WO2011028173A1 - Method of treatment of a patient suffering from an infection or injuries caused or complicated by infection - Google Patents

Abstract

The present invention relates to a method for treatment of a patient suffering from an infection or injuries caused or complicated by infection, comprising administering to said patient a combination of Antithrombin III and Hepatocyte Growth Factor, and optionally an antibiotic effective for treatment of the agent causing said infection.

Description

Method of treatment of a patient suffering from an infection or injuries caused or complicated by infection

Field of the invention

The present invention relates to the field of pharmaceu tical products and their use in therapy. Specifically, it relates to the treatment of infections and complications caused by infection through use of pharmaceutical products comprising Hepatocyte Growth Factor.

Background of the invention

Hepatocyte growth factor (HGF) is a unique growth factor, which is unrelated to other well-known polypeptide mitogens. It is a protein expressed in the mesenchymal cells such as lung macrophages and fibroblasts (Yanagita K. et al. J Biol Chem. 1993, 268), Kupffer cells in the liver and leukocytes (Sakaguchi H. et al. Hepatology 19, 1994, Noji S, et al. Biochem. Biophys Res Commun 173, 1993). HGF is secreted in response to cell damage and appears to be important for the regeneration, of certain organs and healing of wounds (Arakaki N et al. Hepatology 221 , 1995). It is a heterodimer, having disulphide bonded heavy and light chains of approximately 60 and 30 kDa respectively, first synthesized as an inactive precursor (Miyazawa K et al. J Biol Chem 268, 1994). The precursor is cleaved to an active protein in the damaged organ by a specific activator (Naka D et al. J Biol Chem 276, 1992, Nishizaki T et al. J Am. Coll Surg 181 , 1995). HGF acts paracrinally, i.e. it affects adjacen cells, as well as

endocrinally, i.e. it has a long-distance effect (Yanagita K. et al. Biochem Biophys Res Commun 182, 1992, Kono S et al. Biochem Biophys Res Commun 186, 1992). The target cells of HGF are fully developed epithelial cells (Matsumoto K and Nakamura T. J Gastroenterology Hepatology 6, 1991). HGF is produced and is present in high concentrations a sites of organ damage (Nayeri F et al. Scand JInfect Dis, 34, 2002).

The systemic and local production of HGF in various infectious diseases has been studied and high serum HGF concentrations have been observed during acute infectious diseases such as gastroenteritis, sepsis, pneumonia, skin and soft tissue infect ions and pyelonephritis (Nayeri F et al. Scand J Infect Dis, 34, 2002). Simultaneous with enhanced systemic production of HGF, high HGF concentrations have been found in cerebrospinal fluid during meningitis (Nayeri F et al. J Infect Dis, 181 , 2000) . Raised HGF concentrations i exhaled breath condensate (Nayeri F, et al. Respir Med, 96, 2002) in patient s with pneumonia, which had no correlation to serum levels of HGF, indicated a local production of HGF during pneumonia. Furthermore the stability of HGF in serum has been studied (Nayeri F, et al, CYTOLIKE, 2002) . High amounts of HGF in feces during diarrhea have been shown to possibly indicate that patient suffers from a transmittable gastroenteritis. Further, monitoring of HGF levels before and after treatment during infectious diseases have been shown to possibly reveal therapeutic failure at an early stage. It has also been suggested that a

combination of HGF and. antibiotics can be used for treatment of infections and injuries caused or complicated by infections (PCT publication WO 02/ 17964) . A method for purification of HGF is disclosed in WO2007/085626. Anti thrombin III (AT III) is a plasma alpha 2 glycoprotein that accounts for the major anti thrombin activity of normal plasma and also inhibits several other enzymes. It has been used for adjuvant treatment of sepsis for a long time. AT III is commercially available from a number of suppliers, e.g. Octapharma and

Baxter.

AT III counteracts intravascular coagulation and has known anti-inflammatory properties (Moubarak P et al, Shock 1997) . In a review article (Afshari et al Cochrane Database Syst Rev. 2008; 3 : CD 005370), twenty placebo controlled trials evaluating the therapeu tic / side effects of AT III in critically ill patients have been reviewed . Thirteen trials consisted of critically ill participants mainly with sepsis. All the patients with sepsis received antibiotics. The results combining all trials showed no statistically significant effect of AT III on mortality. However the bleeding events significantly increased in the

intervention group. Thus the recent studies do not recommend AT III

substitution to the critically ill patients (Warren et al, 2001 JAMA) . Although very detailed components of the studies were assessed by reviewers, no information about the AT III product used in studies was included. Summary of the invention

The present invention is based on the discovery that some AT III products contain small amounts of biologically active HGF, and that the combination of AT III and HGF has synergistic effects when used in treatment of infections and injuries caused o complicated by infections.

In one aspect, the present invention relates to the combined use of HGF and AT III in therapy of infections and injuries caused or complicated by infections. In one embodiment, HGF and and AT III is further combined with an antibiotic effective for treating said infection.

In one aspect, the invention relates to a method for treatment of a patient suffering from an infection or injuries caused or complicated by infection, comprising administering to said patient a combination of Antithrombin III and Hepatocyte Growth Factor. In one embodiment, the method also comprises the administration of an antibiotic effective for treating said infection.

In one aspect, the invention relates to a pharmaceutical composition comprising a combination of Antithrombin III and Hepatocyte Growth Factor, and optionally an antibiotic, binders, buffers, excipients and /or solvents.

In one aspect, the invention relates to a kit of parts comprising a

pharmaceutical composition comprising Antithrombin III and a pharmaceutical composition comprising Hepatocyte Growth Factor, and optionally a

p h arm a ecu ti c a 1 composition comprising an antibiotic. Such a kit of parts may further comprise instructions for performing a method for treatment according to another aspect of the invention.

Brief description of the figures

Figure 1 : Incubation of antithrombin containing HGF (UF1) with anaerob bacteria Porphyromonas gangivalis in one hour caused decrease in binding affinity of HGF to ligands. Binding for HSPG and anti -HGF is 0 for UFl+P.

gingivali s incubated 1 hour. Figure 2: The binding to HGF to HGF ligands in an ti throm bin- H GF product did not decrease during 24 hours of incubation in room temperature. Definitions

All words and terms used herein should be construed as having the meaning as given to them by a person skilled in the relevant art. For the sake of clarity, some terms are defined more explicitly below. Hepatocyte Growth Factor, abbreviated HGF, is a well-known protein growth factor with a MeSH (U.S. National Library of Medicine's controlled vocabulary) entry created in 1993. The inventor has previously shown that native HGF may loose its biological activity. Several assays for determining the biological activity are available (see e.g. PCT/SE2005/001583). If not otherwise stated, the terms Hepatocyte Growth Factor and HGF relates to biologically active forms of the protein.

Anti thrombin III, abbreviated AT III, is also a well-known protein with a MeSH entry created in 1979. It was formerl called. Anti throm bin II (AT II) which has now been shown to be identical to AT III.

The term "antibiotic" shall be construed as a pharmaceu tical compound effective to treat an infection by a pathogenic microorganism in a patient. Detailed description of the invention

Infection initiates an inflammatory process in the body that affects the circulatory balance in organ. The infective agents build infectious embolus that occludes the capillaries and decreases the blood supply to the organ.

Interactions in the coagulation system initiate different grades of disseminated intravascular coagulopathy. As a result the amounts of lactate in blood is increased which indicates bad circulation and damage on epithelial cells.

Such a mechanism might be responsible for injuries caused by infection in different organ which migh be distinguished by the symptoms in the corresponding organ. When the blood is infected (septicaemia) it is much easier for infection to cause ischemia in several organs. The body defence initiates an inflammatory process as well and together multiple organ dysfunction happens suit of severe sepsis. To overcome such damage the infectious agent should be eliminated and the microcirculation to the organ should be

strengthened and the injured epithelial cells should be repaired. HGF is a potent growth factor that functions on the injured epithelial cells. We have studied the beneficial effects of HGF treatment in complicat ions caused by infection after the infectious agent is properly treated by appropriate antibiotics. We have shown that HGF" had a healing effect on treatment of chronic leg ulcers (WO02/017964). We have succeeded to treat patients with skin ulcers as well as deep ulcers with / without osteomyelitis with HGF.

The inventor has observed an effect of bacteria on the biological activity of HGF in vitro. Incubation o bacteria with endogenous HGF purified from blood inactivated the HGF. Incubation of endogenous HGF with anaerobe bacteria Porfyromonas gingivalis, inactivated HGF already after one hour (Fig 1 ). In the case of aerobe bacteria such as Pseudomonas aeroginosa, Enterococcus fecalis, Enterobacter cloaces, Stafylococcus aureus, Streptococcus pyogenes,

Stafylococcus saprophyticus, Escherichia colt and Klebsiella pneumoniae, HGF was inactivated after incubation during night. Without being bound by theory, this might be the reason why the locally induced HGF at the site of injury could not be activated until the bacteria was eliminated. The present invention is partly based on the beneficial effects of adjuvant treatment of complications caused by sepsis with or without multiple organ failure (heart failure, renal failure, liver dysfunction, ARDS) with HGF and appropriate antibiotics. By studying the products containing HGF (both recombinant and endogenous) we have previously shown that HGF found in healthy blood has a binding character to ligands such as heparan sulfat proteoglycan (HSPG), that differs from HGF found in patients with chronic diseases (Nayeri F J Growth Factors 2008). Plasma-derived products seperated by affinity chromatography using heparin as ligand, such as Antithrombin III, may contain growth factors such as HGF. To obtain a reliable variant of endogenous HGF we have determined the properties of HGF in Antithrombin III products. By establishing a Surface Plasmon Resonance (SPR)-based method (Biacore®) for analysing the biological activity of HGF, the inventor has shown that commercially available AT III products from different suppliers differ from each other in their content of HGF. The biological activity of the HGF contained in different products also differs.

Atenativ® from Octapharma contained HGF, but with no biological activity as measured with the SPR-method. Recently we have assessed Antithrombin III from Baxter and observed that it contained low amounts of very potent HGF with high and stable affinity to essential ligands in the SPR-method. The amounts were detectable by BUS A (Quantikine R&D Systems). By immobilizing Heparan Sulphate Proteoglycan (HSPG) to the purification column (Hi Trap, GE Healthcare) we succeeded to purify HGF from Antithrombin III Baxter® that had affinity to HSPG in the SPR-method (Table 1). Table 1 : AT III Baxter and Atenativ Octapharma purified in column immobilised by heparan sulfate proteoglycan (HSPG) and the elution sample run in Biacore.

D 19: Antibody binding to an internal region of HGF|3 of human origin.

Atenativ 333 120 317 37 original 1: 1

PBS

Eluted from -3 -4 -11 -9

column

diluted x 10

The protein was responsive in the wound assay using CCL-53. 1 cells (Nayeri et al, J Dermatol Sci, 2005). Although the overall mortality in the AT III treated group in the review article (Afshari et al 2008) did not differ significantly from placebo ( risk ratio 95% CI) there was a range in risk ratio (0.2-3.43) and the low risk ratio indicated a lower mortality rate in the intervention group compared to placebo. We observed that AT III products used in the trials were obtained from different companies. The difference in biological activity of HGF found in AT III products: Baxter® and Atenativ® might have caused differences seen in the studies from Schorr 2000, Waydhas 1998 (Atenativ®) and Hai e 1998

( An ti thrombin III Baxter®) (Table 2-3).

Table 2: AT III products used in studies and the risk ratio for overall mortality

Study Journal Risk ratio AT III product

Baudo 1992 Thrombosis 0.25 Immuno AG

Research

Bauda 1998 Intensive Care 0.94 Immuno AG

Medicine

Albert J 1992 Acta Anaesthesiol 0.80 Kabi Pharmacia. Stockholm

Scand

Fourrier 1993 Chest 0.82 Lille regional blood transfusion center, France

Langley 1993 Journal oh 1. 13 No information

Hepatology Diaz- Intensive Care 1. 12 Kybernin-P, Behring,

Cremades Med Barcelona

1994

Smith- Clinical Intensive 1.09 No information

Erichsen Care

1996

Inthorn 1997 Shock 0.81 Atenativ, Pharmacia, Germany

Waydhas Trauma,Injury, 2.00 Atenativ, Pharmacia

1998 Infection and

Critical Care

Schmidt American Journal 2.33 Kybernin, Behringwerke AG

1998 of Respiratory

and Critical Care

medicine

Eisele 1998 Intensive Care 0.61 Centeon Pharma

Med

Haire 1998 Biology of Blood 0.67 AT III Baxter

and Marrow

Transplantation

Schorr 2000 European Journal 1.08 Atenativ, Pharmacia

of Clinical

Investigation

Maki 2000 Thromb Haemost 0 Kybernin HS Behringwerke AG

Warren 2001 JAMA 0.96 Kybernin, Aventis-Behring

Grenander Journal of 3.43 Atenativ, Pharmacia

2001 Neurosurgical

Anesthesiology

Fulia 2003 Biology of the 0.50 Oesterreichisches Institut fur

Neonate Hamoderivate GmbH, Vienna,

Mitchell 2003 Blood 0 Thrombate III, Bayer

coagulation, Corporation, USA

Fibrinolysis and

Cellular Haemostasis

Kobayashi Seminars In 0 Kybernin, Aventis-Behrin§ 2003 Thrombosis And (Marburg, Germany)

Hemostasis

Table 3: the differences between HGF binding affinity in ATI II product from different companies. SPR-method is used.

Monoclonal HSPG Monoclonal HSPG anti HGF anti HGF

Atenativ 50 21 7 15 5

E/ml 1 : 1 PBS

Kybernin 12 - 1 8 _4

50E/ml 1 : 1

PBS

Grifolds 50E 12 0,2 7 - 1

/ml 1 : 1 PBS

Immuno 1853 1419 1640 1347 50E/ml 1 : 1

PBS

ATIII Baxter 1550 1563 3394 214 50E/ml

ATIII Baxter + 37 109 36 -21 Dextran sulfat

Atenativ 434 181 522 254 before freeze- dry process

UF2 613- 686,20264-51

1 : 1 PBS

UF2 + heparin 0,5 5 -4 2 100,000 E

Immuno + -5 -6 - 1 1 -1 1

Heparin

lOmg/ml

100,000 E

Baudo et al (1998) have used AT III Immuno (Table 3) and have reported that patients who received AT III in the group that had developed multiple organ failure had a significant better survival. The outcome was better with AT III in the patients that did not receive heparin products (Warren et al 2001). As seen in Table 2 AT III Immuno has a. very potent binding affinity to ligands which disappeared after addition of 0.1 IE heparin /ml blood (Table 3). Thus we have indirectly shown that HGF is beneficial in treatment of complications caused by sepsis such as multiple organ failure. The Antithrombin III medicine containing active HGF is very stable (Figure 2).

We have previously shown high amounts of HGF in serum of patients with sepsis (Nayeri et al 2002). The amounts of HGF decreased as soon as

appropriate antibiotic was initiated. However patients who died in pneumonia had significant low amounts of HGF in spite of high CRP (Nayeri et al 1998). This might indicate that HGF is essential for healing of injuries caused by serious infection such as multiple organ dysfunction during sepsis.

The infection might be caused by virus, bacteria or fungi and disseminated injury caused by infection might be overcome after the infectious agent has been eliminated by antibiotics and/ or the activated host defence. HGF potentiates the repairing capacity of body on neighbour cells and increases the microcirculation by capillary proliferation. Antithrombin has a well documented effect on coagulopathy during sepsis. The combination of HGF and antithrombin has a synergistic effect on complications of septicaemia such as coagulopathy and multiple organ dysfunction.

Claims

1. An ti thrombin III for use in treatment of an infection or an injury caused or complicated by infection, wherein the treatment further comprises treatment with Hepatocyte Growth Factor.

2. Anti thrombin III for use in treatment of an infection or an injury caused or complicated by infection according to claim 1 , wherein the treatment further comprises treatment with an antibiotic effective for treatment of the agent causing said infection.

3. Hepatocyte Growth Factor for use in treatment of an infection or an injury caused or complicated by infection, wherein the treatment further comprises treatment with Antithrombin III.

4. Hepatocyte Growth Factor for use in treatment of an infection or an injury caused or complicated by infection according to claim 3, wherein the treatment further comprises treatment with an antibiotic effective for treatment of the agent causing said infection.

5. Method for treatment of a patient suffering from an infection or injuries caused or complicated by infection, comprising administering to said patient a combination of Antithrombin III and Hepatocyte Growth Factor, and optionally an antibiotic effective for treatment of the agent causing said infection.

6. Product or method according to any preceding claim, wherein the infection or injury caused or complicated by infection is sepsis or multiple organ

dysfunction.