WO2018031286A1 - Compositions and methods of treatment using glucose analogs - Google Patents

Abstract

The present invention relates to the discovery that the sugar analogs improve survival of subjects with disease.

Description

TITLE OF THE INVENTION

COMPOSITIONS AND METHODS OF TREATMENT USING GLUCOSE

ANALOGS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional Application

No. 62/371,991, filed August 8, 2016, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLYSPONSORED RESEARCH OR

DEVELOPMENT

This invention was made with government support under Grant No. 5T32AR007107-41 awarded by The National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

Sickness behaviors are a collection of very prominent symptoms of acute illness that include anorexia, lethargy, fever, sleepiness, depression, lack of grooming and social withdrawal. It has been long appreciated that sickness behaviors are motivational states, rather than a result of debilitation of physiological functions (Holmes and Miller, 1963, J Exp Med 118:649-58; Miller, 1964, Bull Br Psychol Soc 17: 1-20). Furthermore, sickness behaviors have been conceptualized as well- organized and adaptive programs that promote survival of acute infections (Hart, 1998, Neurosci Biobhav Rev 12: 123-37; Kluger et al, 1975, Science 188: 166-8). However, the mechanisms whereby different sickness behaviors contribute to survival remain largely unknown. This issue is particularly important because the most popular treatment for acute infections is the use of non-steroidal anti-inflammatory drugs (NSAIDs), which work primarily by suppressing the symptoms of sickness behaviors (Pecchi et al, 2009, Physiol Behav 97:279-92).

Understanding the biology of sickness behaviors is also important for improved management of critically ill patients suffering from conditions with complicated and poorly defined pathogenesis, such as sepsis. Indeed, sepsis remains a largely intractable clinical problem with high mortality rates even in modem medical facilities (Angus and van der Poll, 2013, NEJM 369:840-51). It is now increasingly appreciated that the mortality of sepsis can stem from at least three distinct phases of the disease: hemodynamic shock, multiple organ failure and prolonged

immunosuppression (Delano and Ward, 2016, J Clin Invest 126:23-31 ; Hotchkiss et al., 2013, Nat Rev Immunol 13 : 862-74). Furthermore, different forms of sepsis can result from bacterial, fungal and viral infections, each creating a distinct challenge and potentially requiring different management strategies.

Over the last 20 years, multiple randomized controlled trials have attempted to identify new treatments for sepsis, including low-dose steroids, insulin therapy, and alternative volume expanders, all of which failed to improve mortality (Cohen et al, 2014, Lancet Infect Dis 15 :581-614; Marshall, 2014, Trends Mol Med 20: 195-203). It has been long appreciated that the acute anorexia of infection is an aspect of sickness behavior that is conserved from humans to insects (Adamo, 2005, Arch Insect Biochem Physiol 60: 185-97), suggesting a general survival benefit during infectious challenge. Thus, there has been a tremendous effort dedicated to understanding the role of nutrition in septic critical illness (Casaer and Van den

Berghe, 2014, NEJM 370: 1227-36). It was demonstrated in animal models of sepsis in the 1970s that interfering with the anorexia induced by acute Listeria infection using caloric supplementation led to pronounced mortality (Murray and Murray, 1979, Am J Clin Nutr 32:593-6; Wing and Young, 1980, Infect Immun 28:771-6). Importantly, Ayres et al. found that in Drosophila, anorexia was beneficial in some but not all host-pathogen interactions (Ayres and Schneider, 2009, PLoS Biol 7:el000150). Similarly, it was recently shown that the fasting metabolic state was critical to surviving bacterial sepsis (Feingold et al, 2012, Endocrinol 153:2689-700). The mechanism remains elusive, and clinically, the role of nutrition in managing patients with sepsis is unclear at best.

An important new paradigm that can help explain at least some benefits of sickness behavior is the notion of resistance and tolerance: survival of infections can be promoted by either reducing pathogen burden, or by increasing host tolerance to the damage of infection (Ayres and Schneider, 2012, Annu Rev Immunol 30:271-94; Soares et al, 2014, Trends Immunol 35 :483-94). During an infection, multiple physiological processes undergo dramatic alterations with often poorly understood rationale. Depending on the infection, these can include profound changes in metabolism and alterations in hepatic, renal and cardiovascular functions, to name a few. In principle, these responses can be either beneficial, induced as part of a general defense program, or they can be detrimental but unavoidable consequences of infections. Furthermore, the physiological changes that are beneficial for the host survival can contribute to elimination of pathogens (resistance) or to mitigation of tissue damage caused by infection (tolerance). These physiological responses are poorly understood aspects of infection biology, where most of the focus has been devoted to the immune response and microbial pathogenesis.

There are several examples where organismal metabolism has been shown to be regulated in order to deprive necessary substrates for pathogen viability as a strategy of enhancing host resistance (Liu et al., 2012, Cell Host Microbe 11 :227- 39; Nairz et al., 2015, Cell Host Microbe 18:254-61), but how organismal metabolic states such as the starvation state of anorexia contributes to host defense generally, and to host tolerance specifically, is not understood. Indeed, the role of metabolic homeostasis during sepsis, which is directly impacted by nutritional status, is becoming increasingly recognized as critical in surviving sepsis in the clinical setting. In addition to the well-studied, but contentious role of glucose homeostasis in managing sepsis in the intensive care unit (Investigators et al, NEJM 360: 1283-97; van den Berghe et al, 2001, 345: 1359-67), the largest proteomic and metabolomics screen of patients with sepsis to date identified fatty acid, glucose and beta-oxidation pathways as being discriminatory between survivors and non-survivors (Langley et al., 2013, Sci Transl Med 5: 195ra95). It has been appreciated that bacterial sepsis leads to a pro-lipolytic state, which affects the ability of target tissues to utilize glucose via glycolysis and alternative fuel sources such as ketone bodies (KB) and free fatty acids (FFA) via oxidative phosphorylation (OXPHOS) (Agwunobi et al., 2000, J Clin Endocrinol Metab 85:3770-8). A growing body of recent literature in animal models suggests that a shift from glucose to KB/FFA substrate utilization is protective in bacterial sepsis - i.e., temporary starvation is beneficial - and these studies largely rely on pharmacologic targeting of PPARa, the master regulator of the ketogenic program (Budd et al, 2007, Antimicrob Agents Chemother 51 :2965-8; Camara-Lemarroy et al, 2015, Exp Ther Med 9: 1018-22; Varga et al, 2011, Biochimica et Biophysic Acta 1812: 1007-22). In multiple models of septic shock, treatment with PPARa agonists and the switch to an OXPHOS state dependent on oxidation of BHOB (beta hydroxybutyrate)/FFA generally decreased tissue damage although the mechanism remains elusive. By contrast, the role of substrate utilization in severe viral infections is less well understood (Greseth and Traktman, 2014, PLoS Pathog 10: el 004021). These metabolic changes are presumed to be protective but their mechanism of protection remains obscure.

There is thus a need in the art for compositions and methods for treating infections and inflammation. The present invention addresses this unmet need in the art.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides a method of treating an infection in a subject. In one embodiment, the method comprises administering to the subject an effective amount of at least one sugar analog selected from the group consisting of glucose analogs and heptose analogs.

In one embodiment, the glucose analog is 2-deoxy-D-glucose (2DG). In one embodiment, 2DG does not suppress ketogenesis.

In one embodiment, heptose analog is D-manno-heptulose (DMH).

In one embodiment, the infection is selected from the group consisting of sepsis, a bacterial infection and a parasitic infection. In one embodiment, the infection is selected from the group consisting of a listeria infection and a

Plasmodium infection.

In one embodiment, the method further comprises administering at least one additional therapeutic. In one embodiment, the at least one additional therapeutic is selected from an antibiotic and an antiparasitic.

In one embodiment, the subject is a human.

The present invention also provides a method of providing nutritional supplementation to a subject. In one embodiment, the method comprising administering to the subject a composition comprising at least one sugar analog selected from the group consisting of glucose analogs and heptose analogs.

In one embodiment, the glucose analog is 2-deoxy-D-glucose (2DG). In one embodiment, heptose analog is D-manno-heptulose (DMH).

In one embodiment, the subject has an infection selected from the group consisting of sepsis, a bacterial infection and a parasitic infection. In one embodiment, bacterial infection is a listeria infection. In one embodiment, the parasitic infection is a Plasmodium infection.

In one embodiment, the composition does not comprise glucose.

In one embodiment, the method improves survival of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

Figure 1, comprising Figure 1 A through Figure 1G, depicts results of experiments demonstrating glucose caloric supplementation during Listeria monocytogenes infection worsens survival, while 2-Deoxy-D-glucose (2DG) promotes survival. Figure 1A depicts food consumption after infection with 5 x 10⁴ and 5 x 10⁵ CFU wild-type L. monocytogenes. Figure IB depicts survival after infection with 5 x 10⁴ L. monocytogenes. Mice were per os (po) gavaged with 1 Calorie of Abbott Promote (Food) twice a day, 1 Calorie of Glucose twice a day, or PBS vehicle twice a day, and injected i.p. with 5 mg 2DG or PBS. Gavage and injection interventions were initiated 8 hours post infection, po PBS/ip PBS n=20, po Food/ip PBS n= 15 (p=0.0011 vs po PBS/ip PBS), po Glucose/ip PBS n=19 (p=0.004 vs po PBS/ip PBS), po PBS/ip 2DG n=10 (p=0.0085 vs po PBS/ip PBS). Figure 1C depicts survival after L. monocytogenes and indicated treatments. Mice were infected with 5 x 10⁴ CFU L. monocytogenes i.v., then treated with PBS, 20 mg glucose, or 5 mg 2DG i.p. twice a day starting 8 hours after L. monocytogenes infection. Figure ID depicts plasma and tissue harvested L. monocytogenes day 4 after 5 x 10⁴ L.

monocytogenes infection. Plasma IL-6 and IFNy measured by ELISA and Listeria CFUs from spleen and liver are shown. Figure IE depicts flow cytometry analysis of CD45+ cells within the liver 4 days after L. monocytogenes infection and treatment with PBS, glucose, or 2DG Figure IF depicts CFU growth of L. monocytogenes after incubation in brain heart infusion broth with or without 15 rriM 2DG for 18 hours. Figure 1G depicts bone marrow-derived macrophages (BMDM) were infected with 5 x 10⁵ CFU of L. monocytogenes in the presence or absence of 15 mM 2DG for 24 hours. CFU of L. monocytogenes grown from the BMDM cell media supernatant and BMDM cell lysate.

Figure 2, comprising Figure 2A through Figure 2F, depicts results of experiments demonstrating glucose caloric supplementation during LPS sepsis worsens survival, while 2DG promotes survival. Figure 2A depicts survival after 15 mg/kg i.p. LPS and po gavage with 1 Calorie of Abbott Promote (Food) twice a day (BID), or PBS vehicle BID. Gavage interventions were initiated one hour after LPS administration. Figure 2B depicts survival after 15 mg/kg i.p. LPS and po gavage with 1 Calorie of Glucose BID, 1 Calorie of olive oil BID, 1 Calorie of casein BID, or PBS vehicle BID. Gavage interventions were initiated one hour after LPS administration. Figure 2C depicts survival after 15 mg/kg i.p. LPS and po gavage with 1 Calorie of Food BID with i.p. injections of PBS or 5 mg 2DG BID starting an hour after LPS administration. Figure 2D depicts survival after 15 mg/kg i.p. LPS and indicated treatments. IP PBS n=16, IP Glucose n=10 (p<0.0001 vs IP PBS), IP 2DG n=10 (p=0.01 vs IP PBS). Mice were given 15 mg/kg i.p. LPS, then treated with PBS, 20 mg glucose, or 5 mg 2DG given i.p. BID. Injection interventions were initiated one hour after LPS administration. Figure 2E depicts plasma TNFa and IL-6 measured by ELISA. (n= 5-10/group). Figure 2F depicts liver tissue mRNA expression 4 hours after LPS and treatment with PBS (LPS-PBS), glucose (LPSglucose), and 2DG (LPS- 2DG) compared to PBS alone n=3-5/group.

Figure 3 depicts flow cytometry analysis of CD45+ cells within the liver 4 days after L. monocytogenes infection and treatment with PBS, glucose, or 2DG. ***p<0.001, ****p<0.0001.

Figure 4, comprising Figure 4A and Figure 4B, depicts experiments where mice were given 15 mg/kg i.p. LPS, then treated with PBS, 20 mg glucose, or 5 mg 2DG given i.p. twice a day initiated one hour after LPS administration. Figure 4A depicts O2 saturation, respiratory rate, heart rate and body temperature 24 hours after LPS. Figure 4B depicts blood glucose measured at 2, 6, 18 hours after LPS. Plasma troponin-I, ALT, and creatinine levels measured at 24 hours after LPS.

Figure 5, comprising Figure 5A through Figure 5J, depicts results of experiments demonstrating caloric supplementation and glucose utilization is required for surviving influenza infection. Figure 5A depicts food consumption after infection with 450 plaque forming units (pfu) of Influenza strain A/WSN/33. Figure 5B depicts survival after infection with 800 pfu Influenza. Mice were gavaged with 1 Calorie of Abbott Promote (Food) twice a day (BID), or PBS vehicle BID. Gavage and injection interventions were initiated 8 hours post infection. PBS vs Food p = 0.0047 by

Mantel-Cox log rank test. n=10/group. Figure 5C depicts survival after infection with 800 pfu Influenza. Mice were gavaged with 1 Calorie of Abbott Promote (Food) twice a day (BID), 1 Calorie of Glucose BID, or PBS vehicle BID. Mice gavaged with Food were also injected i.p. with PBS (Food) or 5 mg 2DG (Food+2DG) BID. Gavage and injection interventions were initiated 8 hours post infection. PBS vs Glucose p = 0.1058, Food vs Food+2DG p = 0.0001, PBS vs Food+2DG p=0.0256 by Mantel-Cox log rank test. n=10/group. Figure 5D depicts survival after infection with 375 pfu Influenza. Eight hours after infection, mice were injected i.p. with PBS or 5 mg 2DG BID. pO.0001, n=10/group. Figure 5E depicts lung and broncho-alveolar lavage (BAL) viral load 6 days after Influenza infection by pfu and quantitative PCR for WSN nucleoprotein (NP). Mice were infected with 375 pfu of Influenza. Eight hours after infection, mice were injected i.p. with PBS or 5 mg 2DG BID. n=4-5/group. Figure 5F depicts mRNA expression of whole lung tissue at day 6. Figure 5G depicts plasma IFNa measured by ELISA. Figure 5H depicts H&E staining of lung tissue 6 days after Influenza infection. Scale bar = 500 μηι. Figure 51 depicts histologic scoring of lung tissue 6 days after Influenza infection. Figure 5 J depicts C saturation, respiratory rate, heart rate and body temperature after Influenza infection.

Figure 6, comprising Figure 6A through Figure 6E depicts results of experiments. Figure 6A depicts survival after infection with 800 pfu Influenza. Mice were gavaged with 1 Calorie of Abbott Promote (Food), 1 Calorie of Casein, 1 Calorie of Olive Oil twice a day, or PBS vehicle twice a day. Figure 6B depicts mRNA expression of whole lung tissue at day 6 after 375 pfu Influenza. Figure 6C depicts flow cytometric analysis of Lung and BAL on day 6 after 700 pfu Influenza. Figure 6D depicts H&E staining of lung tissue 6 days after Influenza infection. Letters correspond to areas of the lung annotated in Figure 5. Scale bar = 50 μηι. Figure 6E depicts survival after infection with 1x106 L. pneumophila, and treatment with i.p. PBS or 5mg 2DG twice daily starting 8 hours after infection. n=5/group.

Figure 7, comprising Figure 7A through Figure 7E, depicts results of experiments demonstrating inhibition of glucose utilization is lethal in Poly(LC) inflammation. Figure 7A depicts survival of mice after i.v. injection of 30 mg/kg Poly(LC) and treatment with either i.p. PBS, 20 mg Glucose, or 5 mg 2DG BID initiated one hour after Poly(LC) administration. IP PBS n= 15, IP Glucose n=10 (p=0.2207 vs IP PBS), and IP 2DG n=15 (pO.0001 vs IP PBS). Figure 7B depicts survival of B6 wild-type mice and Ifhar-/- mice after i.v. injection of 30 mg/kg

Poly(LC) and treatment with i.p. PBS, 20 mg glucose, or 5 mg 2DG BID initiated one hour after Poly(LC) administration. B6 vs Ifnar-/- p = 0.0027, n=5/group. Figure 7C depicts plasma IFNa measured by ELISA of mice given 30 mg/kg Poly(LC) i.v., then treated with either i.p. PBS, Glucose, or 2DG initiated one hour after Poly(LC) administration. n=5/group. Figure 7D depicts O2 saturation, respiratory rate, heart rate and body temperature measured 18 hours after Poly(I:C) administration. n=3-7/group ***p<0.001, ****p<0.0001. Figure 7E depicts hindbrain mRNA expression 4 hours after Poly(I:C) administration. n=3-5/group. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 8 depicts experiments where mice were given 30 mg/kg Poly(I:C) i.v., then treated with either i.p. PBS, Glucose, or 2DG initiated one hour after Poly(I:C) administration. Blood glucose measured at 2, 6, and 18 hours after Poly (I: C). Plasma troponin-I, ALT and creatinine measured at 24 hours after

Poly (I: C).

Figure 9, comprising Figure 9A through Figure 9E, depicts results of experiments demonstrating inhibition of glucose utilization in Poly(I:C)-induced inflammation enhances ER stress. Figure 9A depicts brain mRNA expression of Gadd34 18 hours after i.v. injection of 30 mg/kg Poly(I:C) and treatment with either i.p. PBS, 20mg Glucose, or 5 mg 2DG BID initiated one hour after Poly(I:C) administration. (n=5/group (one death in Poly(I:C) group). **p<0.01, ****p<0.0001). Figure 9B depicts survival of B6 wild-type mice and Ddit3-/- mice after i.v. injection of 30 mg/kg Poly(I:C) and treatment with i.p. PBS or 5 mg 2DG BID initiated one hour after Poly (I: C) administration. (WT vs Ddit3-/- p =0.0015 WT: Poly (I: C) + 2DG vs Ddit3-/-: Poly(I:C) + 2DG, n=5/group). Figure 9C depicts plasma IFNa measured by ELISA. n=5/group. Figure 9D depicts MEFs treated with vehicle, IFNa, 2DG, or IFNa and 2DG. mRNA expression at 0, 4, and 24 hours after treatment. *p<0.05, **p<0.01, ****p<0.0001. n=3 replicates per group. Data representative of two independent experiments. Figure 9E depicts MEFs treated with vehicle, IFNa, Poly (I: C), and Thapsigargin, in the presence of vehicle, glucose or 2DG for 24 hours. FACS analysis for Annexin V. Two-three replicates per group. Data representative of three independent experiments. ****p<0.0001

Figure 10, comprising Figure 10A through Figure 10D, depicts results of experiments demonstrating glucose utilization in LPS sepsis promotes oxidative stress. Figure 10A depicts plasma NEFA, BHOB, and FGF21 at 0, 2, 6, 20, 24 hours after LPS, with treatment with either i.p. PBS or glucose. ***p<0.001, ****p<0.0001. n=10/group. Figure 10B depicts survival after 8 mg/kg LPS and treatment with glucose. Mice were treated with vehicle, valproic acid (VA) or levetiracetam (Keppra) starting 6 hours after LPS. ****p<0.0001 for Glucose+VA vs Glucose and Glucose+VA vs Glucose+Keppra. n=15 Glucose, n=15 Glucose+VA, n=4

Glucose+Keppra. Figure IOC depicts HEt staining of brain from nice mice given i.p. LPS then initiated with PBS, glucose, and 2DG treatment 1 hour after LPS and staining 24 hours after LPS. Figure 10D depicts TU EL staining of brain sections 24 hours after LPS and treatment with PBS, glucose, or 2DG. Quantification of TUNEL positive cells per high power field (400x magnification). *p<0.05, ***p<0.001.

Figure 11, comprising Figure 11 A through Figure 11C, depicts results of experiment. Figure 11 A depicts whole blood glucose at 0, 2, 6, and 24 hours after LPS with treatments as indicated. Figure 11B depicts representative images of heart, lung, liver, and kidney tissue stained for TUNEL 24 hours after LPS, shown at 400x magnification. Figure 11C depicts brain tissue stained for TUNEL 24 hours after LPS. Representative images of brain tissue stained for TUNEL. Area enclosed by the dotted-line box shown at 400x magnification below tiled image.

Figure 12, comprising Figure 12A through Figure 12H, depicts results of experiments demonstrating the role of ketogenic program in surviving bacterial, but not viral inflammation. Figure 12A depicts survival after 12.5 mg/kg i.p. LPS in WT, Fgf21-/- and Ppara-/- mice. n=10/group. pO.0001 for WT vs Fgf21-/-; p=0.0003 for WT vs Ppara-/-. Figure 12B depicts plasma BHOB measured at 0, 6, 18, and 48 hours after 12.5 mg/kg i.p. LPS in WT, Fgf21-/- and Ppara-/- mice. n=4-6/group. **2 hour WT vs Fgf21-/- p = 0.0082; 2 hour Fgf21-/- vs Ppara-/- p=0.0018; ****p<0.0001 for 18 hour Ppara-/- vs WT and Fgf21-/-. Figure 12C depicts plasma TNFa and IL-6 measured by ELISA in WT and Ppara-/- mice after LPS. n=5/group *p=<0.05. Figure 12D depicts survival after 12.5 mg/kg i.p. LPS in Fgf21-/- and Ppara-/- mice, treated with i.v. 5 ng recombinant mouse FGF21 twice daily starting 6 hours after LPS injection. n=5-6/group. p=0.0491 Fgf21-/- VEH vs Fgf21-/- rmFGF21. p=0.5767 Ppara-/- VEH vs Ppara-/- rmFGF21. Figure 12E depicts survival after 8 mg/kg i.p. LPS in WT and Ppara-/- mice. 2DG treatment was initiated one hour after LPS.

Valproic acid (VA) was initiated 6 hours after LPS. n=4-5/group. p=0.0177 Ppara-/- VEH vs Ppara-/- VA. Figure 12F depicts survival after 400 pfu Influenza in WT and Ppara-/- mice. p=0.0074 WT n=6, Ppara-/- n=8, representative of three independent experiments. Figure 12G depicts viral titers in BAL and lung on day 5 and 7 after 400 pfu Influenza infection. n=4/group. Figure 12H depicts a proposed model of glucose utilization during viral and bacterial-mediated inflammation supporting unique tissue tolerance mechanisms. Figure 13 depicts survival after 375 pfu Influenza in WT and Fgf21-/- mice.

Figure 14 depicts survival of mice challegened with LPS or Poly I:C and treated with 2DG or D Mannoheptulose.

DETAILED DESCRIPTION

The present invention relates to the discovery that the glucose analog 2- Deoxy-D-glucose (2DG) and D-manno-heptulose (DMH) improve survival of subjects with sepsis, a bacterial infection or a parasitic infection, while glucose worsens the subject's mortality. Thus, the present invention is directed to methods and compositions for treating infections and inflammation. In some embodiments, the composition does not inhibit ketogenesis. In one embodiment, the composition comprises a glucose analog. In some embodiments, the composition comprises 2DG or DMH.

In another embodiment, the invention is a method of treating inflammation or infection in a subject. In one embodiment, the subject is administered a composition comprising a glucose analog. In various embodiments, the subject is administered 2DG or DMH. In another embodiment, the infection is sepsis, a bacterial infection or a parasitic infection.

In yet another embodiment, the invention is a method of providing nutritional supplementation to a subject by administering a composition comprising glucose analog. In various embodiments, the subject is administered 2DG or DMH. In some embodiments, the subject has an infection such as a bacterial infection, a parasitic infection, or sepsis.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The skilled artisan will understand that any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention.

As used herein, each of the following terms has the meaning associated with it in this section. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

"About" as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or in some instances ±10%, or in some instances ±5%, or in some instances ±1%, or in some instances ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

A "disease" is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal's health continues to deteriorate.

In contrast, a "disorder" in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal's state of health.

"Effective amount" or "therapeutically effective amount" are used interchangeably herein, and refer to an amount of a compound, formulation, material, or composition, as described herein effective to achieve a particular biological result. Such results may include, but are not limited to, the inhibition of virus infection as determined by any means suitable in the art.

As used herein, an "instructional material" includes a publication, a recording, a diagram, or any other medium of expression which can be used to communicate the usefulness of the compositions and methods of the invention. The instructional material of the kit of the invention may, for example, be affixed to a container which contains the nucleic acid, peptide, and/or composition of the invention or be shipped together with a container which contains the nucleic acid, peptide, and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the instructional material and the compound be used cooperatively by the recipient.

"Parenteral" administration of an immunogenic composition includes, e.g., subcutaneous (s.c), intravenous (i.v.), intramuscular (i.m), or intrasternal injection, or infusion techniques. The terms "subject," "patient," "individual," and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is a human.

As used herein, the term "treatment" or "treating" is defined as the application or administration of a therapeutic agent, i.e., a compound of the invention (alone or in combination with another pharmaceutical agent), to a patient, or application or administration of a therapeutic agent to an isolated tissue or cell line from a patient (e.g., for diagnosis or ex vivo applications), who has a disease or disorder contemplated herein, a sign or symptom of a disease or disorder

contemplated herein or the potential to develop a disease or disorder contemplated herein, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve or affect a diseaser or disorder contemplated herein, at least one sign or symptom of a disease or disorder contemplated herein or the potential to develop a disease or disorder contemplated herein. Such treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Description

The present invention is based on the unexpected discovery that 2- Deoxy-D-glucose (2DG) and D-manno-heptulose (DMH) improve survival and tissue protection through the modulation of metabolic programs in certain inflammatory diseases and disorders. For example, caloric supplementation using glucose increases mortality in sepsis, bacterial infections and parasitic infections by inhibiting ketogenesis, while caloric supplementation using 2DG or DMH improves survival. Accordingly, the present invention provides methods and compositions for treating a bacterial disease or disorder. In one embodiment, the method comprises administering to the subject an effective amount of 2DG, DMH, an analog thereof, or a combination thereof.

Another aspect of the invention provides a method of providing caloric supplementation to a subject by administering a composition of the invention. In one embodiment, the method comprises administering a composition for caloric supplementation, wherein the composition does not comprise glucose, heptose or any analog thereof. In another embodiment, the method comprises administering a composition for caloric supplementation comprising at least one of 2DG and DMH. In some embodiments, the subject has an infectious disease or disorder such as a bacterial infection, a parasitic infection or sepsis. In some embodiments, the composition for caloric supplementation is a food or a medical food.

In yet another aspect, the invention provides a method of improving survival of a patient having a disease or disorder by administering 2DG or DMH. In one embodiment, the patient has a bacterial infection, a parasitic infection or sepsis. In another embodiment, the patient is receiving a nutritional supplement wherein the nutritional supplement does not comprise glucose. In some embodiments, the composition for caloric supplementation is a food or a medical food.

Compositions

In one aspect, the invention provides compositions for treating an infection in a subject. In various embodiments, the present invention includes compositions for improving survival of a subject having disease or disorder, such as an infection or inflammation. In various embodiments, the compositions of the invention do not inhibit ketogenesis.

In one embodiment, the composition of the invention comprises a glucose analog. In another embodiment, the composition of the invention comprises 2-deoxy glucose or derivatives thereof that are converted to 2-DG in a subject, or a related deoxy-substitution of glucose, such as 3-deoxy-D-glucose, 3-O-methylglucose 4-deoxy-D-glucose, 5-deoxy-D-glucose, 5-thio-D-glucose, 6-deoxy-D-glucose, 1,5- anhydro-D-glucitol combinations of other deoxy-glucose substitutions such as 2, n- deoxy-D-glucose (where n=3-5), compounds designated by permutations of the formula n, m deoxy-D-glucose (where n=2-5 and m=integers from 2-5 excluding n). In other embodiments, the composition comprises a sugar that can be metabolized into 2-DG, such as 2-deoxy-D-galactose, as well as disaccharide embodiments such as lactose and sucrose analogues containing 2-DG, and halogenated and other conjugated derivatives of deoxy sugars (as set forth above), such as fluoro-2-deoxy- D-glucose, conjugated deoxy sugars (as set forth above) that are metabolized to 2-DG, and compositions having effects similar to 2-DG. In some embodiments, the composition comprises 2-deoxy -D-glucose (2-DG) or 3-bromopyruvate.

In one embodiment, the composition of the invention comprises a heptose analog. In another embodiment, the composition comprises D-manno- heptulose (DMH). In another embodiment, comprises a derivative of DMH such as perseitol, or sedoheptulose. In other embodiments, the composition comprises a sugar that can be metabolized into DMH, as well as disaccharide embodiments of DMH, and halogenated or other conjugated derivatives of DMH and compositions having similar effects to DMH. In some embodiments, the composition comprises 2DG, DMH or a combination thereof.

As provided herein, compositions comprising 2-DG and methods using said compositions will be understood to encompass preparations of 2-deoxy glucose as the D-stereoisomer, as well as racemic mixtures thereof comprising any combination of D- and L-2-deoxy glucose, provided that the percentage of the D-stereoisomer is greater than zero. Similarly, comprising mannoheptulose and methods of using said compositions will be understood to encompass preparations of mannoheptulose as the D-stereoisomer, as well as racemic mixtures thereof comprising any combination of D- and L- mannoheptulose provided that the percentage of the D-stereoisomer is greater than zero.

2-DG and DMH are available commercially, and preferably are produced according to the standards and guidelines of the pharmaceutical industry and in compliance with all relevant regulatory requirements. 2-DG and DMH can also be synthesized using methods well-established in the art (see, for example, THE MERCK INDEX, 12.sup.th Ed., Monograph 2951, New Jersey: Merck & Co., 1997; Bergmann et al, 1922, Ber. 55: 158; Snowden et al, 1947, JACS 69: 1048; Bolliger et al, 1954, Helv. Chim. Acta 34: 989; Bolliger, 1962, "2-Deoxy-D-arabino-hexose (2-Deoxy-d-glucose)," in METHODS IN CARBOHYDRATE CHEMISTRY, vol. I, (Whistler & Wolfram, eds.), New York Academic Press, pp. 186, 189). The methods and formulations described herein include the use of glucose derivatives, heptose derivatives, crystalline forms (also known as

polymorphs), solvates, amorphous phases, and/or pharmaceutically acceptable salts of glucose derivatives described herein, as well as metabolites and active metabolites of these glucose derivatives having the same type of activity. Solvates include water, ether (e.g., tetrahydrofuran, methyl tert-butyl ether) or alcohol (e.g., ethanol) solvates, acetates and the like. In one embodiment, the compounds described herein exist in solvated forms with pharmaceutically acceptable solvents such as water, and ethanol. In another embodiment, the compounds described herein exist in unsolvated form.

In one embodiment, compounds described herein are prepared as prodrugs. A "prodrug" refers to an agent that is converted into the parent drug in vivo. In one embodiment, upon in vivo administration, a prodrug is chemically converted to the biologically, pharmaceutically or therapeutically active form of the compound. In another embodiment, a prodrug is enzymatically metabolized by one or more steps or processes to the biologically, pharmaceutically or therapeutically active form of the compound.

In another aspect, this invention provides a nutritional package for providing nutrition to subject comprising a nutritional composition for enteral administration and being in at least two forms selected from solid, semi-solid, and liquid forms.

Thus, the present invention provides compositions for nutritional supplementation. In one embodiment, the composition of the invention comprises a glucose analog. In another embodiment, the composition of the invention comprises D-manno-heptulose (DMH) or derivatives thereof that are converted into DMH, 2- deoxy glucose or derivatives thereof that are converted to 2-DG in a subject, or a related deoxy-substitution of glucose, such as 3-deoxy-D-glucose, 4-deoxy-D- glucose, 5-deoxy-D-glucose, 6-deoxy-D-glucose, or any combination thereof. In certain embodiments, the composition does not comprise glucose.

In one embodiment, the composition further comprises a protein source and a carbohydrate source. In another embodiment, the composition further comprises a fat source. Sources of protein include, but are not limited to, casein, soy, whey, and pea protein. Carbohydrate sources include, but are not limited to, dietary fiber.

Dietary fiber passes through the small intestine undigested by enzymes and represents a kind of natural and necessary laxative. Suitable sources of dietary fiber, among others, include soy, oat, and gum arabic. Fat sources may include long chain triglycerides (LCT), including olive oil, corn oil, canola oil, palm kernel oil, sunflower oil, peanut oil, soy lecithin and residual milk fat or medium chain triglycerides. In another embodiment, the composition further comprises vitamins, minerals and trace elements

The compositions of the invention may be incorporated into any formulation known in the art. In one embodiment, the composition of the invention is formulated for oral administration. For example, in some embodiments the nutritional supplement is a nutritional formulation, medical food, a medical beverage, a dietary supplement, or a food additive as a powder for dissolution. In some embodiments, the nutritional supplement is in the form of a complete meal, or part of a meal. In another embodiment, the nutritional supplement is in the form of a pharmaceutical formulation such as in the form of a tablet, pill, sachet or capsule or by tube feeding such as by means of nasogastric, nasoduodenal, esophagostomy, gastrostomy, or jejunostomy tubes, or peripheral or total parenteral nutrition. In an exemplary embodiment, the compositions of the invention may be administered orally as a dietary supplement or medical food.

Combinations

In one embodiment, the composition of the present invention comprises a combination of deoxy glucose derivatives described herein.

For example, in one embodiment the composition comprises a combination comprising at least two of 2-deoxy-D-glucose, 3-deoxy-D-glucose, 4- deoxy-D-glucose, and 5-deoxy-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-galactose, 2-deoxy-D-lactose, 2-deoxy-D-sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5-deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D-glucose, 3, 5-deoxy-D- glucose, 3, 6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6-deoxy-D-glucose and 5,6- deoxy-D-glucose. In one embodiment, the present invention provides a composition, wherein the composition comprises a combination comprising at least three of 2- deoxy-D-glucose, 3-deoxy-D-glucose, 4-deoxy-D-glucose, and 5-deoxy-D-glucose, 6- deoxy-D-glucose, 2-deoxy-D-galactose, 2-deoxy-D-lactose, 2-deoxy-D-sacrose, 2,3- deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5-deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D-glucose, 3, 5-deoxy-D-glucose, 3, 6-deoxy-D-glucose, 4,5-deoxy-D- glucose, 4,6-deoxy-D-glucose and 5, 6-deoxy-D-glucose. In one embodiment, the present invention provides a composition, wherein the composition comprises a combination comprising at least four of a 2-deoxy-D-glucose, 3-deoxy-D-glucose, 4- deoxy-D-glucose, and 5-deoxy-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-galactose, 2-deoxy-D-lactose, 2-deoxy-D-sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5-deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D-glucose, 3, 5-deoxy-D- glucose, 3, 6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6-deoxy-D-glucose and 5,6- deoxy-D-glucose. In one embodiment, the present invention provides a composition, wherein the composition comprises a combination comprising 2-deoxy-D-glucose, 3- deoxy-D-glucose, 4-deoxy-D-glucose, and 5-deoxy-D-glucose, 6-deoxy-D-glucose, 2- deoxy-D-galactose, 2-deoxy-D-lactose, 2-deoxy-D-sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5-deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D- glucose, 3, 5-deoxy-D-glucose, 3, 6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6- deoxy-D-glucose and 5,6-deoxy-D-glucose, and any combination thereof.

In another embodiment, the composition of the present invention comprises a combination of deoxy glucose derivative and at least one additional therapeutic useful for treating inflammation or infections. These additional compounds may comprise compounds of the present invention or other compounds, such as commercially available compounds, known to treat, prevent, or reduce the signs or symptoms of a disease or disorder, such as inflammation and infection.

In one aspect, the present invention contemplates that a glucose derivative of the invention may be used in combination with a therapeutic agent such as an antibiotic, including but not limited tolipopeptide, fluoroquinolone, ketolide, cephalosporin, amikacin, gentamicin, kanamycin, neomycin, netilmicin,

paromomycin, streptomycin, tobramycin, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, ceftaroline, ceftioxide, cefuracetime, imipenem, primaxin, doripenem, meropenem, ertapenem, fiumequine, nalidixic acid, oxolinic acid, piromidic acid pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifioxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, azithromycin, erythromycin, clarithromycin, dirithromycin, roxithromycin, telithromycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, cloxacillin, dicloxacillin, flucloxacillin, mezlocillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, pivampicillin,

pivmecillinam, ticarcillin, sulfamethizole, sulfamethoxazole, sulfisoxazole, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxy tetracycline, tetracycline, linezolid, clindamycin, metronidazole, vancomycin, vancocin, mycobutin, rifampin, nitrofurantoin, and chloramphenicol.

In another aspect, the present invention contemplates that a glucose derivative of the invention may be used in combination with a therapeutic agent such as an antiparasitic, including but not limited to bephenium, diethylcarbamazine, ivermectin, niclosamide, praziquantel, pyrantel, pyrvinium, albendazole,

mebendazole, thiabendazole, benzyl benzoate, benzyl benzoate disulfiram, crotamiton, lindane, malathion , quinine, permethrin, doxycycline, tetracycline, clindamycin, chloroquine, amodiaquine, pyrimethamine, chloroguanide, atovaquone, mefloquine, primaquine, artemisinin, artemether, halofantrine and clindamycin.

In certain embodiments, a composition comprising a combination of therapeutics described herein has an additive effect, wherein the overall effect of the combination is approximately equal to the sum of the effects of each individual therapeutic. In other embodiments, a composition comprising a combination of therapeutics described herein has a synergistic effect, wherein the overall effect of the combination is greater than the sum of the effects of each individual therapeutic.

A composition comprising a combination of molecules comprises individual therapeutics in any suitable ratio. For example, in one embodiment, the composition comprises a 1 : 1 ratio of two individual therapeutics. In one embodiment, the composition comprises a 1 : 1 : 1 ratio of three individual therapeutics. However, the combination is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed. Methods

The invention provides methods of treating an infection in a subject in need thereof. In some embodiments, the method comprises administering to the subject an effective amount of at least one sugar analog selected from at least one glucose analog. In some embodiments, the method comprises administering to the subject an effective amount of at least one sugar analog selected from at least one heptose analog. In some embodiments, the method comprises administering to the subject an effective amount of at least one sugar analog selected from at least one glucose analog and at least one heptose analog For example, in some embodiments, the method treats a bacterial or parasitic infection or inflammation associated with a bacterial or parasitic infection. In one embodiment, the glucose analog or heptose analog does not suppress ketogenesis. In some embodiments, the at least one glucose analog is 2-deoxy-D-glucose, 3-deoxy-D-glucose, 4-deoxy-D-glucose, and 5-deoxy- D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-galactose, 2-deoxy-D-lactose, 2-deoxy-D- sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5-deoxy-D-glucose, 2,6- deoxy-D-glucose, 3,4-deoxy-D-glucose, 3,5-deoxy-D-glucose, 3,6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6-deoxy-D-glucose, 5, 6-deoxy-D-glucose, or any

combination thereof. In another embodiment, the at least one heptose analog is D- manno-heptulose, perseitol, or sedoheptulos. In one embodiment, the method comprises administering to the subject a composition deficient of glucose or carbohydrates.

The present invention also provides a method of providing nutritional supplementation to a patient. In one embodiment, the method comprises administering to the subject a composition comprising at least one sugar analog selected from at least one glucose analog and/or at least one heptose analog. In one embodiment, the at least one glucose analog is 2-deoxy-D-glucose, 3-deoxy-D-glucose, 4-deoxy-D- glucose, and 5-deoxy-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D-galactose, 2-deoxy- D-lactose, 2-deoxy-D-sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy-D-glucose, 2,5- deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D-glucose, 3,5-deoxy-D-glucose, 3,6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6-deoxy-D-glucose, 5, 6-deoxy-D- glucose, or any combination thereof. In another embodiment, the composition does not comprise glucose. In another embodiment, the at least one heptose analog is D- manno-heptulose, perseitol, or sedoheptulos. In one embodiment method comprises administering to the subject a composition deficient of glucose or carbohydrates. In some embodiments, the subject has sepsis, a bacterial infection or a parasitic infection. In another embodiment, the sepsis is bacterial sepsis. In yet another embodiment, the sepsis is not viral sepsis. In another embodiment, the subject is human.

In another embodiment, the method further comprises first diagnosing the subject as having bacterial sepsis. In another embodiment, the method further comprises first diagnosing the subject as not having viral sepsis.

In some embodiments, the subject does not have sepsis, but rather is at risk for sepsis. For example, in one embodiment the method of preventing sepsis comprises administering to the subject a composition comprising at least one sugar analog selected from at least one glucose analog and/or at least one heptose analog. In one embodiment, the at least one glucose analog is 2-deoxy-D-glucose, 3-deoxy-D- glucose, 4-deoxy-D-glucose, and 5-deoxy-D-glucose, 6-deoxy-D-glucose, 2-deoxy-D- galactose, 2-deoxy-D-lactose, 2-deoxy-D-sacrose, 2,3-deoxy-D-glucose, 2,4-deoxy- D-glucose, 2,5-deoxy-D-glucose, 2,6-deoxy-D-glucose, 3,4-deoxy-D-glucose, 3,5- deoxy-D-glucose, 3,6-deoxy-D-glucose, 4,5-deoxy-D-glucose, 4,6-deoxy-D-glucose, 5, 6-deoxy-D-glucose, or any combination thereof. In another embodiment, the composition does not comprise glucose. In another embodiment, the method of preventing sepsis comprises administering to the subject a composition deficient of glucose or carbohydrates. In some embodiments, the subject is at risk for sepsis. Subjects at risk for sepsis, include, but are not limited to those infected with gram- positive and gram-negative organisms such as E. coli, K. pneumoniae, P. aeruginosa, S. pyogenes, S. aureus, or S. epidermidis; surgical pateints, elderly patients, low birth weight infants, burn patients and trauma patients. In another embodiment, the subject displays a symptom indicative of sepsis. Symptoms indicative of sepsis include, but are not limited to weakness, metabolic disturbance, dehydration, tachycardia, tachypnea or hyperpnea, hypotension, hypoperfusion, oliguria, leukocytosis or leukopenia, pyrexia or hypothermia. In another embodiment, the at least one heptose analog is D-manno-heptulose, perseitol, or sedoheptulos.

Exemplary bacterial infections that may be treated by way of the present invention includes, but is not limited to, infections caused by bacteria from the taxonomic genus of Bacillus, Bartonella, Bordetella, Borrelia, Brucella,

Campylobacter, Chlamydia, Chlamydophila, Clostridium, Corynebacterium,

Enterococcus, Escherichia, Francisella, Haemophilus, Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Ureaplasma, Vibrio, and Yersinia. In some embodiments, the bacterial infection is an infection of Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Klebsiella species, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Morexella species, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Proteus species, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, or Yersinia pseudotuberculosis. In one embodiment, the bacterial infection is a Listeria monocytogenes infection.

Exemplary diseases caused by bacterial infections which may be treated using compositions of the present invention, include but are not limited to, bacterially mediated meningitis, sinus tract infections, pneumonia, endocarditis, pancreatitis, appendicitis, gastroenteritis, biliary tract infections, soft tissue infections, urinary tract infections, cystitis, pyelonephritis, osteomyelitis, bacteremia,

Actinomycosis, Whooping cough, Secondary bacterial pneumonia, Lyme disease (B. burgdorferi), Relapsing fever, Brucellosis, Enteritis, bloody diarrhea, Guillain-Barre syndrome, Atypical pneumonia, Trachoma, Neonatal conjunctivitis, Neonatal pneumonia, Nongonococcal urethritis(NGU), Urethritis, Pelvic inflammatory disease, Epididymitis, Prostatitis, Lymphogranuloma venereum (LGV), Psittacosis, Botulism: Mainly muscle weakness and paralysis, Pseudomembranous colitis, Anaerobic cellulitis, Gas gangrene Acutefood poisoning, Tetanus, and Diphtheria. Exemplary parasitic infections that may be treated by way of the present invention includes, but is not limited to, infections caused by Protozoan organisms, tapeworms, Flukes, Roundworms, or Ectoparasites. In one embodiment, the parasitic infection includes, but is not limited to Acanthamoeba spp., Balamuthia mandrillaris, Babesia B. divergens, B. bigemina, B. equi, B. microfti, B. duncani, Balantidium coli, Blastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragilis, Entamoeba histolytica, Giardia lamblia, Isospora belli,

Leishmania spp., Naegleria fowleri, Plasmodium falciparum

Plasmodium vivax,Plasmodium ovale curtisi,Plasmodium ovale

wallikeri,Plasmodium malariae,Plasmodium knowlesi, Rhinosporidium seeberi,

Sarcocystis bovihominis,Sarcocystis suihominis, Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi, Cestoda, Taenia multiceps, Diphyllobothrium latum, Echinococcus granulosus,Echinococcus multilocularis, E. vogeli, E. oligarthrus, Hymenolepis nana,Hymenolepis diminuta, Taenia saginata, Taenia solium, Bertiella mucronata,Bertiella studeri, Spirometra erinaceieuropaei, Clonorchis sinensis; Clonorchis viverrini, Dicrocoelium dendriticum, Fasciola hepatica, Fasciola gigantica, Fasciolopsis buski, Gnathostoma

spinigerum,Gnathostoma hispidum, Metagonimus yokogawai, Metorchis conjunctus, Opisthorchis viverrini,Opisthorchis felineus, Clonorchis sinensis, Paragonimus westermani;Paragonimus africanus;Paragonimus caliensis;Paragonimus

kellicotti;Paragonimus skrjabini;Paragonimus uterobilateralis, Schistosoma sp., Schistosoma mansoni andSchistosoma intercalatum, Schistosoma haematobium, Schistosoma japoni cum, Schistosoma mekongi -, Echinostoma echinatum,

Trichobilharzia regenti,Schistosomatidae, Ancylostoma duodenale,Necator americanus, Angiostrongylus costaricensis, Anisakis, Ascaris sp.Ascaris

lumbricoides, Baylisascaris procyonis, Brugia malayi,Brugia timori, Dioctophyme renale, Dracunculus medinensis, Enterobius vermicularis,Enterobius gregorii, Halicephalobus gingivalis, Loa loa filaria, Mansonella streptocerca, Onchocerca volvulus, Strongyloides stercoralis, Thelazia californiensis,Thelazia callipaeda, Toxocara canis,Toxocara cati, Trichinella spiralis,Trichinella britovi,Trichinella nelsoni,Trichinella nativa, Trichuris trichiura,Trichuris vulpis, Wuchereria bancrofti, Archiacanthocephala,Moniliformis moniliformis, Linguatula serrata,

Oestroidea,Calliphoridae,Sarcophagidae, Cochliomyia hominivorax, Tunga penetrans, Cimex lectularius, and Dermatobia hominis. In one embodiment, the parasitic infection is a Plasmodium infection.

Exemplary diseases caused by parasitic infections which may be treated using compositions of the present invention, include but are not limited to, Acanthocephaliasis, Amoebiasis, Angiostrongyliasis, Anisakiasis, Asian intestinal schistosomiasis, Babesiosis, Balantidiasis, Baylisascariasis, Bedbug, Bertielliasis, Blastocystosis, Calabar swellings, Chagas disease, Chigoe flea, Chinese Liver Fluke, Clonorchiasis, Cryptosporidiosis, Cyclosporiasis, Dientamoebiasis, Dioctophyme renalis infection, Giardiasis, Gnathostomiasis, Granulomatous amoebic encephalitis, Guinea worm , Halicephalobiasis, Halzoun Syndrome, Hookworm, Human Botfly, Lung Fluke, intestinal fluke, Isosporiasis, Lancet liver fluke, Leishmaniasis, lymphatic filariasis, Malaria, Mansonelliasis, Metorchiasis, Myiasis, Onchocerciasis, Parasitic pneumonia, Pinworm , Pork tapeworm, Primary amoebic

meningoencephalitis, Rhinosporidiosis, Sarcocystosis, Schistosomiasis , Screwworm, Cochliomyia, Sleeping sickness, snail fever, Sparganosis,

Strongyloidiasis Thelaziasis, Tapeworm, Toxocariasis, Toxoplasmosis, Trichinosis, Trichomoniasis, and Whipworm. In one embodiment, the parasitic infection causes malaria. Administration/Dosage/Formulations

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the subject either prior to or after the onset of an infection. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present invention to a patient, preferably a mammal, more preferably a human, may be carried out using known procedures, at dosages and for periods of time effective to treat inflammation or infections in the patient. An effective amount of the therapeutic compound necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the patient; the age, sex, and weight of the patient; and the ability of the therapeutic compound to treat the inflammation or infection in the patient. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic compound of the invention is from about 1 and 5,000 mg/kg of body weight/per day. One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic compound without undue experimentation.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

In particular, the selected dosage level depends upon a variety of factors including the activity of the particular compound employed, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds or materials used in combination with the compound, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well, known in the medical arts.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the

pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the therapeutic compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic compound for the treatment of infections in a patient.

In one embodiment, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In one embodiment, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of a compound of the invention and a

pharmaceutically acceptable carrier.

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

In one embodiment, the compositions of the invention are administered to the patient continuously. In another embodiment, the compositions of the invention are administered to the patient in dosages that range from one to five times per day or more. In yet another embodiment, the compositions of the invention are administered to the patient in range of dosages that include, but are not limited to, once every day, every two, days, every three days to once a week, and once every two weeks. It is readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the invention varies from individual to individual depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the invention should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient is determined by the attending physical taking all other factors about the patient into account.

Compounds of the invention for administration may be in the range of from about 1 mg to about 10,000 mg, about 20 mg to about 9,500 mg, about 40 mg to about 9,000 mg, about 75 mg to about 8,500 mg, about 150 mg to about 7,500 mg, about 200 mg to about 7,000 mg, about 3050 mg to about 6,000 mg, about 500 mg to about 5,000 mg, about 750 mg to about 4,000 mg, about 1 mg to about 3,000 mg, about 10 mg to about 2,500 mg, about 20 mg to about 2,000 mg, about 25 mg to about 1,500 mg, about 30 mg to about 1 ,000 mg, about 40 mg to about 900 mg, about 50 mg to about 800 mg, about 60 mg to about 750 mg, about 70 mg to about 600 mg, about 80 mg to about 500 mg, and any and all whole or partial increments therebetween.

In some embodiments, the dose of a compound of the invention is from about 1 mg and about 2,500 mg. In some embodiments, a dose of a compound of the invention used in compositions described herein is less than about 10,000 mg, or less than about 8,000 mg, or less than about 6,000 mg, or less than about 5,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In one embodiment, the present invention is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of a compound of the invention, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms in a patient.

Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and/or aromatic substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic agents.

Routes of administration of any of the compositions of the invention include intravenous, oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual or topical. The compounds for use in the invention may be formulated for administration by any suitable route, such as for oral or parenteral, for example, transdermal, transmucosal (e.g. , sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intranasal and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present invention are not limited to the particular formulations and compositions that are described herein.

Oral Administration

For oral application, particularly suitable are tablets, dragees, liquids, drops, suppositories, or capsules, caplets and gelcaps. The compositions intended for oral use may be prepared according to any method known in the art and such compositions may contain one or more agents selected from the group consisting of inert, non-toxic pharmaceutically excipients that are suitable for the manufacture of tablets. Such excipients include, for example an inert diluent such as lactose;

granulating and disintegrating agents such as cornstarch; binding agents such as starch; and lubricating agents such as magnesium stearate. The tablets may be uncoated or they may be coated by known techniques for elegance or to delay the release of the active ingredients. Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert diluent.

For oral administration, the compounds of the invention may be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., polyvinylpyrrolidone,

hydroxypropylcellulose or hydroxypropylmethylcellulose); fillers (e.g., cornstarch, lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrates (e.g., sodium starch gly collate); or wetting agents (e.g., sodium lauryl sulphate). If desired, the tablets may be coated using suitable methods and coating materials such as OPADRY™ film coating systems available from Colorcon, West Point, Pa. (e.g., OPADRY™ OY Type, OYC Type, Organic Enteric OY-P Type, Aqueous Enteric OY-A Type, OY-PM Type and OPADRY™ White, 32K18400). Liquid preparation for oral administration may be in the form of solutions, syrups or suspensions. The liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methyl cellulose or hydrogenated edible fats); emulsifying agent (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxy benzoates or sorbic acid).

Granulating techniques are well known in the pharmaceutical art for modifying starting powders or other particulate materials of an active ingredient. The powders are typically mixed with a binder material into larger permanent free-flowing agglomerates or granules referred to as a "granulation." For example, solvent-using "wet" granulation processes are generally characterized in that the powders are combined with a binder material and moistened with water or an organic solvent under conditions resulting in the formation of a wet granulated mass from which the solvent must then be evaporated.

Melt granulation generally consists in the use of materials that are solid or semi-solid at room temperature (i.e. having a relatively low softening or melting point range) to promote granulation of powdered or other materials, essentially in the absence of added water or other liquid solvents. The low melting solids, when heated to a temperature in the melting point range, liquefy to act as a binder or granulating medium. The liquefied solid spreads itself over the surface of powdered materials with which it is contacted, and on cooling, forms a solid granulated mass in which the initial materials are bound together. The resulting melt granulation may then be provided to a tablet press or be encapsulated for preparing the oral dosage form. Melt granulation improves the dissolution rate and bioavailability of an active (i.e. drug) by forming a solid dispersion or solid solution. U.S. Patent No. 5,169,645 discloses directly compressible wax- containing granules having improved flow properties. The granules are obtained when waxes are admixed in the melt with certain flow improving additives, followed by cooling and granulation of the admixture. In certain embodiments, only the wax itself melts in the melt combination of the wax(es) and additives(s), and in other cases both the wax(es) and the additives(s) melt.

The present invention also includes a multi-layer tablet comprising a layer providing for the delayed release of one or more compounds of the invention, and a further layer providing for the immediate release of a medication for treatment of G-protein receptor-related diseases or disorders. Using a wax/pH-sensitive polymer mix, a gastric insoluble composition may be obtained in which the active ingredient is entrapped, ensuring its delayed release.

Parenteral Administration

For parenteral administration, the compounds of the invention may be formulated for injection or infusion, for example, intravenous, intramuscular or subcutaneous injection or infusion, or for administration in a bolus dose and/or continuous infusion. Suspensions, solutions or emulsions in an oily or aqueous vehicle, optionally containing other formulatory agents such as suspending, stabilizing and/or dispersing agents may be used.

Additional Administration Forms

Additional dosage forms of this invention include dosage forms as described in U.S. Patents Nos. 6,340,475; 6,488,962; 6,451,808; 5,972,389;

5,582,837; and 5,007,790. Additional dosage forms of this invention also include dosage forms as described in U.S. Patent Applications Nos. 20030147952;

20030104062; 20030104053; 20030044466; 20030039688; and 20020051820.

Additional dosage forms of this invention also include dosage forms as described in

PCT Applications Nos. WO 03/35041 ; WO 03/35040; WO 03/35029; WO 03/35177; WO 03/35039; WO 02/96404; WO 02/32416; WO 01/97783; WO 01/56544; WO

01/32217; WO 98/55107; WO 98/11879; WO 97/47285; WO 93/18755; and WO

90/11757. Controlled Release Formulations and Drug Delivery Systems

In one embodiment, the formulations of the present invention may be, but are not limited to, short-term, rapid-offset, as well as controlled, for example, sustained release, delayed release and pulsatile release formulations.

The term sustained release is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that may, although not necessarily, result in substantially constant blood levels of a drug over an extended time period. The period of time may be as long as a month or more and should be a release which is longer that the same amount of agent administered in bolus form.

For sustained release, the compounds may be formulated with a suitable polymer or hydrophobic material which provides sustained release properties to the compounds. As such, the compounds for use the method of the invention may be administered in the form of microparticles, for example, by injection or in the form of wafers or discs by implantation.

In one embodiment of the invention, the compounds of the invention are administered to a patient, alone or in combination with another pharmaceutical agent, using a sustained release formulation.

The term delayed release is used herein in its conventional sense to refer to a drug formulation that provides for an initial release of the drug after some delay following drug administration and that mat, although not necessarily, includes a delay of from about 10 minutes up to about 12 hours.

The term pulsatile release is used herein in its conventional sense to refer to a drug formulation that provides release of the drug in such a way as to produce pulsed plasma profiles of the drug after drug administration.

The term immediate release is used in its conventional sense to refer to a drug formulation that provides for release of the drug immediately after drug administration.

As used herein, short-term refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes and any or all whole or partial increments thereof after drug administration after drug administration. As used herein, rapid-offset refers to any period of time up to and including about 8 hours, about 7 hours, about 6 hours, about 5 hours, about 4 hours, about 3 hours, about 2 hours, about 1 hour, about 40 minutes, about 20 minutes, or about 10 minutes, and any and all whole or partial increments thereof after drug administration.

Dosing

The therapeutically effective amount or dose of a compound of the present invention depends on the age, sex and weight of the patient, the current medical condition of the patient and the progression of infections in the patient being treated. The skilled artisan is able to determine appropriate dosages depending on these and other factors.

A suitable dose of a compound of the present invention may be in the range of from about 0.01 mg to about 5,000 mg per day, such as from about 0.1 mg to about 1,000 mg, for example, from about 1 mg to about 500 mg, such as about 5 mg to about 250 mg per day. The dose may be administered in a single dosage or in multiple dosages, for example from 1 to 4 or more times per day. When multiple dosages are used, the amount of each dosage may be the same or different. For example, a dose of 1 mg per day may be administered as two 0.5 mg doses, with about a 12-hour interval between doses.

It is understood that the amount of compound dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on.

In the case wherein the patient's status does improve, upon the doctor's discretion the administration of the inhibitor of the invention is optionally given continuously; alternatively, the dose of drug being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a "drug holiday"). The length of the drug holiday optionally varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. The dose reduction during a drug holiday includes from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

Once improvement of the patient's conditions has occurred, a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, is reduced, as a function of the viral load, to a level at which the improved disease is retained. In one embodiment, patients require intermittent treatment on a long-term basis upon any recurrence of symptoms and/or infection.

The compounds for use in the method of the invention may be formulated in unit dosage form. The term "unit dosage form" refers to physically discrete units suitable as unitary dosage for patients undergoing treatment, with each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, optionally in association with a suitable pharmaceutical carrier. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g., about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose.

Toxicity and therapeutic efficacy of such therapeutic regimens are optionally determined in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between the toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. The data obtained from cell culture assays and animal studies are optionally used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage optionally varies within this range depending upon the dosage form employed and the route of administration utilized.

Those skilled in the art recognizes, or is able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures, embodiments, claims, and examples described herein. Such equivalents were considered to be within the scope of this invention and covered by the claims appended hereto. For example, it should be understood, that modifications in reaction conditions, including but not limited to reaction times, reaction size/volume, and experimental reagents, such as solvents, catalysts, pressures, atmospheric conditions, e.g., nitrogen atmosphere, and reducing/oxidizing agents, with art-recognized alternatives and using no more than routine experimentation, are within the scope of the present application.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compositions of the present invention and practice the claimed methods. The following working examples therefore, are not to be construed as limiting in any way the remainder of the disclosure.

Example 1 : Opposing Effects of Fasting Metabolism on Tissue Tolerance in Bacterial and Viral Inflammation

It is described herein that while nutritional supplementation increased mortality of Listeria monocytogenes infection, it protected against lethality of the influenza virus infection. The causative component of food was determined to be glucose, and this effect was largely independent of inflammation or pathogen burden. To study the differential effects of glucose metabolism in bacterial and viral inflammation and sepsis generally, lipopoly saccharide (LPS) and Poly (I: C) models of sepsis were utilized, and it was found that while therapeutic blockade of glucose utilization with 2-deoxy-D-glucose (2DG) protected against LPS-mediated sepsis, it was uniformly lethal with Poly(LC) sepsis, independent of the degree of

inflammation. Further, whereas glucose was necessary for adaptation and survival to the stress of anti-viral inflammation by preventing initiation of ER stress-mediated apoptotic pathways, glucose prevented adaptation to the stress of bacterial inflammation by inhibiting ketogenesis, which was necessary for limiting reactive oxygen species induced by anti-bacterial inflammation. This study implicates mechanistic aspects of how specific metabolic programs are coupled to specific types of inflammation to regulate cellular adaptation and host tolerance to inflammatory damage

The materials and methods employed in these experiments are now described.

Mouse strains and use

Male C57BL/6J mice (The Jackson Laboratory Stock No. 000664) between 7 and 10 weeks of age were used for all animal experiments in this study except where indicated. CHOP knockout mice are strain B6.129S(Cg)- Ddit3tm2.1Dron/J purchased from Jackson Laboratory (Stock No. 005530). PPARa knockout mice are strain B6; 129S4-Pparatml Gonz/J purchased from Jackson Laboratory (Stock No. 008154). FGF21 knockout mice were a generous gift from Dr. David J. Mangelsdorf. For feeding experiments, mice were gavaged PBS control or the equivalent of one calorie of the indicated substance (glucose, casein, olive oil, or Abbott Promote) twice a day starting 8 hours post-infection in infection models and 1 hour post-injection in sterile inflammatory models. For intraperitoneal administration of D-(+)-glucose (Sigma G8270) and 2-deoxy-D-glucose (Sigma D6134), mice were injected intraperitoneally with glucose (20 mg in 100 μΐ water) or 2DG (5 mg in 100 μΐ water) twice a day starting 8 hours post-infection in infection models and 1 hour post-injection in sterile inflammatory models. Valproic acid and levetiracetam were administered intraperitoneally starting 6 hours post-injection at 125 mg/kg and 18 mg/kg in 100 PBS, respectively. FGF21 supplementation was done by retro- orbital injection of recombinant FGF21 (R&D Systems 8409-FG/CF) twice daily at 5 ng in 100 μΐ PBS per injection. Blood oxygen saturation, breath rate, and heart rate were measured by pulse oximetry using the MouseOx Plus (Starr Life Sciences Corp.). Core body temperature was measured by rectal probe thermometry

(Physitemp TH-5 Thermalert).

Mouse infection models

Listeria monocytogenes strain 10403s was grown in BHI broth (BD

241830) as previously described (Auerbuch et al, 2004, J Exp Med 200:527-33). Mice were injected retro-orbitally with 5xl0⁴ CFU of L. monocytogenes. Influenza virus strain A/WSN/33 was originally obtained from the laboratory of Dr. Akiko Iwasaki and was propagated using MDCK cells as described (Okuda et al, 2001, Vaccine 19:3681-91). For influenza infection, mice were anesthetized with a ketamine/xylazine mixture and indicated PFU of influenza in 30 μΐ PBS was administered dropwise. Sterile inflammation models

For the LPS endotoxemia, mice were injected intraperitoneally with the indicated dose of LPS derived from Escherichia coli 055 :B5 (Sigma- Aldrich L2880) diluted in 100 μΐ PBS. For the Poly(LC) viral inflammation model, mice were injected retro-orbitally with 30 mg/kg of high molecular weight Poly(LC) (InvivoGen tlrl-pic-5) diluted in 100 μΐ of normal saline provided by the manufacturer.

Cell culture

Bone marrow derived macrophages (BMDMs) were prepared as previously described (Colegio et al, 2014, Nature 513:559-63). For assaying the effect of 2DG on L. monocytogenes replication in macrophages, BMDMs were plated at a density of 5xl0⁵ in 24-well tissue culture plates. BMDMs were infected with 5xl0⁵ of L. monocytogenes in the presence or absence of 2DG (2.5 mg/ml). After 24 hours, both supernatant and cell lysate were harvested for quantification of bacterial load. Mouse embryonic fibroblasts (MEFs) were cultured in DMEM (Sigma- Aldrich D5796) supplemented with 10% FBS (Gibco 10438-026), 1% penicillin-streptomycin (Gibco 15140-163), 2 mM L-glutamine (Gibco 25030-164), 1 mM sodium pyruvate (Gibco 11360-070), and 0.01 M HEPES (AmericanBio AB06021-00100). For assaying the effect of glucose and 2DG on various cell stress and cytokine treatments, MEFs were plated at a density of lxl 0⁵ cells per well in a 24 well plate. After overnight rest, cells were treated with the indicated chemicals or cytokines in the presence of additional glucose (final concentration 9 g/L), 15 mM 2DG, or vehicle control. Thapsigargin (Cayman Chemical 10522) was administered at 1 μΜ.

Poly(LC) was given at 20 μg/mL. Recombinant mouse IFNa (R&D Systems 12100-1) was used at 1000 U/ml.

Quantification of bacterial and viral loads

L. monocytogenes titers were determined by plating titrated amounts of liver and spleen homogenate on BHI plates as previously described (Auerbuch et al, 2004, J Exp Med 200:527-33). For in vitro assays testing the effect of 2DG on . monocytogenes growth in macrophages, titrated amounts of culture supernatant and cell lysate were plated on BHI plates and grown overnight at 37 °C. To test the effect of 2DG on growth of L. monocytogenes in BHI broth, 5 xlO³ L. monocytogenes was inoculated into 25 ml of BHI with or without 10 mg/ml of 2DG. Flasks were incubated overnight. Titrated volumes of L. monocytogenes containing BHI broth were plated the following day on BHI plates and incubated overnight for

quantification. Influenza titers in the lung and bronchoalveolar lavage fluid were determined as previously described (Okuda et al, 2001, Vaccine 19:3681-91). Plasma cytokine, metabolite, and tissue injury marker analysis

Whole blood was harvested from mice by retro-orbital bleeding and plasma was isolated using lithium heparin coated plasma separator tubes (BD 365985). Plasma TNFa and IL-6 concentration were assayed by sandwich ELISA using capture antibodies (eBioscience 14-7423-85 and 14-7061-85), biotin-conjugated detection antibodies (eBioscience 13-7341-85 and BD 554402), HRP-conjugated streptavidin (BD 554066), and TMB substrate reagent (BD 555214). Plasma IFNa and IL-Ιβ concentrations were assayed using kits according to the manufacturer's protocols (eBioscience BMS6027 and eBioscience 88-7013-22). Plasma Tropinin-I concentration and Alanine Aminotransferase (ALT) activity were assayed using kits according to manufacturers' protocols (Life Diagnostics CTNI-1-HSP and Cayman Chemical 700260). Plasma creatinine was assayed using HPLC by The George M. O'Brien Kidney Center at Yale. Plasma non-esterified fatty acid concentration was measured using a kit according the manufacturer's protocols (Wako Diagnostics 999- 34691, 995-34791, 991-34891, and 993-35191). Plasma β-hydroxybutyrate concentrations were measured using a kit according the manufacturer's protocols (Cayman Chemical 700190).

RNA extraction and quantification

For tissue RNA extraction, tissues were harvested into RNA Bee RNA isolation reagent (AMSBIO) and disrupted by bead-beating (MP Biomedicals). RNA was extracted using the RNeasy Kit according to manufacturer's protocol (Qiagen 74106). For RNA extraction from cultured cells, RNA was harvested using phenol- chloroform extraction according to manufacturer's protocol (AMSBIO). cDNA synthesis was performed using MMLV reverse transcriptase (Clontech 639524) with oligo(dT) primers. qRT-PCR reactions were performed on either a CFX96 Real-Time System or CFX384 Real-Time System (Bio-Rad) using PerfeCTa SYBR Green SuperMix (Quanta Biosciences) and transcript levels were normalized to Rpll3a. Primers used for qRT-PCR are listed in Table 1.

Flow cytometry

Antibodies to the following mouse antigens were used for flow cytometry: CD4-FITC (GK1.5), CDl lc-FITC (N418), B220-PE (RA3-6B2), Grl- PECy7 (RB6-8C5), CD45-APC/eFluor780 (30-Fl l), CD4-B.V.421 (GK1.5), NK1.1- PeCy7 (PK136), CDl lb-B.V.421 (Ml/70) (eBioscience), and TCR-beta-FITC (H57- 597), Ly6C-PE (AL-21), CD8-APC (53-6.7) (BD Biosciences), and EMA (Biotium, Hayward, CA), F4/80-PECy5 (BM8) (Biolegend). Samples were Fc-blocked with functional grade mouse anti-CD 16/32 antibody (93) (eBioscience). Annexin V-FITC and PI were used for apoptosis assays and the manufacturer's protocol was followed (eBioscience). For tissue analyses, at least 1 x 10⁵ cells were acquired on CD45+ cells within the singlet live gate, as defined by size, granularity and pulse-width. Samples were acquired on an LSRII flow cytometer (BD Biosciences), and analyzed using FlowJo (Tree Star Technologies).

In vivo reactive oxygen species staining

24 hours after administration of indicated treatments, mice were injected with 0.2 mg of hydroethidine (HEt) in 100 μΐ PBS. After 30 minutes, mice were anesthetized with a ketamine/xylazine mixture and perfused with 4%

paraformaldehyde. Brains were harvested and 50 micron sections were cut on a vibratome (Lancer Vibratome 1000 Plus). Sections were then stained with DAPI (ThermoFisher Scientific D1306) for 48 hours and visualized immediately.

Positron-emission tomography and analyses

Mice were imaged on the Inveon small animal PET/CT scanner (Siemens Medical Solutions, Malvern, PA) using 5.3±3.9 MBq of 18F-FDG. Mice were scanned for 1 hour under isoflurane anesthesia. Regions-of-interest were delineated in the heart, lung and liver (manually drawn) and brain substructures (using a template (Ma et al, 2005, Neuroscience 134: 1203-15)). Standard uptake values (SUVs) at 40-60 min post-injection were used to assess glucose metabolism.

Histology

All mice were euthanized by carbon dioxide asphyxiation and perfused with PBS or fixative. Tissues were immersion-fixed in either 10% neutral buffered formalin or Bouin's fixative (Ricca Chemical Corporation). Tissues were trimmed, processed, embedded, and sectioned and stained for hematoxylin and eosin by routine methods. Tissues were evaluated by a veterinarian trained in veterinary pathology with extensive expertise in mouse pathology blinded to both experimental and genetic manipulations.

Digital light microscopic images were acquired using a Zeiss Axio Imager Al microscope, an AxioCam MRc5 Camera, and AxioVision 4.8.3.0 imaging software (Carl Zeiss Microimaging, Inc.). The resulting images were optimized using Adobe Photoshop 13.0. lx 64.

TU EL staining was performed as previously described (Babar et al, 2012, PNAS 109:E1695-704). TUNEL images were not captured by CJB. These images were captured on a Leica DMI6000B. All other images were captured by CJB.

Statistical Analysis

Results were statistically analyzed using Student's t test or an analysis of variance (ANOVA) test with multiple comparisons where appropriate using Prism 6.0 (GraphPad Software, Inc). Kaplan Meier survival curves were compared using log-rank Mantel-Cox test. A p value of < 0.05 was considered to be statistically significant.

The results of the experiments are now described.

Anorexia Protects Against L. monocytogenes Infection To begin assessing the role of anorexia in infection, the model of listeriosis was used (Murray and Murray, 1979, Am J Clin Nutr 32:593-6; Wing and Young, 1980, Infect Immun 28:771-6). Upon infection with . monocytogenes, mice decreased their food intake dramatically and that this effect was dose dependent (Figure 1A). Consistent with the previous study (Murray and Murray, 1979, Am J Clin Nutr 32:593-6), gavage of one calorie twice daily (bis in die, BID) starting 8 hours post-infection uniformly killed L. monocytogenes infected mice (Figure IB). For caloric gavage Abbott Promote was used (25%, 23%, and 52% calories from protein, fat and carbohydrates, respectively), enteral nutrition commonly used in the supportive care of critically ill patients with a nearly identical nutritional profile to laboratory Global 2018 Teklad chow (24%, 18%, and 58% calories from protein, fat and carbohydrates, respectively). The caloric content supplemented was only one-fifth of healthy mouse daily food intake. Additionally, gavage with an isocaloric isovolumetric amount of glucose alone was sufficient to cause 100 per cent mortality in infected mice (Figure IB). To exclude contributions from enteroendocrine incretin signaling, glucose was injected intraperitoneally (IP) at a dose iso-osmolar to PBS to exclude hyperosmolar effects, which provided only about 2% of normal daily caloric intake. IP injection of glucose was sufficient to recapitulate the lethal effects of enteral glucose (Figure 1C). To assess if glucose was necessary for lethality, L.

monocytogenes infected mice were injected with 2DG. 2DG fully rescued mice from listeriosis-induced mortality (Figure 1C). This observation is consistent with previous work (Miller et al, 1998, Physiol Behav 65:535-43). Collectively, these data suggest that glucose is the component of food that is necessary and sufficient to mediate lethality in listeriosis when anorexia is blocked by force-feeding.

To investigate whether the survival effect of glucose utilization was mediated through immune clearance of the bacteria or alternative mechanisms, the bacterial titers of spleen and liver were examined four days post-infection. Glucose treatment did not significantly affect bacterial burden, plasma inflammatory cytokines, or tissue infiltration by flow cytometry (Figure ID and Figure 3). In contrast, 2DG-treated mice had significantly decreased bacterial load in both organs as compared to PBS controls, suggesting that 2DG was mediating the clearance of the bacteria (Figure ID). Despite decreased bacterial burden, plasma levels of IFNy and IL6 were also decreased in 2DG- treated animals and 2DG-treated mice also had less immune infiltrate in the liver by flow cytometry (Figure IE and Figure 3). These data were surprising given that glycolytic pathways are necessary to support the immune response (Cheng et al., 2014, Science 345: 1250684; Cramer et al, 2003, Cell 112:645-57; Kelly and O'Neill, 2015, Cell Res 25:771-84; Yang et al, 2014, Nat Commun 5:4436). It was determined if the decrease in bacterial load in 2DG-treated mice was due to the inability of the bacteria to replicate when glucose utilization was inhibited. To address this, effect of 2DG on the growth of L. monocytogenes and on the antimicrobial activity of bone marrow-derived macrophages infected with L. monocytogenes was tested. In both cases, 2DG administration did not affect the growth of the bacteria (Figures IF and 1G). Thus, these data suggest that 2DG did not have a direct toxic effect on Listeria, nor did it have the predicted effect on enhancing immune recruitment and activation which could also explain more effective pathogen clearance. Since the protective effect of anorexia during L. monocytogenes infection was neither mediated through convenetional immune clearance mechanisms of the bacteria nor was it mediated through inhibition of bacterial proliferation, tissue protective mechanisms may be at play.

Inhibition of Glucose Utilization is Protective in Endotoxemia To examine the role of tissue tolerance in mediating the protective effects of anorexia in bacterial sepsis, a commonly used model of murine sepsis was used in which the gram-negative bacterial cell wall component lipopolysaccharide (LPS) is injected into the peritoneum. In this model, pathology and eventual mortality result entirely from an over-exuberant systemic inflammatory response. Gavaging mice with one calorie of enteral nutrition BID starting one hour post LPS injection led to significantly increased mortality, while fluid resuscitation improved survival (Figure 2A) as is also seen in septic patients receiving early goal-directed therapy (Rivers et al, 2001, NEJM 345: 1368-77). To dissect the nutritional components that contribute to mortality, mice were gavaged with isocaloric isovolumetric amounts of glucose, olive oil, or casein in the same regimen as for complete enteral nutrition. Glucose, but not olive oil or casein, significantly increased mortality (Figure 2B). Whether the effects of food intake on susceptibility to sepsis could be reversed with concurrent treatment of 2DG was tested. Indeed, IP injection of 2DG concurrently with gavage of enteral nutrition in endotoxemic mice significantly improved survival rates (Figure 2C). As was the case in L. monocytogenes infection, IP injection of glucose was sufficient to uniformly kill endotoxemic mice while IP injection of 2DG was sufficient to fully rescue them, thereby excluding the effects of enteroendocrine signaling (Figure 2D). Together, these data suggest that the component of nutritional intake that increased susceptibility to endotoxic shock was glucose and that inhibition of glucose utilization during sepsis was protective.

As mentioned previously, the advantage of the LPS sepsis model is that it isolates the immune response, as opposed to direct pathogen toxicity, as the source of tissue damage. In this model, whether the effects of nutrient intake are linked to altered magnitude of the immune response can be assessed. If they are not, then any differential pathological outcomes must be due to tissue tolerance to immunopathology. Plasma from endotoxemic mice treated with PBS, glucose, or 2DG at two, six, and eighteen hours post-LPS injection was collected, circulating levels of TNFa and IL-6 were identical at all time points (Figure 2E). Livers of mice four hours post-LPS treatment were also harvested to look at acute phase response genes by gene expression and there was no difference found between groups (Figure 2F). These findings suggest that modulation of glucose availability and utilization do not affect the magnitude of the immune response in endotoxic shock, but rather the ability of the tissues to tolerate immune-mediated damage. Glucose Utilization Promotes Tissue Damage in Endotoxemia

Mice challenged with LPS and glucose displayed signs and symptoms consistent with tonic-clonic seizure. Prior to death, animals would develop high- amplitude convulsions followed by decerebrate posturing. This is consistent with studies which have demonstrated neurologic deficits, including seizure, and neuronal apoptosis in animals suffering from endotoxic shock (Sayyah et al, 2003, Neurosci 122: 1073-80; Singer et al, 2016, PLoS One l l :e0149136; Song et al, 2014, Cell Mol Neurobiol 34:839-49). Whole blood glucose measurements did not differ significantly between groups over time (Figure S4B). To exclude contributions from carbohydrate- responsive element-binding protein (ChREBP) signaling, which would still be activated with 2DG, another hexokinase inhibitor was utilized which does not activate downstream ChREBP signaling, D-mannoheptulose (DMH) (Li et al, 2010, Biochem Biophys Res Comm 395:395-400), and observed the same protective effects as 2DG. To further clinically characterize the animals, vital signs were monitored (body temperature, blood O2 saturation, respiratory rate, and heart rate) in endotoxemic mice treated with PBS, glucose, or 2DG 24 hours post-LPS injection (Figure 4A). 2DG- treated mice maintained their body temperature significantly better than glucose and PBS treated mice. To assess other evidence of end-organ damage, plasma markers of tissue injury were measured (troponin-I, alanine aminotransferase, and creatinine), which were largely unchanged between treatments, with the exception that glucose treated mice had higher levels of plasma creatinine indicating more severe kidney injury (Figure 4B). Finally, detailed histopathololgic analysis of hematoxylin & eosin (HE) stained slides of brain, heart, lung, liver, kidney, pancreas, stomach, bone marrow, thymus, and spleen for pathologic changes included edema, hemorrhage, inflammation, necrosis, and apoptosis where the observer was blind to experimental manipulation. All three groups of mice had acute lymphoid necrosis/apoptosis consistent with the LPS mouse model and no differences in histopathologic changes were seen except for decreased dark, shrunken neurons in the brains of LPS mice given 2DG compared mice given LPS and PBS or glucose. Together, these data implicate neuronal dysfunction as a possible proximal cause of death in LPS endotoxemia. Inhibition of Glucose Utilization is Lethal in Influenza Infection

Next, it was determined whether viral infections, which induce a very different set of immune cells and cytokines as compared to bacterial infections, were also significantly affected by caloric supplementation. To do so, a commonly used influenza model was employed in which mice are infected intranasally with influenza A/WSN/33. Mice infected with influenza also exhibited anorexia, albeit less severely than in L. moncytogenes infection (Figure 5A). Surprisingly, gavage of one calorie BID of enteral nutrition starting eight hours post-influenza infection actually protected mice from influenza associated mortality (Figure 5B). Gavage of isocaloric isovolumetric glucose partially recapitulated the effect of enteral nutrition, while intraperitoneal injection of 2DG concurrently with feeding completely ablated the survival benefit (Figure 5C). Caloric supplementation with casein and olive oil provided little to no survival benefit (Figure 6A). With a lower dose of influenza infection, 2DG alone was able to uniformly kill flu-infected mice as compared to vehicle control (Figure 5D). These data together indicate that glucose availability and utilization are critical to surviving influenza infection.

Next it was determined whether the effect of caloric supplementation on influenza infection was mediated through immune resistance of virus or tissue tolerance. Differences in viral burden and/or immune activation would implicate an effect of caloric supplementation on host resistance. Six days post-infection, plaque assays were performed using both lung homogenate and bronchoalveolar lavage fluid (BAL) from mice treated with PBS or 2DG. Additionally, gene expression of the influenza gene NP was assayed by qRT-PCR from lung tissue homogenate. In all three cases, no differences in viral load between groups was observed (Figure 5E). Next, antiviral inflammatory mediators were assayed to determine if

immunopathology could account for the lethality caused by 2DG treatment.

Expression of an array of interferon inducible genes as well as Cxcll and 116 were identical between PBS and 2DG-treated groups (Figures 3F and 6B). Similarly, no difference in plasma IFNa levels was found after infection (Figure 5G).

Mortality from influenza infection is often linked to development of pneumonia (Taubenberger and Morens, 2008, Annu Rev Pathol 3:499-522). To determine if 2DG impacted the extent of lung damage, the pathological outcomes of PBS versus 2DG treatment in influenza infection was assessed. Detailed

histopathologic examination of lung showed no differences in edema, hemorrhage, or the nature and extent of inflammatory cell infiltrates in (Figures 5H, 51, and 6D). These findings were verified by performing detailed analyses on BAL composition and parenchymal infiltration by FACS and found no differences between groups (Figure 6C). To identify alternative causes of death, vital signs of PBS or 2DG-treated mice were assessed over the course of influenza infection. 2DG-treated mice did not have differences in oxygen saturation, which is consistent with the lack of differences detected in the lungs of influenza-challenged mice treated with or without 2DG. However, unexpectedly, 2DG-treated mice had decreased heart rate, respiratory rate, and body temperature (Figure 5J). These findings are consistent with a derangement of central autonomic control. To verify that 2DG was not itself causing neuronal dysfunction and lethality, the identical 2DG regimen utilized in influenza was administered to mice infected with another pulmonary pathogen, Legionella pneumophilia, and this did not cause mortality, indicating that the lethal effects of 2DG occurred only in the context of the viral inflammatory state induced by influenza infection (Figure 6E). Collectively, this suggests that the effect of caloric

supplementation on influenza infection is mediated through availability of glucose utilization and its impact on tissue tolerance mechanisms, which are likely impaired in the brain. Inhibition of Glucose Utilization is Lethal in Viral Inflammation

The lethal effects of inhibiting glucose utilization in the influenza infection may be due to effects on tissue tolerance and likely acting on the brain. As the generalizability of the influenza infection model was limited by tissue-tropic and host-pathogen specific effects, the Poly (I: C) model was utilized as a general model of viral inflammation (Smorodintsev et al., 1978, Vopr Virusol 201-6). In this model, 30 mg/kg Poly (I: C) was injected intravenously, and, as in the LPS model, the mice were treated with glucose or 2DG. Administration of 2DG was uniformly lethal within 24 hours of Poly(LC) challenge (Figure 7A). As with the saving effect of 2DG in LPS challenge, differences were not observed between mice challenged with Poly (I:C) and DMH or 2DG, indicating that the ChREBP signaling cascade was not necessary (Figure 14). Since a dose of Poly(LC) that caused lethality by itself could not be generated, the protective effects of glucose supplementation could not be assessed, however, the obersvations in influenza would argue that glucose would not be sufficient to rescue death but rather is necessary for survival (Figure 5). To test if IFNa signaling on target cells is required for the effect of 2DG on Poly(I:C)-induced inflammation, IFNa-receptor (IFNaR) deficient Ifnarl-I-) mice were subjected to Poly(I:C) and 2DG challenge. Ifnarl-I- mice were completely protected (Figure 7B), indicating that IFNa signaling via IFNaR was required for mediating the lethal effects of 2DG.

To examine if the lethal effects of 2DG were mediated by differences in the magnitude of the immune response, plasma cytokines were assessed.

Significant differences in circulating IFNa were not observed (Figure 7C). Again, detailed histopathologic analyses showed no differences in histopathology by treatment group to explain cause of death. To identify the cause of mortality, intermittent vital sign monitoring was performed Mice challenged with Poly(I:C) and 2DG, like mice challenged with influenza, exhibited profound defects in the control of body temperature, respiratory and heart rate, but not oxygen saturation (Figure 7D). Thus, it was reasoned that the neuronal dysfunction and loss of autonomic control could produce this clinical constellation.

To globally assess glucose, and by extension 2DG delivery, uptake and distribution following Poly (I:C) or LPS challenge, mice were subjected to 2-deoxy-2- [18F] fluorodeoxy-D-glucose-positron emission tomography-computed tomography (18FDG-PET-CT) analyses. In these experiments, mice were injected with either LPS or Poly (I:C), and two hours after challenge, injected with 18FDG, and localization was assessed continuously over two hours. Glucose was actively taken up by the brain stem after Poly (I:C) challenge, but not with LPS challenge, whereas LPS induced more glucose uptake in the hypothalamus (Figure 7E). There were no other difference in glucose compartmentalization between Poly (I:C) and LPS (Figure 8). Paradoxically, there were decreased levels of II lb, 116, Tnfa, and the canonical interferon-stimulated gene Mxl in the brain (Figure 8). Consistent with the PET data differences in measures of cardiac, liver, or renal dysfunction were not found (Figure 8 ). These data suggest that the lethal effects mediated by inhibition of glucose utilization after Poly(LC) challenge were likely independent of the magnitude of inflammation but rather dependent on tissue tolerance to immunopathology in the hindbrain downstream of IFNaR signaling.

Inhibition of glucose utilization in viral inflammation drives mortality via a C/EBP-homologous protein (CHOP)-dependent mechanism Because mice displayed signs of neuronal damage in both influenza infection and Poly(I:C)-induced viral inflammation, the effect of inhibition of glucose utilization in viral inflammation was further investigated. It is possible that ER-stress mediated apoptotic pathways, which are integral to the cellular response to viral infection, might link viral inflammation to neuronal damage (Lin et al, 2008, Annu Rev Pathol 3:399-425). In particular, the ER-stress induced transcription factor CHOP was studied, which, upon prolonged or excessive activation, has been well characterized as a driver of apoptosis (Tabas and Ron, 2011, Nat Cell Biol 13: 184- 90). Expression of CHOP protein and its target gene Gadd34 was elevated in the hind-brains of mice treated with Poly (I: C) and 2DG (Figure 9A). To test the role of CHOP directly, mice deficient in CHOP (Ddit3^~ ) were challenged with Poly(I:C) and 2DG and were completely protected in a manner independent of inflammatory magnitude (Figures 5B and 5C). The derangements in autonomic dysfunction were also completely abrogated in CHOP-deficient mice challenged with Poly (I:C) and 2DG (Figure 9D). To test the contribution of CHOP on host tolerance and resistance, the influenza model was utilized. CHOP-deficient mice were significantly protected from influenza and 2DG challenge in a manner independent of pathogen burden or inflammation (Figure 9E and Figure 11 A). Interestingly, CHOP has been implicated in bacterial inflammatory models where its deficiency generally increases tissue injury and morbidity (Endo et al., 2005, J Biohem 138:501-7; Esposito et al, 2013, Am J Physiol Renal Physiol 204:F440-50). To model the effect of IFNa and 2DG on CHOP and cell death in vitro, mouse embryonic fibroblasts (MEFs) as a model target tissue were used. Together, 2DG and IFNa led to sustained and elevated CHOP expression over either treatment alone or vehicle control (Figure 9D). This combined treatment, as well as the combination of Poly(I:C) and 2DG, also led to significantly more cell death as determined by annexin V staining (Figure 9E). These data together suggest that glucose utilization is critical to tissue tolerance of virally -induced inflammation through maintenance of an appropriate ER stress response. Administration of glucose in endotoxemia leads to suppression of ketogenesis. increased neuronal ROS and seizure-induced death in bacterial inflammation

Prolonged fasting results in hypoglycemia accompanied by lipolysis and followed by ketogenesis. Consistent with previous studies, in anorexia following acute LPS challenge, there is a decrease in whole blood glucose and an increase in plasma free fatty acids (FFA), plasma beta-hydroxybutyrate (BHOB), and the 'fasting hormone' fibroblast growth factor 21 (FGF21) (Figure 10A). The switch to this fasting metabolic profile was ablated when glucose was exogenously administered (Figure 10A). There was no appreciable difference in the kinetics of whole blood glucose subsequent to treatment with glucose or 2DG (Figure 4B).

The administration of glucose to LPS-challenged mice led to seizures as evidenced clinically by tonic-clonic activity followed by decerebrate posturing implicating neurotoxicity as a mechanism for death. Therefore, it was asked if anti- epileptic therapies were sufficient to rescue glucose- mediated death, and found that administration of valproic acid (VA), but not levetiracetam (two of the most commonly clinically used anti-epileptic agents) was able to completely rescue LPS- challenged mice treated with glucose (Figure 10B). The anti-epileptic effects of valproic acid are incompletely understood but appear to impact HD AC -inhibition (HDAC-I), GABA transduction, and PI3K and calcium handling (Hsiech et al, 2012, Toxicol 291 :32-42; Kondo et al., 2014, PLoS One 9:el04010; Li et al, 2014, Sci Rep 4:7207). Ketone bodies have also been implicated as HDAC-I of the same class as VA, and have recently been shown to coordinate gene expression programs that confer resistance to ROS-mediated damage (Shimazu et al, 2013, Science 339:211- 4). Thus, it was hypothesized that the suppression of ketone bodies by glucose administration may be inhibiting known HDAC-I -mediated ROS adaptation pathways. To test this, dyhydroethidium staining was utilized to measure ROS in situ in the brains of LPS-challenged mice treated with glucose, and increased ROS in the brains of glucose-treated mice was observed (Figure IOC). More TU EL-positive nuclei was observed in sections of mouse brain in glucose-treated mice and compared to 2DG-treated mice or PBS-treated mice challenged with LPS (Figure 10D). All groups had TUNEL-positive nuclei in areas of lymphoid cell death (thymus, spleen), but there were no TUNEL-positive nuclei in any other tissues surveyed (heart, lung, liver, and kidney). The brain was the only tissue where differences in TUNEL- positive nuclei were seen between groups (Figure 13). Together, these data suggest that the enhanced lethality seen subsequent to glucose supplementation in endotoxemia is likely mediated through increased ROS and subsequent neuronal death. Inhibition of the ketogenic program in bacterial inflammation, but not viral inflammation, results in mortality

To test the role of ketogenesis in bacterial and viral inflammation, mice deficient in PPARa and FGF21 were subjected to both LPS and influenza. Both PPARa and FGF21 deficient mice displayed enhanced mortality (Figure 12A). It was verified that PPARa-deficient mice have severely impaired ketogenesis following LPS challenge, and no significant changes in the level of BHOB was observed in FGF21 -deficient animals, consistent with findings observed in the fasting state (Potthoff et al, 2009, PNAS 106: 10853-8) (Figure 12B). Consistent with other data, an increase in systemic cytokines was not detected, and if anything, IL-6 level was decreased in PPARa deficient mice (Figure 12C). It was hypothesized that either the lack of FGF21 or the lack of alternative fuel sources, which were both suppressed after glucose supplementation, was the causative lesion mediating death. Since FGF21 is a known downstream target of PPARa (Feingold et al, 2012, Endocrinol 153:2689- 700; Inagaki et al, Cell Metab 5:415-25), it was tested if defective FGF21 production was the causative lesion in PPARa-deficiency. PPARa-deficient and FGF21 -deficient mice were reconstituted with recombinant FGF21 given intravenously. While FGF21 was sufficient to rescue FGF21 -deficient mice, it was not sufficient to rescue PPARa- deficient mice (Figure 12D), arguing that other aspects of the ketogenic program were necessary to mediate survival of LPS sepsis. Finally, VA, but not 2DG, was able to rescue PPARa mice challenged with LPS (Figure 12E), indicating that some aspect of VA action - likely its HDAC-I activity - was sufficient to rescue the lack of ketone bodies in PPARa mice, and also that the protective effects of 2DG required an intact ketogenic program, presumably because 2DG acts upstream of ketogenesis.

Since ketotic pre-conditioning has been shown to improve other neurologic conditions such as epilepsy (Levy et al, 2012, Cochrane Database Sys Rev, CD001903), it was tested if ketotic pre-conditioning would improve survival to LPS. Mice which were pre-fasted, on ketogenic diets for 3 days, or pre-treated with valproic acid displayed no difference or enhanced mortality to LPS. The possibility that ketoacidosis was driving death was excluded. These data indicate that the activation of the ketogenic program and subsequent HDAC-I must be temporally coupled to evolution of the inflammatory challenge.

Because the viral and bacterial models had polar dependencies on glucose, they may also have polar dependences on ketogenesis. To test the effect of PPARa deficiency in viral inflammation, PPARa-deficient mice were subjected to influenza challenge. Whereas PPARa deficiency was lethal following LPS challenge, it was protective in influenza infection, in a manner independent of pathogen control (Figures 12F and 12G). This protective effect was not observed in FGF21 -deficient animals (Figure 13). Together, these data show that whereas impairment of ketogenesis, whether through genetic deletion of PPARa or glucose administration, was lethal in bacterial inflammation, it was either neutral or protective in viral inflammation, in a manner independent of the magnitude of inflammation. Differential need for metabolic fuels as a function of infection

The current study was initiated to dissect the physiologic purpose for anorexia during acute infection, and uncovered a surprising differential role for fasting metabolism in maintaining tissue tolerance in different infectious states. It is increasingly appreciated that immune and inflammatory responses must be coupled to specific metabolic programs to support their energetic demands (Buck et al, 2014, J Exp Med 212: 1345-60; Galvan-Pena and O'Neill, 2014, 5:420). In this study, it was observed that metabolism on an organismal level appears to be coordinated to support tolerance to different inflammatory states. While glucose utilization is required for survival in models of viral inflammation, it was lethal in models of bacterial inflammation. Concordantly, whereas ketogenesis was required for survival in bacterial inflammation, it was dispensable in the case of viral inflammation.

Unexpectedly, these effects on mortality were largely independent on the degree of inflammation and pathogen clearance. In the case of viral inflammation, which was modeled with Poly(LC), lethality subsequent to inhibition of glucose utilization appeared to be mediated by IFNa signaling on target tissues - likely the brain - which require glucose to mitigate the ER stress response and CHOP-mediated cell death. In the case of bacterial inflammation, which was modeled with LPS, lethality subsequent to glucose administration appeared to be mediated by suppression of ketogenesis, which lead to impaired resistance to ROS-mediated damage on the brain (Figure 12H). Thus, these results suggest that distinct inflammatory responses may be coupled with specific metabolic responses in order to support unique tissue tolerance mechanisms, that, when uncoupled, lead to enhanced immunopathology leading to death. Host responses to infectious challenge involve both host resistance and host tolerance mechanisms. Whereas host resistance promotes pathogen clearance, host tolerance relies on adaptation to a given level of pathogen, and by extension, a given level of inflammatory response (Raberg et al, 2009, Phios trans R Sco Lond B Biol Sci 318:812-4; Raberg et al, 2007, Science 318:812-4; Schneider and Ayres, 2008, Nat Rev immunol 8: 889-95). Disease morbidity and mortality can be a result of either inadequate or impaired host resistance, characterized by high pathogen burden, or as a result of impaired host tolerance. Immunopathology falls into the latter category, and insufficient tissue protection is likely to be an important determinant in conditions characterized by excessive inflammation, such as sepsis (Figueiredo et al, 2013, Immunity 39:874-84; Larsen et al, 2010 Sci Transl Med 2:51ra71). Tissue protection is likely a function of cellular stress adaptation pathways, which allow cells to adapt and survive noxious states such as increased free radicals and accumulation of unfolded proteins (Figueiredo et al., 2013, Immunity 39:874-84; Larsen et al., 2010 Sci Transl Med 2:51ra71). When these adaptation pathways are overwhelmed, cells undergo apoptotic cell death (Boison et al, 2013, Epilepsy Curr 13:219-20; Tabas and Ron, 2011, Nat Cell Biol 13: 184-90). Thus, one important determinant of host tolerance may be related to the ability of cellular adaptation programs to tolerate noxious states that would be found in infections. Since different infections generate different types of inflammatory responses and different noxious states, one would predict that different cellular adaptation programs would need to be activated - and would be dominant - in different infection contexts.

It is shown herein that interfering with the normal ketogenic state following LPS-mediated inflammation was lethal, likely by interfering with ROS adaption programs in the brain. These findings are consistent with observations that PPARa agonism and inhibition of glucose utilization are generally protective in bacterial sepsis models (Budd et al, 2007, Antimicrob Agents Chemother 51 :2965-8; Camara-Lemarroy et al, 2015, Exp Ther Med 9: 1018-22; Yoo et al, 2013, Biochem Biophys Res Commun 436:366-71). However, unlike many of these studies, large differences in the magnitude of inflammation was not observed, and it is possible that this is because 2DG and glucose were administered after and not before infectious or inflammatory challenge. Thus, these observations are likely unrelated to the body of literature that supports a role for Hifla and PKM2 and aerobic glycolysis in generating the LPS inflammatory response (Liu et al, 2016, PNAS 113: 1564-9; Yang et al, 2014, Nat Commun 5:4436). Consistent with the inability to detect large differences in inflammatory parameters in sterile inflammatory models, large differences in pathogen burden were not detected in the live infection models where glucose administration led to lethality in L. monocytogenes infection in the absence of increased pathogen burden or detectable systemic cytokines. Consistent with the idea that the immune and inflammatory responses were not compromised with inhibition of glycolysis, decreased L. monocytogenes burden in 2DG-treated animals were observed, an observation that has previously been noted and remains mechanistically elusive. ROS-mediated cytotoxicity is a well-appreciated phenomenon in bacterial sepsis (Hoetzenecker et al., 2012, Nat Med 18: 128-34; Kolls, 2006, J Clin Invest 116:860-3), and ROS-detoxification pathways have been implicated in mitigating tissue damage and mortality. Shimazu et al recently reported that BHOB functioned as an HDAC-1 inhibitor, and that this lead to transcription of ROS-detoxification pathways (Shimazu et al, 2013, Science 339:211-4). The data described herein provides evidence that the fasting state is required to maintain resistance to oxidative stress in LPS sepsis.

Interestingly, although anorexia is a common response in both bacterial and viral infections, the opposite consequence of fasting metabolism was observed in the models of bacterial and viral inflammation. While in bacterial infection and LPS induced inflammation effect of glucose had a detrimental, a protective effect of 2DG, and a requirement for ketogenesis in order to maintain tolerance, in viral infection and Poly(I:C)-induced inflammation these effects were the opposite. 2DG administration led to lethality in Poly (LC)-induced inflammation in a manner that was independent of the magnitude of IFNa expression, and 2DG-mediated lethality required intact IFNaR signaling. Concordant with a tolerance mechanism, the live infection model with influenza did not demonstrate an increased viral burden, and unexpectedly showed modest but significant improvement in lung pathology. Viral infections are known to stimulate the unfolded protein response as a cytoprotective mechanism but also as a resistance mechanism to limit the amount of viral protein translation, and this has been shown to be in part mediated via the PERK-eIF2a-ATF4-CHOP unfolded protein response pathway (Janssens et al, 2014, Nat Immunol 15:910-9). When this pathway is engaged, the cell can either adapt to the ER stress, or induce apoptosis through CHOP if the ER stress cannot be managed (Tabas and Ron, 2011, Nat Cell Biol 13: 184-90). The data presented herein suggests that glucose utilization is required for the cytoprotective response in the setting of viral inflammation, as inhibition of glucose utilization lead to cell death, which was dependent on CHOP. The precise mechanism whereby IFNa signaling converges with glucose utilization programs remains to be fully elucidated, but recent studies demonstrated that interferon signaling leads to changes in glucose uptake, which is important for antiviral response (Burke et al, 2014, J Virol 88:3485-95). Thus, whereas alternative fuel substrate availability is coupled to and necessary for adaptation to bacterial inflammation, glucose availability is coupled to and necessary for cellular adaptation to viral inflammation. The logic of their coupling is likely related to the substrate- dependence of the cellular adaptation programs that are engaged. These findings are consistent with previous studies which show that synergistic lethality in mice co- infected with influenza and legionella occurred in a manner independent of pathogen burden (Jamieson et al, 2013, Science 340: 1230-4), and it is interesting to speculate here that perhaps the cause of lethality in this co-infection model is a result of metabolic incompatibility in the setting of both a viral and bacterial infection.

Given the conservation of cellular adaptation and metabolic programs, these findings likely have clinical implications. The role of nutrition in managing patients with sepsis is unclear at best, and multiple studies have failed to show differences in survival from feeding, including the most recent study which attempted to ask if lower caloric supplementation would change outcomes compared to normal supplementation (Arabi et al., 2015, NEJM, 372:2398-408). There have been a series of studies exploring different feeding formulations with different caloric or micronutrient contents (Casaer and Van den Berghe, 2014, NEJM 370: 1227-36). However, an example where different feeding formulations were targeted at different types of infections, as opposed to different types of organ failure (Seron-Arbeloa et al., 2013, J Clin Med Res 5 : 1-11), or where post-hoc analyses was directed at pathogen class could not be found. This study implicates a differential need for metabolic fuels as a function of infection (or inflammation) class, and sheds light on the biology behind the old adage "starve a fever, stuff a cold."

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed is:

1. A method of treating an infection in a subject, the method comprising administering to the subject an effective amount of at least one sugar analog selected from the group consisting of glucose analogs and heptose analogs.

2. The method of claim 1, wherein the glucose analog is 2-deoxy-D- glucose (2DG).

3. The method of claim 1, wherein the heptose analog is D-manno- heptulose (DMH).

4. The method of claim 1, wherein the infection is selected from the group consisting of sepsis, a bacterial infection and a parasitic infection.

5. The method of claim 4, where 2DG does not suppress ketogenesis.

6. The method of claim 4, wherein the infection is selected from the group consisting of a listeria infection and a Plasmodium infection.

7. The method of claim 1, wherein the method further comprises administering at least one additional therapeutic.

8. The method of claim 7, wherein the at least one additional therapeutic is selected from an antibiotic and an antiparasitic.

9. The method of claim 1, wherein the subject is a human.

10. A method of providing nutritional supplementation to a subject, the method comprising administering to the subject a composition comprising at least one sugar analog selected from the group consisting of glucose analogs and heptose analogs.

11. The method of claim 10, wherein the glucose analog is 2-deoxy-D- glucose (2DG).

12. The method of claim 10, wherein the heptose analog is D-manno- heptulose (DMH).

13. The method of claim 10, wherein the subject has an infection selected from the group consisting of sepsis, a bacterial infection and a parasitic infection.

14. The method of claim 13, wherein the bacterial infection is a listeria infection.

15. The method of claim 13, wherein the parasitic infection is a Plasmodium infection.

16. The method of claim 13, wherein the composition does not comprise glucose.

The method of claim 10, wherein the method improves survival of