WO2008104200A1

WO2008104200A1 - Use of apyrase for the treatment of a pathology resulting from endotoxin activity

Info

Publication number: WO2008104200A1
Application number: PCT/EP2007/001986
Authority: WO
Inventors: Rudi Brands
Original assignee: Gelato Corporation N.V.
Priority date: 2007-03-01
Filing date: 2007-03-01
Publication date: 2008-09-04

Abstract

The present invention relates to the use of Ectonucleoside phosphohydrolase 1 (apyrase) for the preparation of a medicament for the treatment or prophylaxis of pathological conditions such as exposure to agents like bacterial toxins, e.g., endotoxin, being the most potent pro-inflammation inducer, resulting from endotoxic activity such as acute or chronic systemic inflammation or a disruption of the vascular endothelial homeostasis.

Description

USE OF APYRASE FOR THE TREATMENT OF A PATHOLOGY RESULTING FROM ENDOTOXIN

ACTIVITY

The present invention relates to the use of Ecto- nucleoside phosphohydrolase 1 (apyrase) for the preparation of a medicament for the treatment or prophylaxis of pathological conditions resulting from endotoxic activity such as acute or chronic systemic inflammation or a disruption of the vascular endothelial homeostasis.

Such pathological conditions may result from exposure to agents like bacterial toxins, e.g., endotoxin, being the most potent pro-inflammation inducer. The present invention also relates to a pharmaceutical composition comprising apyrase for the treatment of pathological conditions resulting from endotoxic activity.

Endotoxins are a component of the cell wall of Gram negative bacteria. The term endotoxin refers to the lipopolysaccharide (LPS) complex associated with the outer membrane of all Gram negative bacteria. These bacteria include commensal and/or opportunistic pathogenic species, such as E. coli, Salmonella, Shigella, Pseudomonas, Neisseria, Helicobacter and Haemophilus, as well as many other common species . Lipopolysaccharides comprise a lipid portion and a polysaccharide portion. The lipid portion, also denoted as lipid A, is highly conserved amongst Gram negative bacterial species. This means that despite considerable genetic differences between the various Gram negative species, the lipid A portion of LPS in most of these species is substantially or completely identical. The polysaccharide portion of lipopolysaccharides differs however amongst the various Gram negative species and even between different strains of the same species.

The lipid A portion of LPS is not generally antigenic, i.e. it does not usually provoke the production of antibodies and immune defenses specific to it. Despite its low antigenicity, it is the lipid A portion which is essentially responsible for the toxicity of LPS. Lipid A, if stripped from LPS and injected intravenously, will produce all the effects of an intact LPS injection and almost all the effects of injecting the corresponding live bacteria themselves .

Because the Lipid A portion is highly conserved across species of bacteria, the reaction to different varieties of LPS from different bacteria is roughly similar (in the same host) , however the Lipid A of some species is considerably more toxic than that of others.

The polysaccharide portion of LPS is quite antigenic, i.e. it provokes a specific immune response. The polysaccharide portion contains a core and an "O-specific chain" .

The O-specific chain is the portion of LPS most responsible for its immune recognition. Minor variations in the structure of the O-polysaccharide make enormous differences to the virulence of bacterial infections, i.e. the capacity of the bacteria to multiply, infect and virulence .

While it is the function of the immune system to recognize the O-polysaccharide and to mount defenses against the invading bacteria or their toxins, it is the Lipid A fragments that produces the harmful and potentially lethal effects of an infection. Paradoxically, destroying the microbe increases the harm it does because of cell death and subsequent disintegration, LPS is released in large quantities from the destroyed pathogen. Lipid A, once released into, for example, the vascular system and tissue spaces, invokes a powerful non-specific immune reaction.

It is this reaction, if on a large enough scale, that can potentially cause septic shock and death. The systemic reaction to LPS occurs even with minute doses entering the bloodstream in the range of picograms or more per kilogram body weight.

Under normal circumstances, the endothelial barrier is the primary defense against LPS although other mechanisms such as scavengers and detoxifying molecules are known. From this, it follows that any condition affecting the integrity of the endothelial barrier such as severe burns, antibiotic use/abuse, severe trauma, intense or endurance exercise, ischemia, reperfusion damage of the gut (and other organs) and several common gastrointestinal pathologies are potentially dangerous by readily allowing LPS to cross the gut barrier into the blood.

The effects of the systemic presence of LPS on macrophages, neutrophils, B and T cells and the cascades of cytokines and inflammatory mediators has been studied in enormous detail.

LPS exposure leads to an increase in circulating levels of nitrate and nitrite, which are stable by-products of nitric oxide. In sepsis, LPS induces inducible nitric oxide synthase (iNOS) expression leading to an increased synthesis of nitric oxide (NO) . NO is a potent vasodilator and its continuous overproduction during inflammation, along with the production of various cytokines, leads to vasodilation. Inhaled endotoxin inevitably affects the lungs first, and gut-derived endotoxin inevitably affects the liver first. However, and wherever it is presented, once bound, LPS is a potent initiator of immune responses. Phagocytes ingest LPS and the LPS signaling induces gene transcription for cytokines and iNOS . Phagocytes produce large amounts of cytokines in response, typically TNF-alpha and interleukins . These cytokines leave the phagocytes and enter the general circulation and tissue spaces. Control of the production, release and response to these and other cytokines determines the clinical course of the LPS exposure. The production of NO, TNF-alpha and other interleukins after pro-inflammatory exposure to even picograms amounts of LPS can literally cause the vascular collapse, inflammation, coagulation and multiple organ failure being characteristic of severe sepsis.

Toxic quantities of LPS open the blood brain barrier and expose the CNS to abnormal blood components, leading to catastrophic meningeal and/or brain inflammation. This is the essential mechanism of the pathology seen in meningitis. LPS depletes membrane glutathione (GSH) and severely depletes ascorbate, as well as other key antioxidants. Drastic reductions in antioxidant concentrations in multiple tissues are characteristic of sepsis. Still today, despite advances in sepsis care, approximately 400,000-500,000 people develop sepsis in Europe and the USA each year. Half of these people show signs of shock. Over half of the people who develop shock will die despite all treatment efforts. LPS is a potent initiator of immune cascades. Because of this it is extremely difficult to detect it accurately and it is extremely difficult to interrupt the LPS signaling process to a clinically useful extent. Anti-LPS drugs have not shown as much clinical efficacy as has been hoped. In severely ill people, it is virtually impossible to stop significant LPS translocation into the blood stream from the gut. Gut-derived LPS is also a major route of exposure for patients undergoing surgery, burns patients, trauma patients and in general critically ill patients and is proposed to adversely effect the progress of chronic inflamation diseases like RA and asthma. This of course is the general route of exposure for people with gastrointestinal pathologies.

LPS may also be derived from blood-borne bacteria or from some other tissue infection. Efforts to control infection with antibiotics are documented to increase LPS loads, leading to the paradoxical tension between controlling the infection and limiting the damage it does from LPS release .

Because of the extreme toxicity of LPS, many studies have been performed with the aim of detoxifying the endotoxin thereby preventing the induction of the potentially lethal systemic responses to LPS.

One approach is to neutralize LPS by binding it to another compound such as an antibody. However, since the molecule after binding remains potentially toxic and in vivo binding is almost never irreversible, there remains a high risk of potentially lethal endotoxin exposure.

Another approach is the enzymatic detoxification of LPS. A major contributor to the toxicity of LPS is the presence of two phosphate groups on the molecule. Dephosphorylation of the molecule will drastically reduce or even abolish the toxicity of the molecule. Hence, enzymes capable of dephosphorylating LPS are potentially powerful pharmaceuticals for the treatment of endotoxin induced conditions . To this end, the use of alkaline phosphatase (AP) has been suggested for the treatment of endotoxin induced conditions .

However, there remains still a need for alternatives for the enzymatic dephoshorylation of endotoxin and specifically the Lipid A moiety thereof and/or the attenuation of the effects invoked by LPS at a local level .

The present invention has as object to provide such alternative for the treatment of endotoxin induced conditions.

This object of the present invention is met by providing the use of ecto-nucleoside phosphohydrolase (apyrase) for the preparation of a medicament for the treatment and/or prophylaxis of an pathological condition in a mammal resulting from endotoxic activity.

Ecto-nucleoside Triphosphate Diphosphohydrolase 1 or apyrase, which terms are interchangeably used herein, is a nucleotide metabolizing enzyme belonging to a family of acid anhydride hydrolases. Examples of other enzymes belonging to this family are GTP phosphohydrolase, pyrophosphatase and thiamin-triphosphatase .

The ecto-enzyme was first identified in 1949 and in 1963 partially purified from potato. The enzyme is also known under its registry number EC 3.6.1.5. or as CD39. Apyrases are naturally occurring transmembrane glycoproteins that can activate intracellular pathways upon activation. Apyrases are found in a large number of microbial species such as E.coli, Aspergillus fumigatus and Kluyveromyces lactis, plants such as Arabidopsis thaliana, Glycine max and Oryza sativa, insects such as Drosophila melanogaster and mammals like Rattus norvegicus, Mus musculus and Homo sapiens. An apyrase enzyme comprises three domains, an extracelluar, a transmembrane and an intracellular domain. The extracellular domain comprises a conserved catalytic region responsible for the catalytic activity of the extracellular enzyme.

The catalytical domain catalyzes the hydrolysis of ATP to yield AMP and orthophosphate . Such activity can thus be characterized as an ATP-diphosphatase or ATP diphosphohydrolase. It can also act on ADP, again yielding AMP and orthophosphate. This activity can be characterized as an ADPase or ADP phosphohydrolase . Based on the combined enzymatic activities of the catalytic domain, the enzyme can also be regarded as an ATP-ADPase.

Reported physiological functions of apyrases address their possible involvement in maintenance of hemostasis and inhibition of platelet aggregation through hydrolysis of extracellular ADP, which is released from activated thrombocytes upon vascular injury.

This ADP is known to function in recruitment and induction of platelet aggregation if not cleared by endothelial cell plasma membrane apyrase.

The present invention is based on the discovery that apyrase, and specifically exogenous apyrase, i.e., non- membrane associated apyrase, is also capable of providing inactivation or attenuation of a bacterial endotoxin and/or its provoked effects.

According to a preferred embodiment of the present invention, apyrase, and preferably exogenous apyrase, is used for the treatment and/or prophylaxis of an endotoxin induced inflammatory reaction which can be acute or chronic.

Specific examples of inflammatory diseases which can preferably be treated using apyrase, and specifically exogenous apyrase, are LPS-mediated diseases amongst which sepsis, peritonitis, pancreatitis, pneumonia, inflammatory bowel disease, rheumatoid arthritis, vascular diseases, atherosclerosis, fibrosis, asthma, barrier-integrity loss, acute myocardial infarction, wound healing and Alzheimer' s disease. In these diseases, (bacterial) toxins have an aggravating effect on the disease conditions.

The apyrase according to the present invention can be a naturally occurring apyrase such as an apyrase found in microbial species such as E.coli, Aspergillus fumigatus and Kluyveromyces lactis, in plants species such as Arabidopsis thaliana, Glycine max and Oryza sativa, in insects species such as Drosophila melanogaster and in mammals like Rattus norvegicus, Mus musculus and Homo sapiens.

A preferred source of apyrase is Homo sapiens. This source is preferred since the use of human apyrase will inherently prevent or reduce adverse immune reactions triggered after administrating the apyrase during prophylaxis and/or treatment.

Another preferred source of apyrase is potato. Potato, and preferably genetically modified potato producing a non-membrane bound apyrase (ecto-apyrase) , allows the isolation of large quantities of apyrase using a relative simple and economic method.

The present invention relates to the use of an apyrase having 70%, preferably 80%, more preferably 90%, most preferably 95% sequence identity with the amino acid sequence depicted SEQ ID No: 1 (Fig. 1) .

The present invention also relates to the use of a nucleic acid sequence encoding 70%, preferably 80%, more preferably 90%, most preferably 95% of the amino acid sequence as depicted in SEQ ID No: 1.

As a preferred alternative to a naturally occurring apyrase, apyrase can be a recombinant apyrase and preferably a non-membrane associated apyrase. Such recombinant protein can readily be obtained using well known cloning methods using for example the known apyrase sequence depicted in SEQ ID no: 1. For example, a coding sequence of apyrase can be identified and isolated from different sources using probes derived from known apyrase DNA sequences, preferably of human origin, to screen cDNA or genomic libraries.

As an alternative, using PCR primers based on the known apyrase DNA sequences, preferably of human origin, nucleic acid amplification techniques can be used for the identification, isolation and cloning of the coding sequence of an apyrase.

Once isolated, the coding sequence can be placed under the control of appropriate regulation signals and transformed in a suitable host allowing the expression and isolation of the enzyme.

Appropriate regulation signals are a promoter, one or more enhancer sequences, a transcription and/or translation initiation site and a transcription and/or translation termination site.

In a particularly preferred embodiment of the present invention, the present apyrase is a soluble or exogenous apyrase. In most cases, this would mean a deletion of the transmembrane and intracellular domain while leaving the catalytic part of the extracellular part of the enzyme functionally in tact. But also an other construction yielding a soluble recombinant protein comprising the catalytic domain of apyrase can be envisaged such as a fusion protein, in frame deletion of only the transmembrane domain, amino acid modifications such as substituting hydrophobic amino acids for hydrophilic ones, etc. According to one aspect, the present invention relates to a pharmaceutical composition for treatment or prophylaxis of an pathological condition in a mammal resulting from endotoxic (LPS) activity comprising a therapeutically effective amount of ecto-nucleoside phosphohydrolase (apyrase) and one ore more pharmaceutically acceptable excipients .

Preferred therapeutically effective amounts of ecto- nucleoside phosphohydrolase (apyrase) are between 10 to 400 IU/kg body weight. One IU unit is defined as the amount of apyrase capable of liberating 1 micromole of inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 ⁰C.

The present invention will further be described in the following examples which show certain preferred embodiments of the present invention. However, these examples are not intended in any way to limit the scope of the present invention which is only determined by the attached claims.

In the examples, reference is made to the following figures wherein:

Fig. 1 shows the amino acid sequence of the GDA1/CD39 catalytic domain (aa 86 to 540) of human apyrase

(accession number CAB40415)

Fig. 2 shows attenuation of a LPS insult in mice after administration of apyrase;

Fig. 3 shows attenuation of proinflammatory MCP-I induction in a mouse myocardial unsult model after adminsitration of apyrase;

Fig. 4 shows attenuation of a LPS insult in mice after administration of recombinant human apyrase.

Examples Example 1

Apyrase is capable of in vivo attenuation of LPS mediated inflammation

In an LPS challenge model, mice (C57B16) were i.p. administered both CD39 (apyrase) (4.5 units) and alkaline phosphatase (rec IAP 0502, 1.5 units) and LPS (5 micrograms). As shown in fig. 2, the administration of apyrase or alkaline phosphatase resulted in a comparable attenuation of inflammation as demonstrated by a stabilization of body temperature. LPS (5 micrograms) administered i.p. alone, resulted in lower body temperature which recovered after 5 hours. (In mice reduced body temp is generally observed after LPS administration) . Both alkaline phosphatase and apyrase (potato-apyrase) injected mice show normalized body temperatures .

Example 2

Apyrase reduces inflammation after acute myocardial infarction (AMI) induction in mice

Apyrase Clinical grade apyrase was obtained from Sigma (Sigma Aldrich, St. Louis, MO) . One IU unit is defined as the amount of apyrase capable of liberating 1 micromole of inorganic phosphate from ATP or ADP per minute at pH 6.5 at 30 ⁰C.

Clinical grade bovine intestinal alkaline phosphatase (BIAP) was obtained from Biozyme (Blaenavon, UK) . One unit is defined as that amount of BIAP able to hydrolyse 1 micromole of p-nitrophenyl phosphate per minute using a Tris-glycine buffer at pH 9.6 at 25 ⁰C. Animal treatment

Specific pathogen free female BALB/c mice (23-27 gram) were purchased from Charles River (Sulzfeld, Germany) . Mice were acclimatized for 1 week under barrier conditions in filter-topped macrolon cages with drinking water and standard food pellets ad libitum.

Animals were divided into two groups: an AMI group treated with BIAP (n=4), an AMI group treated with apyrase (n=4) and an AMI control group treated with vehicle alone (n=4) .

BIAP and apyrase was injected into the tail vein just before anaesthezation as a single intravenous dose of 5 IU in 100 μl PBS (approximately 100 times above plasma levels) .

Control mice were injected with an equal volume of PBS. The study was approved by the animal ethics committee of the Faculty of Veterinary Medicine, University Utrecht.

Myocardial infarction

Mice were anaesthetized by inhalation of a mixture of O₂ air and 4% isoflurane, endotracheally intubated, and mechanically ventilated. Myocardial infarction was induced as described previously (Salto-Tellez, M., et al . , Myocardial infarction in the C57BL/6J mouse: A quantifiable and highly reproducible experimental model. Cardiovascular Pathology, 2004. 13(2) : p. 91-97.)

Briefly, The LAD coronary artery was exposed via a left thoractomy and double ligated with an 8.0 prolene suture. Animals were sacrificed 4 hours after myocardial infarction after which blood was collected. Heart, lung, liver and kidneys were removed and fixed in 4% paraformaldehyde in PBS .

Enzyme-linked Immunosorbent Assay (ELISA) Blood samples were centrifuged and serum was collected for determination of mice mast cell protease-1 (MMCP-I) . Mouse mast cell protease-1 (MMCP-I) ELISA was from Moredun (Midlothian, Scotland, UK) and performed according to the manufacturer's instructions.

Statistics

All data presented are mean ± SEM. Statistical analysis was performed using Student's t-test for unpaired data (GraphPad Prism) .

Results

The results as depicted in fig. 3. As can be seen in fig. 2, apyrase attenuates proinflammatory MCP-I induction compared to the control group in response to a myocardial infarction .

Example 3

Recombinant human apyrase (NTPase-1 , CD-39-1) has inflammatory activity in a LPS challenge model

In an LPS challenge model, mice (C57B16) were i.p. administered recombinant human apyrase (NTPase-1, CD39-1) , recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase and were subsequently challenged with LPS. Mice only injected with the carrier served as control group. As shown in fig. 4, administration of recombinant human apyrase (NTPase-1, CD39-1), recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase resulted in a comparable attenuation of inflammation as demonstrated by a stabilization of body temperature.

LPS administered alone, resulted in lower body temperature which recovered after 5 hours. (In mice reduced body temp is generally observed after LPS administration) . recombinant human apyrase (NTPase-1, CD39-1) , recombinant human placental alkaline phosphatase or bovine intestinal alkaline phosphatase injected mice show comparable normalized body temperatures .

Claims

1. Use of an ecto-nucleoside phosphohydrolase (apyrase) for the preparation of a medicament for the treatment or prophylaxis of an pathological condition in a mammal resulting from endotoxic (LPS) activity.

2. Use according to claim 1, wherein the pathological condition is an inflammatory condition.

3. Use according to claim 2, wherein the inflammatory condition is an acute or chronic systemic inflammatory condition.

4. Use according to claim 2 or claim 3, wherein the inflammatory conditions is chosen from the group consisting of sepsis, peritonitis, pancreatitis, pneumonia, inflammatory bowel disease, rheumatoid arthritis, vascular diseases, atherosclerosis, fibrosis, asthma, barrier-integrity loss, acute myocardial infarction, wound healing and Alzheimer's disease .

5. Use according to any of the claims 1 to 4 , wherein the ecto-nucleoside phosphohydrolase (apyrase) is soluble.

6. Method for treating a mammal for a pathological condition resulting from endotoxic (LPS) activity comprising adminsitrating a therapeutic effective amount of an ecto- nucleoside phosphohydrolase (apyrase) into said mammal.

7. Method according to claim 6, wherein the pathological condition is an inflammatory condition.

8. Method according to claim 7, wherein the inflammatory condition is an acute or chronic systemic inflammatory condition.

9. Method according to claim 7 or claim 8, wherein the inflammatory conditions is chosen from the group consisting of sepsis, peritonitis, pancreatitis, pneumonia, inflammatory bowel disease, rheumatoid arthritis, vascular diseases, atherosclerosis, fibrosis, asthma, barrier- integrity loss, acute myocardial infarction, and Alzheimer's disease .

10. Method according to any of the claims 6 to 9, wherein the ecto-nucleoside phosphohydrolase (apyrase) is soluble.

11. Pharmaceutical composition for treatment or prophylaxis of an pathological condition in a mammal resulting from endotoxic (LPS) activity comprising a therapeutically effective amount of ecto-nucleoside phosphohydrolase (apyrase) and one ore more pharmaceutically acceptabele excipients.

12. Pharmaceutical composition according to claim 11, wherein the therapeutically effective amount of ecto- nucleoside phosphohydrolase (apyrase) is 10 to 400 IU/kg body weight .