Peptides for treatment of inflammation and shock Number:7,094,760 from the United States Patent and Trademark Office (PTO) owispatent Submandibular peptides have been isolated and characterised and provide pharmaceutical compositions for the treatment or prevention of anaphylactic reactions, endotoxic reactions and SIRS-induced shock.<H inflammation, treatment, Peptides, for, tr, 7094760.html, Patent, pto, 7094760

Title: Peptides for treatment of inflammation and shock

Abstract: Submandibular peptides have been isolated and characterised and provide pharmaceutical compositions for the treatment or prevention of anaphylactic reactions, endotoxic reactions and SIRS-induced shock.

Patent Number: 7,094,760 Issued on 08/22/2006 to Mathison, et al.

Inventors: Mathison; Ronald (Calgary, CA), Davison; Joseph S. (Calgary, CA), Befus; Dean (Edmonton, CA), Moore; Graham (Calgary, CA)
Assignee: Salpep Biotechnology, Inc. (Calgary, CA)

Appl. No.: 10/773,229

Filed: February 9, 2004

Related U.S. Patent Documents


Application Number	Filing Date	Patent Number	Issue Date
09051395		6852697
PCT/CA97/00568	Aug., 1997

Foreign Application Priority Data


Aug 13, 1996 [GB]			9617021.2

Current U.S. Class: 514/16 ; 514/17; 514/18; 530/328; 530/329; 530/330; 530/331

Current International Class: A61K 38/08 (20060101)

Field of Search: 514/2,16,17,18 530/324,328,330,331

References Cited [Referenced By]

U.S. Patent Documents


4880779	November 1989	Gallaher
6586403	July 2003	Mathison et al.

Foreign Patent Documents


WO 92/11858	Jul., 1992	WO

Other References

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Primary Examiner: Gupta; Anish
Attorney, Agent or Firm: Buchanan Ingersoll, PC

Parent Case Text

This application is a divisional of application Ser. No. 09/051,395, filed on May 8, 1998 now U.S. Pat. No. 6,852,697, which is a national stage filing under 35 U.S.C. .sctn. 371 of International Application No. PCT/CA97/00568, filed on Aug. 13, 1997, the entire contents of which are incorporated herein in its entirety.

Claims

We claim:

1. A method for treating an inflammatory reaction in a mammal comprising administering to the mammal a peptide of the formula: X.sup.1--R.sup.1--X.sup.2--R.sup.2 wherein X.sup.1 is an aromatic amino acid residue; X.sup.2 is any amino acid residue; and R.sup.1 is NH.sub.2--or an amino acid sequence X.sup.3--X.sup.4--X.sup.5 wherein X.sup.3 is an aliphatic amino acid residue having a side chain hydroxyl group and X.sup.4 and X.sup.5 are the same or different and are any amino acid residue and wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues, or a fragment or derivative of said peptide of the formula R.sup.1--X.sup.1--X.sup.2--R.sup.2 effective to treat an inflammatory reaction.

2. The method of claim 1 wherein X.sup.1 is phenylalanine; R.sup.1 is NH.sub.2--; and R.sup.2 is an aliphatic amino acid residue.

3. The method of claim 1 wherein X.sup.1 is phenylalanine; X.sup.2 is Glu or Ala; R.sup.2 is selected from the group consisting of Gly, Gly-Gly and Gly-Gly-Gly; and R.sup.1 is NH.sub.2--or X.sup.3--X.sup.4--X.sup.5 wherein X.sup.3 is Thr, X.sup.4 is Asp or Ala and X.sup.5 is Ile or Ala.

4. The method of claim 1 wherein X.sup.1 is phenylalanine; X.sup.2 is Glu; R.sup.1 is NH.sub.2--; and R.sup.2 is selected from the group consisting of Gly, Gly-Gly and Gly-Gly-Gly.

5. The method of claim 1 wherein the peptide is selected from the group consisting of: (a) Thr-Asp-Ile-Phe-Glu-Gly-Gly (Sequence ID NO:8); (b) Thr-Ala-Ile-Phe-Glu-Gly-Gly (Sequence ID NO:3); (c) Thr-Asp-Ala-Phe-Glu-Gly-Gly (Sequence ID NO:4); (d) Thr-Asp-Ile-Phe-Ala-Gly-Gly (Sequence ID NO:6); (e) Phe-Glu-Gly-Gly-Gly (Sequence ID NO:9); (f) Phe-Glu-Gly-Gly (Sequence ID NO:11); (g) Phe-Ala-Gly-Gly-Gly (Sequence ID NO: 12); and (h) Phe-Glu-Sarcosine.

6. The method of claim 1 wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are selected from the group consisting of glycine, sarcosine, azetidine, nipecotic acid and pipecotic acid.

7. The method of claim 1 wherein at least one amino acid of said peptide is a D amino acid.

8. The method of claim 1 wherein the peptide is Phe-Glu-Gly.

9. The method of claim 1 wherein the peptide is DPhe-DGlu-Gly.

10. The method of claim 1 wherein the inflammatory reaction is associated with a disorder selected from the group consisting of a rheumatic disorder, inflammatory bowel disease, post-ischemic inflammation and systemic inflammatory response syndrome.

11. A method for reducing the infiltration of neutrophils into an inflammatory site in a mammal comprising administering to the mammal a peptide of the formula: X.sup.1--R.sup.1--X.sup.2--R.sup.2 wherein X.sup.1 is an aromatic amino acid; X.sup.2 is any amino acid residue; and R.sup.1 is NH.sub.2--or an amino acid sequence X.sup.3 --X.sup.4 --X.sub.5 wherein X.sup.3 is an aliphatic amino acid residue having a side chain hydroxyl group and X.sup.4 and X.sup.5 are the same or different and are any amino acid residue and wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues, or a fragment or derivative of said peptide of the formula X.sup.1--R.sup.1--X.sup.2--R.sup.2 effective for reducing the infiltration of neutrophils into an inflammatory site.

12. The method of claim 11 wherein X.sup.1 is phenylalanine; R.sup.1 is NH.sub.2--; and R.sup.2 is an aliphatic amino acid residue.

13. The method of claim 11 wherein X.sup.1 is phenylalanine; X.sup.2 is Glu or Ala R.sup.2 is selected from the group consisting of Gly, Gly-Gly and Gly-Gly-Gly; and R.sup.1 is NH.sub.2-- or X.sup.3--X.sup.4--X.sup.5 wherein X.sup.3 is Thr, X.sup.4 is Asp or Ala and X.sup.5 is Ile or Ala.

14. The method of claim 11 wherein X.sup.1 is phenylalanine; X.sup.2 is Glu; R.sup.1 is NH.sub.2--; and R.sup.2 is selected from the group consisting of Gly, Gly-Gly and Gly-Gly-Gly.

15. The method of claim 11 wherein the peptide is selected from the group consisting of: (a) Thr-Asp-Ile-Phe-Glu-Gly-Gly (Sequence ID NO:8); (b) Thr-Ala-Ile-Phe-Glu-Gly-Gly (Sequence ID NO:3); (c) Thr-Asp-Ala-Phe-Glu-Gly-Gly (Sequence ID NO:4); (d) Thr-Asp-Ile-Phe-Ala-Gly-Gly (Sequence ID NO:6); (e) Phe-GIu-Gly-Gly-Gly (Sequence ID NO:9); (f) Phe-Glu-Gly-Gly (Sequence ID NO:11); (g) Phe-Ala-Gly-Gly-Gly (Sequence ID NO: 12); and (h) Phe-Glu-Sarcosine.

16. The method of claim 11 wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are selected from the group consisting of glycine, sarcosine, azetidine, nipecotic acid and pipecotic acid.

17. The method of claim 11 wherein at least one amino acid of said peptide is a D amino acid.

18. The method of claim 11 wherein the peptide is Phe-Glu-Gly.

19. The method of claim 11 wherein the peptide is DPhe-DGlu-Gly.

20. A method for inhibiting activation of neutrophils in a mammal comprising administering to the mammal a peptide of the formula: R.sup.1--X.sup.1--X.sup.2--R.sup.2 wherein X.sup.1 is an aromatic amino acid; X.sup.2 is any amino acid residue; and R.sup.1 is NH.sub.2--or an amino acid sequence X.sup.3--X.sup.4--X.sup.5 wherein X.sup.3 is an aliphatic amino acid residue having a side chain hydroxyl group and X.sup.4 and X.sup.5 are the same or different and are any amino acid residue and wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues, or a fragment or derivative of said peptide of the formula X.sup.1--R.sup.1--X.sup.2--R.sup.2 effective for inhibiting activation of neutrophils.

21. The method of claim 20 wherein X.sup.1 is phenylalanine; X.sup.2 is Glu; R.sup.1 is NH.sub.2--; and R.sup.2 is selected from the group consisting of Gly, Gly-Gly and Gly-Gly-Gly.

22. The method of claim 20 wherein the peptide is Phe-Glu-Gly or DPhe-DGlu-Gly.

23. The method of claim 20 wherein the peptide is Phe-Glu-Gly-Gly-Gly.

24. The method of claim 1 wherein the peptide is Phe-Glu-Gly-Gly-Gly.

25. The method of claim 11 wherein the peptide is Phe-Glu-Gly-Gly-Gly.

26. The method of claim 1 wherein X.sup.2 is an acidic amino acid residue; R.sup.1 is NH.sub.2--; and R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues.

27. The method of claim 11 wherein X.sup.2 is an acidic amino acid residue; R.sup.1 is NH.sub.2--; and R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues.

28. The method of claim 20 wherein X.sup.2 is an acidic amino acid residue; R.sup.1 is NH.sub.2--; and R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues.

Description

FIELD OF THE INVENTION

This invention relates to peptides which modulate anaphylactic, endotoxic and inflammatory reactions in mammals.

BACKGROUND OF THE INVENTION

In the description which follows, references are made to certain literature citations which are listed at the end of the specification.

Infections, trauma (e.g. falls, automobile accidents, gun and knife wounds), cardiovascular accidents (e.g. aneurysms and ischemic events often associated with surgery) and endogenous inflammatory reactions (e.g. pancreatitis and nephritis) often lead to profound dysfunction of the homeostatic mechanisms involved in regulating cardiovascular and immune system function.

Several conditions such as ischemia and infections, through inappropriate or excessive activation of the immune system, may result in cardiovascular dysfunction that develops over a period of hours to days. Compromised cardiovascular function increases morbidity and is life threatening.

Systemic inflammatory response syndrome (SIRS) is diagnosed largely on observed physiological changes such as increase (fever) or decrease in body temperature (hypothermia), increased heart rate (tachycardia), increased respiration rate (tachypnea), elevated or diminished white blood cell counts and inadequate perfusion of tissues and organs. Changes in blood pressure are not included in the definition because they occur late in the course of the syndrome. Decreases in blood pressure reflect the development of shock, and contribute to multiple organ failure, a leading cause of death in these patients. This definition of sepsis syndrome includes a large number of patients who exhibit similar physiological signals but have no evidence of any type of infection; other insults which induce a sepsis-like condition include pancreatitis, burns, ischemia, multiple trauma and tissue injury (often due to surgeries and transplants), haemorrhagic shock and immune-mediated organ dysfunction.

SIRS is the 13th leading cause of death in the United States of America. On average, 40% of sepsis syndrome patients are dead within 28 days of admission to intensive care.

The standard therapies for SIRS and septic shock involve administration of antibiotics to bring the infection under control and fluid/colloid therapy to maintain circulating blood volume. Frequently, drugs which help maintain blood pressure, such as dopamine and vasopressin, are also administered.

Recent strategies for developing more targeted therapies for the treatment of sepsis have been disappointing. In addition, many molecules in the new generation of anti-septic agents are very expensive and most possibly produce untoward immunological and cardiovascular reactions which make them contra-indicated in some cases of non-bacteremic shock.

Hypersensitivity to environmental antigens can, in severe cases, result in anaphylactic shock, which is treated by the administration of fluids and vasoactive agents to restore blood pressure.

There remains a need for inexpensive and effective agents for treatment of cardiovascular shock, sepsis, systemic inflammatory response syndrome and anaphylaxis.

The salivary glands are classically viewed as accessory digestive glands which mediate their actions through exocrine secretion, although appreciation has grown recently for the importance of their endocrine secretions (1 3). The exocrine secretion of biologically active peptides from the salivary glands is essential for the health of the mouth (4), whereas the endocrine secretions of these glands help maintain the structure and function of a large variety of internal tissues and organs such as the digestive tract (5 7), the mammary glands (8), the liver (9,10), and the reproductive tract (11,12). Rosinski-Chupin et al. (13,14) have described a protein from the male rat submandibular gland which is androgen-regulated and which they believed to have a male-specific function in the rate.

Another important action of salivary endocrine secretions is the modulation of the immune system, with effects being observed on lymphocyte (15,16), mast cell (17) and neutrophil (18,19) functions. The submandibular glands also regulate inflammatory reactions associated with the late-phase pulmonary inflammation induced by allergen in sensitized rats (20 22), and their removal exacerbates the severity of the hypotensive responses induced by intravenously administered lipopolysaccharide (LPS) (23). Removal of the submandibular glands, however, does not affect arterial blood pressure in the normal state (23).

In accordance with one embodiment, the invention provides a peptide of the formula: R.sup.1--X.sup.1--X.sup.2--R.sup.2

wherein X.sup.1 is an aromatic amino acid residue; X.sup.2 is any amino acid residue; R.sup.1 is NH.sub.2--or an amino acid sequence X.sup.3--X.sup.4--X.sup.5 wherein X.sup.3 is an aliphatic amino acid residue having a side chain hydroxyl group and X.sup.4 and X.sup.5 are the same or different and are any amino acid residue and

wherein R.sup.2 is a sequence of 1 to 3 amino acid residues which are the same or different and are aliphatic amino acid residues.

In accordance with a further embodiment, the invention provides a peptide having the amino acid sequence

Ser-Gly-Glu-Gly-Val-Arg (Sequence ID NO:1).

In accordance with a further embodiment, the invention provides a method for treating or preventing SIRS-induced hypotension in a mammal comprising administering to the mammal an effective amount of a described peptide or of an effective fragment or derivative of the peptide.

In accordance with a further embodiment, the invention provides a method for treating or preventing anaphylactic hypotension in a mammal comprising administering to the mammal an effective amount of a described peptide or of an effective fragment or derivative of said peptide.

In accordance with a further embodiment, the invention provides a method of reducing or preventing an anaphylactic reaction in a mammal comprising administering an effective amount of a described peptide or of an effective fragment or derivative of said peptide to the mammal.

In accordance with a further embodiment, the invention provides a method of reducing or preventing an endotoxic reaction in a mammal comprising administering an effective amount of a described peptide or 11 or an effective fragment or derivative of said peptide to the mammal.

In accordance with a further embodiment, the invention provides a method for treating an inflammatory disorder in a mammal comprising administering to the mammal an effective amount of a described peptide or of an effective fragment or derivative of the peptide to the mammal.

In accordance with a further embodiment, the invention provides an antibody which specifically recognises an epitope of a peptide of the invention.

In accordance with a further embodiment, the invention provides a method of determining the peptide SGP-T or the peptide SGPS in a biological fluid comprising obtaining a sample of the biological fluid and determining the peptide in the fluid by immunoassay employing an antibody which specifically epitope of a peptide of the invention.

Certain embodiments of the invention are described, reference being made to the accompanying drawings, wherein:

FIG. 1 shows the effect of SGP-T concentration on the average decrease over a 1 hour period in mean arterial blood pressure (MABP) induced by intravenous LPS (Salmonella typhosa, 3.5 mg/kg), evaluated in pentobarbital anesthetized unoperated (.box-solid.) and sialadenectomized (.tangle-solidup.) rats. * p<0.05

FIG. 2 shows the average decrease in MABP in rats (sensitised 1 month previously with 3000 larvae of the nematode Nippostrongylus brasiliensis) challenged by injection of worm antigen (100 worm equivalents), after the following treatments:

A: no drug

B: 10 .mu.g SGP-T

C: 35 .mu.g SGP-T

D: 100 .mu.g SGP-T. * p<0.05

FIG. 3 shows % treated rats with disrupted MMC's (solid bars) or diarrhoea (hatched bars) after the indicated pretreatments, followed by challenge with egg albumin (EA) or bovine serum albumin (BSA).

FIG. 4 shows number of neutrophils migrating into carrageenin-soaked sponges implanted in rats treated with various indicated concentrations of SGP-T.

FIG. 5 shows superoxide anion production, expressed as n moles 0.sub.2.sup.-/min/10.sup.7 cells, in neutrophils from carrageenin-soaked sponges implanted in rats treated with various indicated concentrations of SGP-Tcarr=carrageenin-soaked sponge from rat with no SGP-T treatment; sal=saline soaked sponge (control) from rat with no SGP-T treatment.

FIG. 6 shows superoxide anion production by human (.box-solid.) or rat (.tangle-solidup.) neutrophils treated in vitro with SGP-T.

FIG. 7 shows the effect of SGP-T and various analogues and fragments on antigen-induced jejunal segment contraction (from ovalbumin (OA)- sensitized rats). Con: saline control; T: SGP-T; A1: ADIFEGG (Sequence ID NO:2); A2 (Sequence ID NO:3): TAIFEGG (Sequence ID NO:3); A3: TDAFEGG (Sequence ID NO:4); A4: TDIAEGG (Sequence ID NO:5); A5: TDIFAGG (Sequence ID NO:6); X6: TDIFE (Sequence ID NO:7); X7: TDIFEGG-NH2 (Sequence ID NO:8); X8: FEGGG (Sequence ID NO:9). Ure/OA Ratio (X axis) is the ratio of the contractile response to the cholinergic agonist, urecholine (URE), divided by the contractile response to OA. * P<0.05. The dotted line indicates the control (Con) response across the figure.

FIG. 8 shows the effect of SGP-T and various analogues on antigen-induced jejunal segment contraction (from OA- sensitized rats). Cont: control; T: SGP-T; FEG: FEG; Sar: FE-Sarcosine; CIT: FE-Citrulline; Pro: FE-proline. Ure/OA Ratio (X axis) is the ratio of the contractile response to the cholinergic agonist, urecholine (URE), divided by the contractile response to OA. * P<0.05. The dotted line indicates the control (Con) response across the figure.

FIG. 9 shows the effect of SGP-T and various analogues on antigen-induced anaphylactic hypotension. X axis: ventricular peak systolic pressure (VPSP). Con saline control; feG: D-phenylalanine, D-glutamate, glycine. * P<0.05

FIG. 10 shows the effect of feG on antigen-induced anaphylactic hypotension. X axis: VPSP

FIG. 11 shows the incidence of disrupted intestinal myoelectric activity (disrupted MMC's) and diarrhoea in rats treated with egg albumin alone (EA), bovine serum albumin alone (BSA), SGP-T prior to OA (SGP-T), FEG prior to OA (FEG), feG prior to OA (feG) and feG orally prior to OA (feG oral).

FIG. 12 shows changes in ventricular peak systolic pressure (VPSP) over time in rats treated with saline (.box-solid.), 10 .mu.g/kg SGP-T (.tangle-solidup.) or 100 .mu.g/kg SGP-T (.diamond-solid.) prior to challenge with antigen. * P<0.05, n.gtoreq.4

FIG. 13 shows the antigen-induced decrease in VPSP in rats after pre-treatment with various doses of SGP-T. * P<0.05.

FIG. 14 shows MABP 10 min before (Before) or 60 minutes after (After) LPS injection in rats treated 30 minutes before LPS with FEG (hatched bars) or vehicle (open bars).

FIG. 15 shows the effect of SGP-T at various concentrations (solid bars) on endotoxin-induced fever (X axis: .DELTA.Tb=change in body temperature (.degree. C.)) relative to pre-endotoxin body temperaturs. Open bars: control * P<0.05 n=4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to peptides obtained from mammalian submandibular glands which modulate anaphylactic reactions and endotoxic and inflammatory reactions in mammals.

Anaphylactic reactions in mammals are severe and harmful reactions to an allergen to which a mammal has become sensitized or hypersensitized. These reactions are severe or excessive manifestations of an immunological protective mechanism designed to protect the mammal against foreign antigens. Anaphylactic reactions can occur in response to such allergens as insect bites and stings, plant pollens, plants such as poison ivy, food allergens, such as nuts and seafood, animal dander and house dust and have a role in the etiology of asthma, rhinitis, urticaria, eczema and certain gastrointestinal disorders.

An anaphylactic reaction to an antigen can be associated with hypotension, increased intestinal motility, diarrhea, bronchial constriction and edematous swelling. Anaphylactic hypotension may be severe enough to cause anaphylactic shock, which may be life-threatening.

Endotoxic reactions in mammals develop consequent to the activation of the immune system in an attempt to rid the body of a bacterial infection. Unlike anaphylactic reactions, which develop rapidly in a matter of minutes, endotoxic reactions increase in severity over a period of days. Pro-inflammatory mediators such as cytokines, which are released as part of the body's response to an infection, stimulate the respiratory and cardiovascular systems. In some patients, the fight against the infection fails and the pro-inflammatory mediators begin to have deleterious effects on the patient, promoting the progressive collapse of the cardiovascular system, poor organ perfusion and further tissue damage. If these severe inflammatory reactions are not arrested, endotoxic shock can ensue.

A clinically useful paradigm has been developed that helps define patients that could possibly progress into sepsis or sepsis-like conditions, and eventually shock. This paradigm is called the systemic inflammatory response syndrome (SIRS).

SIRS is defined clinically as the presence of two or more of the following criteria:

1) a body temperature greater than 38.degree. C. or less than 36.degree. C.;

2) a heart rate greater than 90 beats/minute;

3) a respiratory rate greater than 20 breaths/minute;

4) a white blood cell count greater than 12 million/ml or less than 4 million/ml.

Some 68% of all patients entering the hospital possess SIRS, and thus are at risk for the development of sepsis or a sepsis-like condition and shock. A confirmed infectious process (i.e. positive blood cultures) are required for the rigorous diagnosis of sepsis. Nonetheless, some infection-negative patients progress to a stage of severe sepsis, which is defined by the presence by one of the following conditions:

1) a reduction of systolic blood pressure to less than 90 mm Hg;

2) a systemic manifestation of poor tissue perfusion as reflected by lactic acidosis, low urine output or acute alteration of mental state.

An SIRS patient can progress directly to severe sepsis, in the absence of a definable infectious agent. Some of the conditions that favour progression to the severe sepsis stage include: pancreatitis, burns, and cerebral or spinal injuries. Patients with SIRS can proceed to SIRS-induced hypotension and SIRS-induced shock. A patient is considered to be in shock if he or she remains hypotensive (i.e. systolic blood pressure below 90 mm Hg) following the administration of 500 ml of fluid.

The cervical sympathetic trunk-submandibular gland (CST-SMG) axis has recently been identified as a neuroendocrine axis that modulates pulmonary inflammatory and cardiovascular responses provoked by sensitizing antigen (20,21) and endotoxins (23), respectively. The cervical sympathetic nerves modulate such. inflammatory reactions, for example that induced by intravenous administration of gram negative bacterial endotoxin, by regulating the release, from the submandibular glands (3), of factors which reduce the severity of the reaction (23).

The present inventors have obtained from rat submandibular glands, and characterized, two novel peptides which have profound effects on a variety of cardiovascular and immunological perturbations and are likely candidates for the agents by which the submandibular gland exerts its influence on anaphylactic or endotoxic reactions.

In accordance with one embodiment, the present invention provides submandibular gland peptides S and T (SGP-S and SGP-T), which have the following amino acid sequences:

SGP-S: NH.sub.2-Ser-Gly-Glu-Gly-Val-Arg-COOH (SGEGVR: Sequence ID NO:1);

SGP-T: NH.sub.2-Thr-Asp-Ile-Phe-Glu-Gly-Gly-COOH (TDIFEGG Sequence ID NO:8).

The invention also includes effective fragments and derivatives of these peptides which retain at least one biological activity of the peptide from which they are derived. The terms "derivative" extends to any functional and/or chemical equivalent of the peptides of the invention and includes peptides having one or more amino acid substitutions, peptides incorporating unnatural amino acids and peptides having modified side chains. The invention also includes homologues of these peptides in other species, including human. Such homologues may be identified using antibodies to the peptides disclosed herein, as will be understood by those of ordinary skill in the art.

The biological effects of SGP-T include reduction or prevention of endotoxic hypotension, at doses as low as 1 .mu.g peptide/kg body weight; reduction or prevention of anaphylactic hypotension in foreign protein-sensitised mammals; a protective effect against ventricular dysfunction during systemic anaphylaxis; attentuation of antigen-induced perturbations of gastrointestinal motility in ovalbumin-sensitised animals; a significant reduction of in vitro antigen-induced smooth muscle contraction, in muscle from ovalbumin-sensitized animals; a significant reduction of the fever provoked by bacterial endotoxin; an approximately 50% inhibition of neutrophil migration; a prevention of carrageenin-induced inhibition of superoxide production by phorbal myristate (PMA) and formyl-Met-Leu-Phe. (fMLP);

The invention enables pharmaceutical compositions comprising the peptide SGP-T or an effective fragment or derivative thereof, for the treatment or prevention of anaphylactic reactions, including anaphylactic shock, and endotoxic reactions, including endotoxic shock.

These compositions may also be used for the treatment or prevention of systemic inflammatory response syndrome (SIRS) and the treatment of inflammation or any disorder ameliorated by down regulation of neutrophil function. Such disorders include rheumatic diseases, inflammatory bowel disease and post-ischemic lesions subsequent to stroke or cardiac infarct.

The peptides of the invention modulate and reduce the severity of anaphylactic reactions, including cardiovascular and intestinal anaphylactic reactions, anaphylactic shock and SIRS-induced reactions, including endotoxic shock and SIRS-induced shock.

The invention also enables methods for preventing or treating anaphylactic reactions, including anaphylactic shock, endotoxic reactions, including endotoxic shock, SIRS, inflammation and disorders ameliorated by down regulation of neutrophil function.

Examination of fragments and derivatives of SGP-T indicates that various amino acid substitutions may be effected without loss of biological activity, as shown in Tables 2, 3 and 4.

SGP-T and various full length derivatives thereof are active in inhibiting anaphylactic and endotoxic hypotension.

The fragment FEG shows the same activity as SGP-T in inhibiting anaphylactic hypotension but does not inhibit endotoxic hypotension. The anti-anaphylactic hypotension activity of FEG is maintained if D amino acids are substituted for F and G.

Although the mechanism of action of the peptides of the invention is still unknown, these results suggest that the anti-endotoxic activity and the anti-anaphylactic activity of SGP-T may be mediated by different cell receptors.

SGP-T does not prevent an initial anaphylactic reaction (see FIG. 12 which shows the same initial blood pressure drop in SGP-T-treated animals and controls) but it does prevent a sustained anaphylactic reaction. This later-phase inhibitory effect could occur by one or both of two mechanisms: 1) prevention of the release of mediators whose synthesis is stimulated by the binding of the allergen to the immunoglobulin E receptors on mast cells. Such mediators include leukotrienes, platelet activating factor and prostaglandins; 2) prevention of the reaction of either the preformed or the newly formed mediators with effector cells contributing to the hypotension or disturbances in intestinal motility. Practice of the invention is not, however, dependent on the mechanism by which these peptides act.

The invention also enables methods for preventing and/or treating inflammation, anaphylactic reactions, including anaphylactic shock, endotoxic reactions including endotoxic shock, and SIRS, by administration of an effective amount of SGP-T or an effective fragment or derivative thereof.

The invention further enables pharmaceutical compositions comprising the peptide SGP-S or an effective fragment or derivative thereof, for the treatment or prevention of endotoxic reactions, including endotoxic shock.

The invention also enables methods for preventing or treating endotoxic reactions, including endotoxic shock, by administration of an effective amount of the peptide SGP-S or of an effective fragment or derivative thereof.

Peptides SGP-T and SGP-S and effective fragments or derivatives thereof may be administered prophylactically to patients at risk of developing shock before they progress into shock, or may be administered after shock has developed, to combat hypotension.

Peptide SGP-T and effective fragments or derivatives thereof may be administered prophylactically to prevent anaphylactic reactions in susceptible subjects, for example individuals who experience anaphylactic reactions to insect bites or stings, to pollens, to particular foods or to occupational allergens such as grain dusts or latex, or used to arrest the progression of an already initiated anaphylactic reaction to a more severe or more systemic reaction.

Preparation of Peptides

Peptides in accordance with the invention or fragments or derivatives thereof may be prepared by any suitable peptide synthetic method.

Chemical synthesis may be employed, for example standard solid phase peptide synthetic techniques may be used. In standard solid phase peptide synthesis, peptides of varying length can be prepared using commercially available equipment. This equipment can be obtained, for example, from Applied Biosystems (Foster City, Calif.). The reaction conditions in peptide synthesis are optimized to prevent isomerization of stereochemical centres, to prevent side reactions and to obtain high yields. The peptides are synthesized using standard automated protocols, using t-butoxycarbonyl-alpha-amino acids, and following the manufacturer's instructions for blocking interfering groups, protecting the amino acid to be reacted, coupling, deprotecting and capping of unreacted residues. The solid support is generally based on a polystyrene resin, the resin acting both as a support for the growing peptide chain, and as a protective group for the carboxy terminus. Cleavage from the resin yields the free carboxylic acid. Peptides are purified by HPLC techniques, for example on a preparative C18 reverse phase column, using acetonitrile gradients in 0.1% trifluoroacetic acid, followed by vacuum drying.

Peptides may also be produced by recombinant synthesis. A DNA sequence encoding the desired peptide is prepared and subcloned into an expression plasmid DNA. Suitable mammalian expression plasmids include pRC/CMV from Invitrogen Inc. The gene construct is expressed in a suitable cell line, such as a Cos or CHO cell line and the expressed peptide is extracted and purified by conventional methods. Suitable methods for recombinant synthesis of peptides are described in "Molecular Cloning" (25). Derivatives of a peptide may be prepared by similar synthetic methods. Examples of side chain modifications contemplated by the present invention include modification of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH.sub.4; amidation with methylacetimidate; acetylation with acetic anhydride; carbamylation of amino groups with 2, 4, 6, trinitrobenzene sulfonic acid (TNBS); alkylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5'-phosphate followed by reduction with NaBH.sub.4.

The guanidino group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via acylisourea formation followed by subsequent derivatization, for example, to a corresponding amide.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid-, t-butyglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers or amino acids.

Examples of conservative amino acid substitutions are substitutions within the following five groups of amino acids:

Group 1: F Y W

Group 2: V L I

Group 3: HKR

Group 4: M S T P A G

Group 5: D E

Fragments or derivatives of the peptides of the invention may be screened for their effectiveness by one of the assay systems described herein. The assay selected will depend on the biological activity of interest in each case. For example, peptide fragments or derivatives may be screened for their effectiveness in inhibiting intestinal anaphylaxis by the method described in Example 7 or for their effectiveness in inhibiting endotoxic hypotension by the method described in Example 2.

The peptides of the invention may be administered therapeutically by injection or by oral, nasal, buccal, sub-lihgual, rectal, vaginal, transdermal or ocular routes in a variety of formations, as is known to those in the art.

For oral administration, various techniques can be used to improve stability, based for example on chemical modification, formulation and use of protease inhibitors. Stability can be improved if synthetic amino acids are used, such as peptides or betidamino acids, or if metabolically stable analogues are prepared.

Formulation may be, for example, in water/oil emulsion or in liposomes for improved stability. Oral administration of peptides may be accompanied by protease inhibitors such as aprotinin, soybean trypsin inhibitor or FK-448, to provide protection for the peptide. Suitable methods for preparation of oral formulations of peptide drugs have been described, for example, by Saffran et al., (26), (use of trasylol protease inhibitor); Lundin et al. (27) and Vilhardt et al., (28).

Due to the high surface area and extensive vascular network, the nasal cavity provides a good site for absorption of both lipophilic and hydrophilic drugs, especially when coadministered with absorption enhancers. The nasal absorption of peptide-based drugs can be improved by using aminoboronic acid derivatives, amastatin, and other enzyme inhibitors as absorption enhancers and by using surfactants such as sodium glycolate, as described in Amidon et al., (29).

The transdermal route provides good control of delivery and maintenance of the therapeutic level of drug over a prolonged period of time. A means of increasing skin permeability is desirable, to provide for systemic access of peptides. For example, iontophoresis can be used as an active driving force for charged peptides or chemical enhancers such as the nonionic surfactant n-decylmethyl sulfoxide (NDMS) can be used.

Transdermal delivery of peptides is described in Amidon et al. (29) and Choi et al. (30).

Peptides may also be conjugated with water soluble polymers such as polyethlene glycol, dextran or albumin or incorporated into drug delivery systems such as polymeric matrices to increase plasma half-life.

More generally, formulations suitable for particular modes of administration of peptides are described, for example, in Remington's Pharmaceutical Sciences (31).

The particular dosage required in a given subject can be determined by the attending physician. A starting dosage in the range of 1 .mu.g peptide/kg body weight to 100 .mu.g/kg can be employed, with adjustment of the dosage based on the response of a particular subject, as understood by those of ordinary skill in the art.

Antibodies

The peptides of the invention may be coupled to a carrier protein to increase immunogenicity for antibody production. For example, the peptides of the invention may be coupled to bovine serum albumin or keyhole limpet haemocyanin.

In order to prepare peptides for production of polyclonal antibodies, fusion proteins containing a selected peptide, such as peptide 15 or peptide 42, can be synthesized in bacteria by expression of corresponding DNA sequences in a suitable cloning vehicle. Fusion proteins are commonly used-as a source of antigen for producing antibodies. Two widely used expression systems for E. coli are lacZ fusions using the pUR series of vectors and trpE fusions using the pATH vectors. The peptides can then be purified, coupled to a carrier protein if desired, and mixed with Freund's adjuvant (to help stimulate the antigenic response of the animal) and injected into rabbits or other appropriate laboratory animals.

Following booster injections at weekly intervals, the rabbits or other laboratory animals are bled and their serum isolated. The serum can be used directly or the polyclonal antibodies purified prior to use by various methods including affinity chromatography.

As will be understood by those skilled in the art, monoclonal antibodies may also be produced using the peptides of the invention. A selected peptide, coupled to a carrier protein if desired, is injected in Freund's adjuvant into mice. After being injected three times over a three week period, the mice spleens are removed and resuspended in phosphate buffered saline (PBS). The spleen cells serve as a source of lymphocytes, some of which are producing antibody of the appropriate specificity. These are then fused with a permanently growing myeloma partner cell, and the products of the fusion are plated into a number of tissue culture wells in the presence of a selective agent such as HAT. The wells are then screened by ELISA to identify those containing cells making binding antibody. These are then plated and after a period of growth, these wells are again screened to identify antibody-producing cells. Several cloning procedures are carried out until over 90% of the wells contain single clones which are positive for antibody production. From this procedure a stable line of clones which produce the antibody is established. The monoclonal antibody can then be purified by affinity chromatography using Protein A Sepharose, ion-exchange chromatography, as well as variations and combinations of these techniques. Truncated versions of monoclonal antibodies may also be produced by recombinant techniques in which plasmids are generated which express the desired monoclonal antibody fragment in a suitable host.

Human neutrophils appear to possess receptors for the peptides of the invention (see, for example, FIG. 6) and it is likely that these peptides, or homologues thereof, occur in humans.

Antibodies to SGP-T and SGP-S, or their human homologues, may be used to determine the circulating concentration of these peptides. By knowing the circulating levels of the SGP-T and SGP-S peptides, a diagnostic tool becomes available to predict the susceptibility of the patient to progress from systemic inflammatory response syndrome SIRS) to the development of septic or nonseptic shock. Low circulating levels of these peptides would indicate increased susceptibility to progression from SIRS to shock, whereas high circulating levels would be indicative of reduced susceptibility to the development of shock.

In addition, knowledge of the circulating levels of SGP-T and/or SGP-S in humans may be used to monitor and adjust dosage levels of the peptides in treatment. Furthermore, this information could be used to predict alternative therapeutic approaches in the management of patients, for example treatment with antibiotics, aggressive volume replacement therapy or the administration of vasoactive agents such as dopamine or vasopressin.

In addition, determination of circulating levels of SGP-T may be useful in determining the reactivity state of neutrophils and monocytes in inflammatory conditions such as arthritis or inflammatory bowel disease. Low circulating levels may indicate the need for neutrophil suppression therapy using the peptides of the invention.

EXAMPLES

The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.

Methods of protein and peptide biochemistry and immunology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.

Example 1

Isolation, Purification and Sequencing of SGP-T and SGP-S

The purification procedures involved homogenization of rat submandibular glands in 0.1N HCL, centrifugation of the extract at 15,000 g for 1 h, sequential molecular weight cut-off filtration of the supernatant with Amicon 30,000, Centricon 10,000 and 3,000 filters followed by 5 steps of reverse phase high performance liquid chromatography (RP-HPLC) using 20 to 50% acetonitrile. At each step of the purification, biologically active fractions were identified by monitoring their ability to reduce LPS-induced hypotension in sialadenectomized rats. The fractions of submandibular gland extracts generated during purification were assayed by measuring endotoxin induced hypotension in sialadenectomized rates. The extracted glands were lyophilized at each step of the purification to remove acid and/or organic solvents. All fractions were dissolved in 0.9% saline (pH 7.0) at a concentration equivalent of 0.2 .mu.g of original submandibular gland/.mu.l, with each rat receiving 1 .mu.l/kg body weight of the extracts.

Two procedures were used to extract anti-shock activities from submandibular glands. Initially, the glands were homogenized in distilled water containing 50,000 units/mil of Aprotinin (Sigma) and 5 mM benzamidine (Sigma) to inhibit serine proteases (protease inhibitor extraction). One g of frozen submandibular gland in 5 ml of extraction medium was blended for 2 min at high speed in a Waring blender, and the mixture was disrupted further with a ground glass homogenizer. The homogenate was centrifuged at 15,000 g for 1 h with a sorval RC5C maintained at 4.degree. C., and the supernatant, designated the crude homogenate was frozen at -70.degree. C. for subsequent purification. This crude homogenate was then fractionated using several molecular weight exclusion filtrations, which consisted of Diaflow Ultrafilters 30,000 and 10,000 (Amicon Div., W.R. Grace & Co., Danvers, Mass.), performed under 70 psi of nitrogen at 20.degree. C., and Centricon 3000 filters (Amicon Div., Beverly, Mass.) which were centrifuged with a Sorval RC5C centrifuge at 4.degree. C. for 4 h at 7,500 g. The supernatant was then eluted through Sep-Pak columns (Waters Chromatographic Div., Millipore Corp., Milford Corp., Milford, Mass.) with 50% acetonitrile, and after drying the eluant was run on preparative RP-HPLC (20% to 50% acetbnitrile). Further HPLC purification was performed using analytical RP-HPLC columns was performed using analytical RP-HPLC (Vydac C18, 5 .mu.m, 4.6 mm.times.25 cm). The protein eluting from the HPLC columns was detected monitoring absorbance at 214 nM, a wavelength which detects peptide bonds.

With the protease inhibitor extraction procedures the biological activity was not conserved consistently through to the analytical HPLC stage. This instability was probably due to the limited half-life of the kallikrein inhibitors and the rich array of proteases found in salivary glands. Thus, an acid extraction procedure was applied. The submandibular glands were homogenized in 0.1N HCl to destroy all enzymatic activity, and the extract was purified using the procedures described for the protease inhibition extraction. Using this acid extraction procedure biological activity was conserved through all steps of the purification.

The two peptides were sequenced at the Protein/Peptide Synthesis Unit of The University of Calgary and the Alberta Peptide Institute at The University of Alberta, Edmonton, Alberta.

The peptides were then synthesized, using standard solid phase synthesis Sephadex G-10 and HPLC purified and their amino acid compositions confirmed.

Example 2

Effects of SGP-T and SGP-S on Experimental Endotoxemia

The animal model of endotoxic shock used involves intravenous injection of endotoxin (3.5 mg/kg of lipopolysaccharide (LPS) from Salmonella typhosa) into pentobarbital-anaesthetized Sprague-Dawley rats, to produce a fall in mean arterial blood pressure within 3 to 5 minutes. The endotoxin was injected slowly over 1 min. via the jugular vein, and mean arterial blood pressure in mm mercury (MABP) was monitored continuously for 60 min, with the average blood pressure being calculated at 15, 30, 45 and 60 min. Studies were performed on unoperated rats and rats with their submandibular glands removed (sialadenectomized).

The results shown in Table 1 are the averages of three different experiments using the protocol defined above. The data shown represent the average decrease in MABP (dec MABP) and the % Decrease in MABP over the 60 minutes following LPS administration. SGP-S (100.mu.g/kg) and SGP-T (100 .mu.g/kg) were administered 90 min. before LPS. The sialadenectomized rats exhibited a more severe hypotensive response to LPS than unoperated rats. The average MABP for the 60 minutes following LPS injection (MABPaft) was significantly less for the sialadenectomized rats (68.03.+-.3.4 mm Hg) than for the unoperated rats (88.03.+-.3.6 mm Hg). Neither SGP-T nor SGP-S, when given prior to the LPS challenge, had appreciable effects on MABP.

In unoperated rats, SGP-T reduced the drop in MABP elicited by LPS, and this effect was independent of time of administration of the peptide. Overall, SGP-T reduced by 60% the decrease and the percent decrease in MABP induced by endotoxin, relative to pre-LPS values. SGP-S, on the other hand, had no effect on the shock induced by endotoxin in unoperated rats.

In sialadenectomized rats, SGP-S (but not SGP-T) significantly reduced the drop in MABP after LPS, the decrease in MABP after LPS relative to MABP bef, and the percent decrease in MABP relative to MAEP bef.

The dose-response relationship of the inhibitory effect of SGP-T and SGP-S on endotoxin-induced hypotension was also examined and the results are shown in FIG. 1. It can be seen that SGP-T, given intravenously 1.5 hours before LPS, dose-dependently inhibited the decrease in blood pressure induced by the LPS in sialadenectomized rats. SGP-T at doses of 1, 3.5 and 10 .mu.g/kg significantly prevented the LPS-provoked drop in blood pressure. In contrast, saline controls (SGP-T zero) exhibited an average MABP drop of 55 mm Hg. Doses of SGP-T higher than 10 .mu.g/kg were less effective in preventing the shock. The optimal dose of SGP-T for reducing LPS-induced hypotension in unoperated rats was higher than in operated animals.

Example 3

Effect of SGP-T and SGP-S on Anaphylaxis

Cardiovascular Anaphylaxis

The effect of SGP-T on antigen-induced anaphylactic hypotension was examined in Sprague-Dawley rats sensitized 5 weeks previously with the nematode parasite Nippostrongylys brasiliensis. Under pentobarbital anesthesia, worm antigen (100 worm equivalents) was injected and arterial blood pressure was followed for 1 hour. The results are'shown in FIG. 2. Whereas rats treated with saline vehicle (SGP-T zero) experienced a drop in blood pressure after antigen challenge of approximately 30 mm Hg, SGP-T given 10 minutes prior to induction of anaphylaxis dose-dependently protected against the anaphylactic hypotension.

Intestinal Anaphylaxis

SGP-T was also effective in preventing intestinal anaphylaxis. FIG. 3 shows that in rats sensitized to the antigen hen egg albumin, instillation of the antigen (EA) into the jejunum after saline pretreatment promoted diarrhoea and disruption of normal fasting gut motility (migrating myoelectric complexes; MMCs). SGP-T, given intravenously at doses as low as 10 .mu.g/kg significantly attenuated these anaphylactic reactions. A dose of 100 .mu.g/kg totally prevented the manifestation of anaphylaxis.

A similar protection against intestinal anaphylaxis was observed in vitro using isolated intestinal (jejunal) segments obtained from egg albumin sensitized rats. SGP-T, at doses as low as 6.8.times.10.sup.-7M, reduced antigen induced contractions of these isolated intestinal tissues by 60% (data not shown).

Example 4

Modulation of Neutrophil Function by SGP-T

The subcutaneous implantation of a carrageenin-soaked sponge in rats is a model used to evaluate agents that modulate neutrophil chemotaxis, as carrageein is a potent chemotactic agent and the sponge serves as a collecting reservoir.

Under halothane anaesthesia the sponge was removed from its subcutaneous site and the fluid was squeezed from it into a test tube. Following centrifugation at 2,000 g for 10 min, the exudate was decanted and the remaining cells were suspended in 4 ml of 0.9% saline, and their number determined using a Coulter Counter. One ml of the remaining cell suspension was extracted for use in the superoxide assay.

Superoxide Assay

Neutrophils (10.sup.6) were suspended in PBS buffer of the following composition: NaCl 137 mM, KCl 2.7 mM, Na.sub.2HPO.sub.4, 8.1 mM, KH.sub.2PO.sub.4 1.47 mM, CaCl.sub.2 1.19 mM, MgCl.sub.2 0.54 mM, glucose 7.5 mM and cytochrome C 1.5 mM (Sigma Chemical Co. St. Louis, Mich.) incubated at 37 C. Each sample was read along with a reference sample containing 1440 units of superoxide dismutase (Sigma) in a spectrophotometer (Hitachi, U200 spectrophotometer). The rate of superoxide production in response to 10.sup.-7M phorbol myristate acetate (PMA) or 10.sup.-5 M N-formyl-methionyl-leucyl-phenylalanine (fMLP) was then inferred from the slope. (Derian et al., (1996), Biochemistry 35(4): 1265 9).

Intravenous injections of SGP-T inhibited neutrophil influx into a carrageenin-soaked sponge in a dose-dependent manner (FIG. 4). When the ability of neutrophils obtained from the carrageenin-soaked sponges to generate superoxide anion was evaluated, those obtained from saline-treated rats were totally refractory to f MLP and phorbol myristate acetate (PMA). In contrast, neutrophils collected from rats that received intravenous (via the penile vein) SGP-T 4 hr. before implantation of the sponge were able to generate substantial amounts of superoxide (FIG. 5).

Although the reasons for the lack of a superoxide response in carrageenin exposed neutrophils are unknown, receptor desensitization or uncoupling of the NADPH complex that generates the superoxide are possible explanations. SGP-T abrogates this desensitisation phenomena. By attenuating neutrophil chemotaxis, and by conserving the oxidative capacity of neutrophils, SGP-T provides a new anti-inflammatory agent. Treatment with SGP-T would limit an excessive movement of neutrophils into an inflammatory site, prevent an excessive and intensive generation of supe