEC-6 Goova MT, Taguchi A, Yan SF, Yan SD, Stern D M and cells, activating phosphorylation of p38 increases nearly ten 55 Schmidt A M. RAGE is a multiligand receptor of the fold within 5-10 min of LPS treatment, and returns to basal immunoglobulin Superfamily: implications for homeosta levels 30 min after addition of LPS (19). In order to examine sis and chronic disease. Cell Mol Life Sci 59: 1117-1128, the effect of semapimod on activation of p38 by LPS, IEC-6 2002. cells were pretreated with different doses of the drug (0.1-10 9. Bustin M. Revised nomenclature for high mobility group uM) for 1 hr prior to stimulation with 5ug/ml LPS for 10 min. 60 (HMG) chromosomal proteins. Trends Biochem Sci 26: The protein lysates were then collected and Western blotting 152-153, 2001. analysis for phospho-p38 and p38 (loading control) was per 10. Caplan MS, Lickerman M. Adler L. Dietsch GN and Yu formed as described in Methods. The present inventors found A. The role of recombinant platelet-activating factor that semapimod dose dependently inhibited the phosphory acetylhydrolase in a neonatal rat model of necrotizing lation of p38 with a maximal effect at concentrations higher 65 enterocolitis. Pediatr Res 42: 779-783, 1997. than 1 uM (FIG. 6a). The effect of semapimod on p38 acti 11. 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Yang H. Wang Hand Tracey K.J. HMG-1 rediscovered as a cytokine. Shock 15: 247-253, 2001. 49. Zamora R, VodovotzY, Aulak KS, Kim PK, Kane JM, III, Alarcon L. Stuehr DJ and Billiar T R. A DNA microarray study of nitric oxide-induced genes in mouse hepatocytes: implications for hepatic heme oxygenase-1 expression in ischemia/reperfusion. Nitric Oxide 7: 165-186, 2002. 25 50. Zamora R, VodovotZY and Billiar T R. Inducible nitric oxide synthase and inflammatory diseases. Mol Med 6: and/or the mesylate-salt thereof, or a combination of the 347-373, 2000. compound and said salt, for treating or reducing the 51. Zill H, Gunther R, Erbersdobler HF, Folsch U Rand Faist incidence of intestinal injury caused by formula feeding V. RAGE expression and AGE-induced MAP kinase acti 30 and exposure to hypoxia to a Subject in need thereof. vation in Caco-2 cells. Biochem Biophy's Res Commun 288: 2. The method of claim 1, wherein the subject is a human 1108-1111, 2001. Subject. From the description herein, it will be clear to those skilled 3. The method of claim 1, wherein the subject is a human in the art that changes and modifications can be made to the infant. embodiments described herein without departing from the 35 4. The method of claim 1, wherein the compound is in the invention. It should be understood, therefore, that the inven salt form. tion is not limited to only the disclosed embodiments. 5. The method of claim 1, wherein the compound, salt thereof, or a combination of the compound and a salt thereof, The invention claimed is: is administered in the form of a composition further compris 1. A method, comprising administering a compound hav 40 ing at least one carrier. ing the following formula: k k k k k