WO2008088864A2

WO2008088864A2 - Matrix metalloproteinase-9-related methods

Info

Publication number: WO2008088864A2
Application number: PCT/US2008/000667
Authority: WO
Inventors: Richard L. Whelan; Irena Kirman
Original assignee: The Trustess Of Columbia University In The City Of New York
Priority date: 2007-01-18
Filing date: 2008-01-18
Publication date: 2008-07-24
Also published as: WO2008088864A3; WO2008088864A9

Abstract

This invention provides method for decreasing the degradation of insulin-like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery. This invention further provides a method for decreasing the degradation of insulin-like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery. This invention further provides a method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery. Finally, this invention further provides a method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery.

Description

MATRIX METALLOPROTEINASE-9-REIATED METHODS

Throughout this application, various publications are referred to by Arabic numerals within parentheses. Full citations for these publications are presented immediately before the claims. Disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

Background of the Invention

Colorectal adenocarcinoma is a leading cause of cancer- related death. Segmental colorectal resection remains the standard treatment. The rate of tumor recurrence after surgery is relatively high. Approximately one-third of patients are expected to experience recurrences post- operatively during the first 5 years. In this unfortunate subpopulation, despite resection, viable tumor cells persist after surgery in the abdominal cavity, in the bloodstream, or in tissue microfoci. Experimental data suggested that surgical trauma stimulates tumor growth early after surgery (1) . Surgical trauma, in theory, may accelerate the growth of tumor cells via surgery-associated immunosuppression or as a result of alterations in the balance of cell growth stimulatory/cell growth inhibitory substances. Cell growth is regulated in part by the insulin-like growth factors (IGF) 1 and 2 as well as by IGF-binding protein-3 (IGFBP-3), which is an IGF-binding protein. The inhibitory growth effects of IGFBP-3 are exerted not only through binding and limiting the availability of IGFs, but also via its own direct pro- apoptotic effect for various types of tumor cells.

It was previously demonstrated in humans that open surgery (OS) , but not laparoscopically assisted surgery (LS) , induces depletion of cell growth regulatory protein IGFBP-3 (9). Rapid IGFBP-3 depletion during abdominal surgery is thought to be mediated by proteases. Experimental studies have shown that IGFBP-3 proteolysis can be mediated by matrix metalloproteinases (MMP) : MMP-I, MMP-2, MMP-3, and MMP-9 (6, 13). Metalloproteinase concentrations have been shown to increase during periods of active inflammation in the setting of chronic autoimmune disorders as well as during acute inflammatory reactions (14, 22) . The major source of MMPs is inflammatory cells such as neutrophils and granulocytes. Lymphocytes also have been shown to produce MMP-9 (23) . Leukocytes, in response to mitogenic stimuli, increase proteinase production. Thus, plant lectins and lipopolysaccharide have been shown to stimulate the production of proteinases by human leukocytes (5, 16) . Surgical trauma is known to activate leukocytes, as evidenced by the release of multiple proinflammatory cytokines in the early postoperative period. An increased level of MMP-9 has been found in the blood plasma and in surgical wound fluid (21) . Therefore, the possibility of a surgery-related increase in the postoperative production of MMPs exists. Summary of the Invention

This invention provides method for decreasing the degradation of insulin-like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject

(a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

This invention further provides a method for decreasing the degradation of insulin-like growth factor binding protein 3

(IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) -producing cells in the subject and/or inhibit the release of MMP-9 from such cells so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

This invention further provides a method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery comprising administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma so as to thereby reduce the likelihood of tumor development or recurrence in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

Finally, this invention provides a method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery comprising administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) -producing cells in the subject and/or inhibit the release of MMP-9 from such cells so as to reduce the likelihood of tumor development or recurrence in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after the surgery, or any combination of (a) , (b) or (c) .

Brief Description of the Figures

Figures 1A-1C

Gelatinases in patients with colon cancer undergoing open (OS) or laparoscopically assisted (LS) surgery. A. Profile of plasma gelatinases in zymography: samples (lanes 1-5) and recombinant human matrix metalloproteinase-9 (MMP- 9) (lane 6) . The predominant plasma 92-kDa gelatinase corresponds to a proform of MMP-9 monomer, whereas a high- molecular-weight protease is its dimmer. B. MMP-9 activity in plasma samples from OS and LS patients. OS: Patient A preoperatively (pre-OP) (lane 1) and on postoperative day (POD) 1 (lane 2), patient B pre-OP (lane 3) and POD 2 (lane 4) . LS: Patient C at pre-OP (lane 7) and on POD 1 (lane 6), and patient D at pre-OP (lane 7) and on POD 2 (lane 8) . Recombinant human MMP-9 (lane 9) . C. Western blot analysis confirms that a 92-kDa gelatinase is MMP-9. Lane 1: recombinant human MMP-9. Lanes 2 and 3: MMP-9 from human plasma.

Figure 2

Matrix metalloproteinase-9 (MMP-9) enzyme-linked immunoassay (ELISA) in plasma samples from patients with colon cancer undergoing open (OS) or laparoscopically assisted (LS) surgery. MMP-9 ELISA assay was performed on preoperative (pre-OP) and postoperative days (POD) 1 to 3 samples, as described in the materials and methods section. A statistically significant difference was found between POD 1 and pre-OP values in OS, but not in LS, patients. *p < 0.003. Figure 3

Tissue inhibitor of metalloproteinase-1 (TIMP-I) enzyme- linked immunoassay in plasma samples from patients with colon cancer undergoing open or laparoscopically assisted surgery. TIMP-I ELISA assay was performed on preoperative (pre-OP) and postoperative day (POD) 1 to 3 samples, as described in the materials and methods section. **p < 0.0003. *p < 0.01.

Detailed Description of the Invention

Terms

"Administering" an agent can be effected or performed using any of the various methods and delivery systems known to those skilled in the art. The administering can be performed, for example, intravenously, orally, nasally, via the cerebrospinal fluid, via implant, transmucosally, transdermally, intramuscularly, and subcutaneously . The following delivery systems, which employ a number of routinely used pharmaceutically acceptable carriers, are only representative of the many embodiments envisioned for administering compositions according to the instant methods.

Injectable drug delivery systems include solutions, suspensions, gels, microspheres and polymeric injectables, and can comprise excipients such as solubility-altering agents (e.g., ethanol, propylene glycol and sucrose) and polymers (e.g., polycaprylactones and PLGA' s). Implantable systems include rods and discs, and can contain excipients such as PLGA and polycaprylactone.

Oral delivery systems include tablets and capsules. These can contain excipients such as binders (e.g., hydroxypropylmethylcellulose, polyvinyl pyrilodone, other cellulosic materials and starch), diluents (e.g., lactose and other sugars, starch, dicalcium phosphate and cellulosic materials), disintegrating agents (e.g., starch polymers and cellulosic materials) and lubricating agents (e.g., stearates and talc). Transmucosal delivery systems include patches, tablets, suppositories, pessaries, gels and creams, and can contain excipients such as solubilizers and enhancers (e.g., propylene glycol, bile salts and amino acids), and other vehicles (e.g., polyethylene glycol, fatty acid esters and derivatives, and hydrophilic polymers such as hydroxypropylmethylcellulose and hyaluronic acid) .

Dermal delivery systems include, for example, aqueous and nonaqueous gels, creams, multiple emulsions, microemulsions, liposomes, ointments, aqueous and nonaqueous solutions, lotions, aerosols, hydrocarbon bases and powders, and can contain excipients such as solubilizers, permeation enhancers (e.g., fatty acids, fatty acid esters, fatty alcohols and amino acids) , and hydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone) . In one embodiment, the pharmaceutically acceptable carrier is a liposome or a transdermal enhancer.

Solutions, suspensions and powders for reconstitutable delivery systems include vehicles such as suspending agents (e.g., gums, zanthans, cellulosics and sugars), humectants (e.g., sorbitol), solubilizers (e.g., ethanol, water, PEG and propylene glycol), surfactants (e.g., sodium lauryl sulfate, Spans, Tweens, and cetyl pyridine) , preservatives and antioxidants (e.g., parabens, vitamins E and C, and ascorbic acid) , anti-caking agents, coating agents, and chelating agents (e.g., EDTA).

"Agent" shall mean any chemical entity, including, without limitation, a glycomer, a protein, an antibody, a lectin, a nucleic acid, a small molecule, and any combination thereof .

"Antibody" shall include, by way of example, both naturally occurring and non-naturally occurring antibodies.

Specifically, this term includes polyclonal and monoclonal antibodies, and antigen-binding fragments (e.g., Fab fragments) thereof. Furthermore, this term includes chimeric antibodies (e.g., humanized antibodies) and wholly synthetic antibodies, and antigen-binding fragments thereof.

"Subject" shall mean any organism including, without limitation, a mammal such as a mouse, a rat, a dog, a guinea pig, a ferret, a rabbit and a primate. In the preferred embodiment, the subject is a human being.

Embodiments of the Invention

This invention provides a method for decreasing the degradation of insulin-like growth factor binding protein 3

(IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject

(a) prior to, (b) during or (c) after, the surgery, or any combination of (a), (b) or (c) .

In one embodiment, the subject is a human. In another embodiment, the agent is a PPAR-alpha agonist or a PPAR- gamma agonist. Examples of PPAR-alpha and PPAR-gamma agonists include, without limitation, Ragaglitazar, Pioglitazone, Rosiglitazone and Bezafibrate. In another embodiment, the agent is fenofibrate. In another embodiment, the agent is a protease inhibitor, such as a tissue inhibitor of metalloproteinase-1 (TIMP-I) . Other protease inhibitors include, without limitation, tissue inhibitor of metalloproteinase-2 (TIMP-2), tissue inhibitor of metalloproteinase-3 (TIMP-3), aproteinin and leupeptin.

In one embodiment, the effective amount of the agent is from about 1 mg/day/kg body weight to about 500 mg/day/kg body weight. In another embodiment, the effective amount of the agent is about 100 mg/day/kg body weight. In further embodiments, the effective amount of the agent is from about 1 mg/day/kg body weight to about 10 mg/day/kg body weight, from about 10 mg/day/kg body weight to about 100 mg/day/kg body weight, from about 100 mg/day/kg body weight to about 500 mg/day/kg body weight, from about 50 mg/day/kg body weight to about 200 mg/day/kg body weight, from about 10 mg/day/kg body weight to about 200 mg/day/kg body weight and from about 1 mg/day/kg body weight to about 200 mg/day/kg body weight.

In one embodiment, the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery. In another embodiment, the agent is administered to the subject prior to surgery. In another embodiment, the agent is administered during the surgery. In another embodiment, the agent is administered after the surgery. In another embodiment, the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery. In another embodiment, the agent is administered intravenously or orally. In a further embodiment, the open surgery is open abdominal surgery. In yet a further embodiment, open surgery means surgery wherein the incision length is at least about 10 cm.

In one embodiment, decreasing the level of MMP-9 in plasma means decreasing such level by at least 5%. In another embodiment, decreasing the level of MMP-9 in plasma means decreasing such level by at least 10%, 20% or 50%.

In another embodiment, decreasing degradation of IGFBP-3 means decreasing such degradation by at least 5%. In another embodiment, decreasing degradation of IGFBP-3 means decreasing such degradation by at least 10%, 20% or 50%.

This invention further provides a method for decreasing the degradation of insulin-like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) -producing cells in the subject and/or inhibit the release of MMP-9 from such cells so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

In one embodiment, the subject is a human. In another embodiment, the agent is an anti-MMP-9 antibody. In another embodiment, the agent is an antibody that recognizes an MMP-9-producing cell, such as a monocyte. Other MMP-9-producing cells include, without limitation, granulocytes and lymphocytes.

In one embodiment, the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery. In another embodiment, the agent is administered to the subject prior to surgery. In another embodiment, the agent is administered during the surgery. In another embodiment, the agent is administered after the surgery. In another embodiment, the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery. In another embodiment, the agent is administered intravenously or orally.

In one embodiment, the method further comprises administering to the subject an amount of a second agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma, wherein the second agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) , and does not decrease the level of MMP-9-producing cells in the subject or inhibit the release of MMP-9 from such cells. The second agent can be, for example, aproteinin or any other MMP-9 inhibitor.

In one embodiment, the subject is a human. In another embodiment, the agent is a PPAR-alpha agonist or a PPAR- gamma agonist. In another embodiment, the agent is fenofibrate. In another embodiment, the agent is a protease inhibitor, such as a tissue inhibitor of metalloproteinase-1 (TIMP-I) .

In one embodiment, the effective amount of the agent is from about 1 mg/day/kg body weight to about 500 mg/day/kg body weight. In another embodiment, the effective amount of the agent is about 100 mg/day/kg body weight.

In one embodiment, the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery. In another embodiment, the agent is administered to the subject prior to surgery. In another embodiment, the agent is administered during the surgery. In another embodiment, the agent is administered after the surgery. In another embodiment, the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery. In another embodiment, the agent is administered intravenously or orally. In one embodiment, the tumor is a colorectal tumor. In another embodiment, the tumor is any solid tumor. In one embodiment, reducing the likelihood of tumor development or recurrence in a subject means reducing such likelihood by at least 5%. In another embodiment, reducing the likelihood of tumor development or recurrence in a subject means reducing such likelihood at least 10%, 20% or 50%.

In one embodiment, the subject is a human. In another embodiment, the agent is an anti-MMP-9 antibody. In another embodiment, the agent is an antibody that recognizes an MMP-9-producing cell, such as a monocyte.

In one embodiment, the method further comprises administering to the subject an amount of a second agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma, wherein the second agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) , and does not decrease the level of MMP-9-producing cells in the subject or inhibit the release of MMP-9 from such cells. In another embodiment, the tumor is a colorectal tumor.

Wherever applicable, each embodiment above (i.e., relating to PPAR agonists, protease inhibitors, agents and therapeutically effective amounts of agents, open surgery, MMP-9-producing cells, decreasing MMP-9 levels, IGFBP-3 levels and likelihood of tumor development and recurrence, and tumor types) set forth with respect to one of the instant methods applies mutatis mutandis to each other instant method.

This invention will be better understood from the Experimental Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter. Experimental Details

Experimental Section 1

Synopsis

It was previously demonstrated that insulin-like growth factor binding protein-3 (IGFBP-3) is depleted in plasma for 1 to 3 days after major open surgery (OS), but not after laparoscopic surgery (LS) . After surgery, IGFBP-3 cleavage occurs rapidly and is likely attributable to altered plasma proteolytic activity. This study assessed plasma proteolysis after both open and closed colorectal surgery in order to identify a protease/protease inhibitor system affected by surgery.

Materials and Methods

Patients

88 patients (39 men and 49 women ages 66 ± 14 years) with a diagnosis of colorectal adenocarcinoma stages I to III. Of these 88 patients, 34 underwent segmental colectomy via a standard laparotomy (i.e., OS), whereas 54 underwent a laparoscopically assisted colectomy (i.e., LS). Patients receiving immunosuppressants and those who had undergone recent chemotherapy or transfusions were excluded from the study. The mean incision length was 18.7 ± 5.9 cm in the OS group and 5.4 ±2.0 cm in the LS group. No statistically significant differences were found in the breakdown of right and left hemicolectomies and sigmoid/rectosigmoid resections between the OS ( 12% : 32% : 56% ) and LS (9%:53%:38%) groups. The percentages of patients in each surgical group with a final diagnosis of stage I, II or III disease were comparable. There was no statistical difference in operative times between the OS (208 ± 61.4 min) and LS

(242.8 ± 78.2 min) groups. All the patients, after full explanation, consented to participate in this institutional review board-approved study, and to have blood samples drawn both preoperatively and postoperatively. Peripheral blood was collected in heparinized tubes from al the patients preoperatively (pre-OP) and on postoperative days

(POD) 1 to 3. Plasma was isolated and stored at -80⁰C until the analyses were performed.

Zymography

Plasma samples (3 μl/sample) diluted with loading buffer were electrophoretically separated on gelatine zymogram precast gels (Invitrogen, Carlsbad, CA, USA). After separation, the samples were renatured according to the manufacturer's instructions, stained with Coomassie Blue

(Bio-Rad Laboratories, Hercules, CA, USA) , and counterstained with 0.1% methylene blue solution (LabChem,

Pittsburgh, PA, USA). The image was evaluated by scanning.

MMP-9 Enzyme-Linked Immunoassay

Enzyme-linked immunoassay (ELISA) 96-well plates (Corning Incorporated, Corning, NY, USA) were coated with a monoclonal antibody to human MMP-9 (R&D Systems, Minneapolis, MN, USA) . After several washes, the plates were blocked with a 3% milk solution, and serial dilutions of human plasma were applied in duplicates with a starting dilution of 1:10. Subsequently, the plates were washed and then incubated with polyclonal biotinylated antibodies to human MMP-9 (R&D Systems) and streptavidin-peroxidase (BD Pharmingen, San Jose, CA, USA) . The reaction was developed with teteramethylbenzidine solution (Sigma Chemical, St. Louis, MO, USA) and stopped with a sulfuric acid solution (Sigma) . A recombinant human MMP-9 (R&D Systems) was used as a standard. The reaction was evaluated using an ELX800 microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) .

MMP-9 Western Blot Analysis

The specificity of the ELISA and zymography findings was confirmed in western blot analyses assisted by immunomagnetic separation. Briefly, streptavidin- conjugated Dynabeads (Bynal Biotech ASA, Oslo, Norway) were coated with biotinylated polyclonal anti-MMP9 antibody (R&D Systems) and incubated with plasma samples. Subsequently, the products of this immunomagnetic isolation were electrophoretically separated and transferred to a nitrocellulose membrane. The membrane then was blocked with 3% milk, incubated first with the monoclonal mouse antibody to human MMP-9 (R&D Systems) , and then incubated with peroxidase-labeled antimouse antibody (Pierce, Rockford, IL, USA) . Finally, the membrane was developed via chemiluminescent reaction using West Pico Luminol Supersignal solution (Pierce). Tissue Metalloproteinase-I ELISA

The method is similar to that described above for the MMP-9 ELISA. Initially, 96-wel plates were coated with a monoclonal antibody to human tissue inhibitor of metalloproteinase-1 (TIMP-I) (R&D Systems) . After several washes, the plates were blocked with a 3% milk solution, and several dilutions of plasma were added. Next, the plates were washed and then incubated with polyclonal biotinylated antibody to human TIMP-I (R&D Systems) as well as streptavidin-peroxidase (BD Pharmingen, San Jose, CA, USA) . The reaction was developed with tetramethylbenzidine solution (Sigma Chemical) and stopped with a sulfuric acid solution (Sigma Chemical) . Recombinant human TIMP-I was used as a standard. The reaction was evaluated using an ELxδOO microplate reader (Bio-Tek Instruments).

Statistical Analysis

The results are expressed as mean ± standard deviation. The difference between pre- and postoperative values within a group was analyzed using the Wilcoxon' s test. A p value of 0.05 or less was considered statistically significant.

Results

Gelatinases in the Plasma of Patients with Colon Cancer

Zymography analysis showed the presence of two major gelatinases: 92 kDa, which corresponds to a proform of MMP- 9, and 72 kDa, the molecular weight of an MMP-2 proform (Fig. IA) . Several subsequent tests have shown that the intensity of the 92 kDa protease notably changes after surgery, whereas the translucent band of the 72 kDa protease remains constant. Therefore, the focus of these subsequent studies was the 92 kDa gelatinase, MMP-9 proform. In OS patients, MMP-9-related proteolytic activity, as judged by zymography, increased most dramatically on POD 1 and to a lesser extent on POD 2 (Fig. IB) . In the majority of LS patients, no obvious changes in MMP-9 activity were found (Fig. IB) . On POD 3, MMP-9 activity returned to preoperative values in both groups. Western blot analysis confirmed that a 92 kDa gelatinase was indeed MMP-9 (proform) , which was shown to as a single band (Fig. 1C). For accurate measurement of MMP-9 concentration, an ELISA assay was developed.

MMP-9 Levels

In OS patients, the mean MMP-9 concentration on POD 1 (349.6 ± 236.0 ng/ml) was significantly higher than the mean pre-OP value (216.5 ± 198.1 ng/ml; p < 0.003). No statistically significant changes were found on POD 2 (247.5 ± 215.9 ng/ml) or POD 3 (255.6 ±245.1 ng/ml) (Fig. 2) . The most dramatic increase in MMP-9 levels (fivefold and greater) tended to occur in patients with larger surgical incisions (≥ 20 cm) , although no statistically- significant correlation was found between the increase in MMP-9 concentration and any of the clinical parameters assessed.

In the LS group, the concentration of MMP-9 was comparable in samples taken preoperatively (259.9 ± 242.6 ng/ml), on POD 1 (246.1 ± 254.0 ng/ml) , on POD 2 (230.8 ± 172.3 ng/ml), and on POD 3 (212.4 ± 213.9 ng/ml) (Fig. 2). However, 30% of LS patients had a 1.5-fold or more increase in MMP-9 levels after surgery. The majority of these patients had an advanced stage of disease (stage II or III), although no direct correlation between the change in MMP-9 levels and the clinical parameters was found.

TIMP-I Levels

The finding of a significant increase in MMP-9 levels after OS prompted a search for natural MMP-9 inhibitory proteins. A likely candidate was thought to be TIMP-I. The concentration of TIMP-I was determined in plasma samples from OS and LS patients. In OS patients, a dramatic increase in the level of TIMP-I was found on POD 1 (245.7 ± 136.0 ng/ml), as compared with the mean pre-OP value (105.5 ±78.2 ng/ml; p < 0.0003). A lesser but still significant elevation in TIMP-I concentration also was found on POD 2 (141.6 ± 62.7 ng/ml; p < 0.01) (Fig. 3) in the OS group. The LS patients showed a less impressive, yet significant, increase in mean TIMP-I levels on both POD 1 (147.5 ± 96.8 ng/ml; p < 0.01) and POD 2 (154.3 ± 148.2 ng/ml; p < 0.01), as compared with the mean pre-OP value (109.4 ± 96.5 ng/ml). On POD 3, the concentration of TIMP-I (132.4 ± 81.7 ng/ml) did not significantly differ from pre-OP levels .

Discussion

In this study, plasma protease activity in the perioperative period was assessed via zymography. The protease most affected by surgical trauma was MMP-9. The activity of MMP-2, the other principal protease found in both pre- and postoperative plasma samples, was not appreciably altered by colon resection. Subsequently, the plasma concentration of MMP-9 was determined via ELISA for both OS and LS patients.

On POD 1 after open colorectal resection, a significant increase in the plasma level of MMP-9 was noted, as compared with preoperative levels. On POD 2 and 3, smaller, nonsignificant MMP-9 changes were noted in the OS group. After laparoscopically assisted colorectal resection, MMP-9 levels preoperatively and at all three postoperative time points were similar.

It may be that MMP-9, known to be an IGFBP-3 cleaving enzyme, is the protease responsible for the previously reported surgery-related IGFBP-3 depletion noted early after major abdominal procedures (10). Furthermore, MMP-9 is capable of cleaving a number of other important proteins. For example, MMP-9 has been shown to modulate the activity of chemokines (25) and to cleave metastasis suppressor gene product kisspeptin (KiSS-I) protein/metastin (20), insulin (4), transforming growth factor β (TGF-β) binding protein (3), interleukin-2 receptor (IL-2Rα) (17), matrix proteins (11), and other vital molecules. Therefore, increased plasma MMP-9 levels in OS patients after surgery may prove to be a risk factor not only for tumor reccurences, by virtue decreased IGFBP-3 levels, but also for other insulin-dependent and immune functions. In addition, elevated MMP-9 levels after surgery may increase chances of anastomotic leakage attributable to alterations in the integrity of extracellular matrix proteins (18). Moreover, one mechanism of surgical stress-accelerated tumor metastasis is thought to be MMP production (24) . Both MMP-9 production and its overexpression are dependent on leukocytes (15). Tumor cells may induce release of MMP-9 from human mononuclear cells (19). Inhibition of MMP has been shown to delay the growth of tumors and prolonged survival of animals in experimental studies (2, 12). These findings suggest that release of MMP during surgery may have important implications for patients with colon cancer.

In the current study, although MMP-9 levels were similar before and after surgery for the LS group as a whole, in fact, 30% of the laparoscopic cancer patients had increased MMP-9 levels after surgery. This was more often the case for patients with advanced disease. Other reasons for increased MMP-9 levels in the closed surgery setting remain to be elucidated. Further studies are needed to determine the clinical importance, if any, of the short-lived increase in plasma MMP-9 levels noted after colectomy. Likewise, additional studies are needed to identify MMP regulatory agents in this setting.

The increase in MMP-9 levels noted after OS was short lived. The POD 2 and 3 levels were similar to preoperative levels. Surgery may be associated with alterations in the concentrations of MMP inhibitors that might account for the rapid return of MMP-9 levels to baseline noted in the OS patients. To explore this possibility, plasma levels of TIMP-I, a well-characterized MMP inhibitor, were assessed in OS and LS patients. The early postoperative period was accompanied by an increase in TIMP-I concentration after both open and closed colectomy. However, a more profound and persistent increase was noted in the OS group. On POD 1 in the OS group, TIMP-I levels were 233% higher than the mean preoperative level, whereas in the LS group, a 135% increase over baseline was noted (significant difference for both groups) . On POD 2, TIMP-I levels were about 135% greater than the mean preoperative level for the patients in both OS and LS groups (significant difference for both groups) . On POD 3 in the OS group, a significant increase in TIMP-I levels (mean value, 167% of the mean preoperative level) was noted, wherein as in the LS group, a mild but nonsignificant elevation in TIMP levels was recorded.

The increase in TIMP-2 production after surgery may serve as a feedback mechanism that regulates MMP-9 levels. Serum TIMP-I concentration is reported to be elevated in patients with colorectal cancer (7), although the clinical significance of elevated levels in this patient population remains unclear. In patients with lung cancer, however, increased TIMP-I levels have been associated with a poor prognosis (26) . With regard to renal cell carcinoma, increased expression of MMP-9, MMP-2, TIMP-I and TIMP-2 were noted to be associated with other indicators of poor prognosis (8 ) .

In conclusion, open, more so than laparoscopically assisted, colectomy induces a significant increase in the concentration of plasma MMP-9, which suggests that systemic release of this protease is associated with the invasiveness of the open surgical procedure. The short- lived effect of OS trauma on MMP-9 release may be explained by the opposing action of its natural inhibitor, TIMP-I.

Experimental Section 2

Synopsis

It was previously demonstrated that a significant decrease in the plasma level of intact insulin-like growth factor binding protein 3 (IGFBP-3) occurs following major open surgery in humans and it is postulated that this decrease may have an important effect on postoperative tumor growth. In contrast, the vast majority of patients that undergo laparoscopic surgery do not demonstrate an intact IGFBP-3 decrease after surgery. The goal of this experiment was to create an animal model which would allow further study of the effect of surgical trauma on IGFBP-3. An additional goal was to determine whether MMP-9, a known protease inhibitor of IGFBP-3, is responsible for the degradation of IGFBP-3 observed after open surgery.

Methods

30 mice were divided into three groups. Sham Laparotomy (SL), CO₂ Pneumoperitoneum (PP) and Anesthesia Control (AC).

All mice were hIGFBP-3 transgenics on a CD-I background.

48 hours prior and 24 hours following the procedure, blood was drawn retroorbitally . Intact IGFBP-3 levels were measured using a combination Western blot analysis and ELISA at each time point. Serum and intracellular levels

(mononuclear cell lysates) of MMP-9 were measured at each time point using zymography. Results

Plasma levels of intact IGFBP-3 were significantly lower post SL when compared to preoperative levels. A mean decrease of 76.6% was found after laparotomy (P<0.05). Zymography analysis demonstrated significantly higher MMP- 9-related proteolytic activity post SL when compared to preoperative levels (78.5 RU vs. 42.3 RU, p<0.05). In the PP and AC groups, no significant change was found between the preoperative and postoperative levels of intact plasma IGFBP-3 or MMP-9. Mononuclear intracellular levels of MMP- 9 were significantly lower post SL when compared to preoperative levels (3 RU vs. 37 RU) . Post-procedure intracellular levels of MMP-9 were not significantly decreased in the PP or AC groups.

Conclusion

Plasma levels of intact IGFBP-3 were found to be significantly decreased following SL. This decrease was not seen following PP. Depletion of intact IGFBP-3 following SL correlated with a rapid release of MMP-9 from mononuclear cells and an increase in circulating serum MMP-

9 levels. This suggests that MMP-9 may play an important role in IGFBP-3 proteolysis post surgical trauma and that circulating mononuclear cells are an important source.

This provides a reliable animal model in which to further study the mechanism of IGFBP-3 proteolysis following surgical trauma and its effect on postoperative tumor growth. Experimental Section 3

It was recently demonstrated that a matrix metalloproteinase-9 (MMP-9) induced depletion of insulin- like growth factor binding protein-3 (IGFBP-3) may be partially responsible for the increased tumor growth following surgical trauma observed in animal models. Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors found to have an important role in various diseases including cancer. It has been shown that PPAR alpha agonists inhibit MMP-9 secretion from monocytes in vitro. It is therefore hypothesized that administration of a PPAR alpha agonist in the perioperative period may inhibit the synthesis and release of MMP-9 and the subsequent depletion of intact IGFBP-3 normally seen following surgical trauma.

Methods

A total of 20 mice were included in this study. All the mice were IGFBP-3 transgenic mice on a CD-I background. 10 mice were assigned to receive fenofibrate - 100mg/kg

(dissolved in 70% ethanol) (PPAR alpha agonist) once a day for 5 days and 10 mice received placebo (70% ethanol) according to the same time schedule. On day #4, all mice underwent a sham laparotomy (midline incision from xiphoid to pubis) . Retroorbital blood drawing took place prior to the administration of the drug/placebo, and 24 hours following laparotomy (day #5) . Intact IGFBP-3 levels were measured using a combination of Western blot analysis and ELISA at each time point (48 hours pre-op and 24 hours post-op) . Serum levels of MMP-9 were measured at each time point using zymography.

Results

Mice receiving placebo had a significant decrease in their intact IGFBP-3 levels 24 hours following laparotomy (5403 pg/ml pre-op vs. 2260 pg/ml post-op: p<0.05). This decrease was associated with a significant increase in plasma MMP-9 levels (34.56RU pre-op vs. 83.92RU post-op; p<0.05). In contrast, the mice receiving fenofibrate perioperatively had no significant increase in circulating MMP-9 activity (56.87RU pre-op vs. 54.65RU post-op; p>0.05) and no significant decrease in the levels of intact IGFBP-3 following laparotomy (5274 pg/ml vs. 5687 pg/ml; p>0.05).

Conclusion

PPAR alpha agonist fenofibrate inhibits the release of MMP- 9 and the depletion of intact IGFBP-3 normally seen following surgical trauma in mice. This inhibition may play a role in reversing the tumor-enhancing effects of surgical therapy seen in previous animal models. Although further studies are necessary, these results suggest that the perioperative administration of fenofibrate may be useful in the inhibition of tumor growth in the postoperative period. References

1. Allendorf, J. D., et al., "Increased tumor establishment and growth after laparotomy vs. laparoscopy in a murine model," Arch. Surg. 130:649- 653 (1995) .

2. Aparicio, T. et al., "Matrix metalloproteinase inhibition prevents colon cancer peritoneal carcinomatosis development and prolongs survival in rats," Carcinogenesis 20: 1445-1451 (1999).

3. Dallas, S. L., et al . , Proteolysis of latent transforming growth factor-beta (TGF-beta) -binding protein-1 by osteoclasts: a cellular mechanism for release of TGF-beta from bone matrix," J. Biol. Chem. 277: 21352-21360 (2002) .

4. Descamps, F.J., et al., "Gelatinase B is diabetogenic in acute and chronic pancreatitis by cleaving insulin," FASEB J. 17: 887-889 (2003).

5. Dubois, B., et al., "Regulation of gelatinase B (MMP- 9) in leukocytes by plant lectins," FEBS Lett. 427: 275-278 (1998).

6. Fowlkes, J. L., et al., "Matrix metalloproteinases degrade insulin-like growth factor-binding protein-3 in dermal fibroblast cultures," J. Biol. Chem. 269: 25742-25746 (1994) . 7. Holten-Andersen M.N., et al., "Total levels of tissue inhibitor of metalloproteinases 1 in plasma yield high diagnostic sensitivity and specificity in patients with colon cancer," Clin. Cancer Res. 8: 156-164 (2002).

8. Kallakury, B.V., et al . , "Increased expression of matrix metalloproteinases 2 and 9 and tissue inhibitors of metalloproteinases 1 and 2 correlate with poor prognostic variables in renal cell carcinoma," Clin. Cancer Res. 7: 3113-3119 (2001).

9. Kirman, I., et al., "Plasma from patients undergoing major open surgery stimulates in vitro tumor growth: lower insulin-like growth factor-binding protein 3 levels may, in part, account for this change," Surgery 132: 186-192 (2002) .

10. Kirman, I., et al . , "Open surgery induces a dramatic decrease in circulating intact IGFBP-3 in patients with colorectal cancer not seen with laparoscopic surgery," Surg. Endosc. 19: 55-59 (2004).

11. Kridel, S.J., et al., "Substrate hydrolysis by matrix metalloproteinase-9, " J. Biol. Chem. 276: 20572-20578

(2001) .

12. Lozonschi, L., et al., "Controlling tumor angiogenesis and metastasis of C26 murine colon adenocarcinoma by a new matrix metalloproteinase inhibitor, KB-R7785, in two tumor models," Cancer Res. 59: 1252-1258 (1999). 13. Manes, S., et al., "The matrix metalloproteinase-9 regulates the insulin-like growth factor-triggered autocrine response in DU-145 carcinoma cells," J. Biol. Chem. 274: 6935-6945 (1999).

14. Matsuyama, A., et al., "Matrix metalloproteinases as novel disease markers in Takayusu arteritis," Circulation 108: 1469-1473 (2003).

15. Nielsen, B. S., et al., "92 kDa type IV collagenase (MMP-9) is expressed in neutrophils and macrophages but not in malignant epithelial cells in human colon cancer," Int. J. Cancer 65: 57-62 (1996).

16. Shankavaram, U. T., et al., "Lipopolysaccharide induction of monocyte matrix metalloproteinases is regulated by the tyrosine phosphorylation of cytosolic phospholipase A2," J. Leukoc. Biol. 64: 221-227

(1998) .

17. Sheu, B.C., et al., "A novel role of metalloproteinases in cancer-mediated immunosuppression," Cancer Res. 61: 237-242 (2001).

18. Stumpf, M., et al., "Changes of the extracellular matrix as a risk factor for anastomotic leakage after large bowel surgery," Surgery 137: 229-234 (2005).

19. Swallow, CJ. , et al . , "Metastatic colorectal cancer cells induce matrix metalloproteinase release by human monocytes," Clin. Exp. Metastasis 14: 3-11 (1996). 20. Takino, T., et al., "Cleavage of metastasis suppressor gene product KiSS-I protein/metastin by matrix metalloproteinases," Oncogene 22: 4617-4626 (2003).

21. Tarlton, J. F., et al., "Postsurgical wound progression monitored by temporal changes in the expression of matrix metalloproteinase-9, " Br. J. Dermatol. 137:506- 516 (1997) .

22. Tchetverikov, I., "Matrix metalloproteinases-3, -8, -9 as markers of disease activity and joint damage progression in early rheumatoid arthritis," Ann. Rheum. Dis. 62: 1094-1099 (2003).

23. Trocme, C, et al., "Human B lymphocytes synthesize the 92-kDa gelatinase matrix metalloproteinase-9," J. Biol. Chem. 273: 20677-20684 (1998).

24. Tsuchiya, Y., et al . , "Increased surgical stress promotes tumor metastasis," Surgery 133: 547-555

(2003) .

25. Van Den Steen, P. E., et al., "Gelatinase B/MMP-9 and neutrophil collagenase/MMP-8 process the chemokines human GCP-2/CXCL6, ENA-78/CXCL5 and mouse GCP-2/LIX and modulate their physiological activities," Er. J. Biochem. 270: 3739-3749 (2003).

26. Ylisirnio, S., et al., "Elevated serum levels of type I collagen degradation marker ICTP and tissue inhibitor of metalloproteinase (TIMP) 1 are associated with poor prognosis in lung cancer," Clin. Cancer Res. 7: 1633-1637 (2001).

Claims

What is claimed is:

1. A method for decreasing the degradation of insulin- like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

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

3. The method of claim 1, wherein the agent is a PPAR- alpha agonist or PPAR-gamma agonist.

4. The method of claim 1, wherein the agent is fenofibrate .

5. The method of claim 1, wherein the agent is a protease inhibitor.

6. The method of claim 5, wherein the protease inhibitor is a tissue inhibitor of metalloproteinase-1 (TIMP-I).

7. The method of claim 1, wherein the effective amount of the agent is from about 1 mg/day/kg body weight to about 500 mg/day/kg body weight.

8. The method of claim 1, wherein the effective amount of the agent is about 100 mg/day/kg body weight.

9. The method of claim 1, wherein the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery.

10. The method of claim 1, wherein the agent is administered to the subject prior to the surgery.

11. The method of claim 1, wherein the agent is administered to the subject during the surgery.

12. The method of claim 1, wherein the agent is administered to the subject after the surgery.

13. The method of claim 1, wherein the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery.

14. The method of claim 1, wherein the agent is administered intravenously or orally.

15. A method for decreasing the degradation of insulin- like growth factor binding protein 3 (IGFBP-3) in a subject following open or laparoscopic surgery which comprises administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) -producing cells in the subject and/or inhibit the release of MMP-9 from such cells so as to thereby decrease the degradation of IGFBP-3 in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a), (b) or (C) .

16. The method of claim 15, wherein the subject is a human .

17. The method of claim 15, wherein the agent is an anti- MMP-9 antibody.

18. The method of claim 15, wherein the agent is an antibody which recognizes an MMP-9-producing cell.

19. The method of claim 18, wherein the MMP-9-producing cell is a monocyte.

20. The method of claim 15, wherein the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery.

21. The method of claim 15, wherein the agent is administered to the subject prior to the surgery.

22. The method of claim 15, wherein the agent is administered to the subject during the surgery.

23. The method of claim 15, wherein the agent is administered to the subject after the surgery.

24. The method of claim 15, wherein the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery.

25. The method of claim 15, wherein the agent is administered intravenously or orally.

26. The method of claim 15, wherein the method further comprises administering to the subject an amount of a second agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma, wherein the second agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) , and does not decrease the level of MMP-9-producing cells in the subject or inhibit the release of MMP-9 from such cells.

27. A method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery comprising administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP- 9) in the subject's plasma so as to thereby reduce the likelihood of tumor development or recurrence in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) .

28. The method of claim 27, wherein the subject is a human .

29. The method of claim 27, wherein the agent is a PPAR- alpha agonist or PPAR-gamma agonist.

30. The method of claim 27, wherein the agent is fenofibrate.

31. The method of claim 27, wherein the agent is a protease inhibitor.

32. The method of claim 31, wherein the protease inhibitor is a tissue inhibitor of metalloproteinase-1 (TIMP-I).

33. The method of claim 27, wherein the effective amount of the agent is between about 1 mg/day/kg body weight and about 500 mg/day/kg body weight.

34. The method of claim 27, wherein the effective amount of the agent is about 100 mg/day/kg body weight.

35. The method of claim 27, wherein the agent is administered to the subject (a) prior to, (b) during and (c) after, the surgery.

36. The method of claim 27, wherein the agent is administered to the subject prior to the surgery.

37. The method of claim 27, wherein the agent is administered to the subject during the surgery.

38. The method of claim 27, wherein the agent is administered to the subject after the surgery.

39. The method of claim 27, wherein the agent is administered to the subject daily commencing about one month prior to the surgery and continuing until about three weeks after the surgery.

40. The method of claim 27, wherein the agent is administered intravenously or orally.

41. The method of claim 27, wherein the tumor is a colorectal tumor.

42. A method for reducing the likelihood of tumor development or recurrence in a subject following open or laparoscopic surgery comprising administering to the subject an amount of an agent effective to decrease the level of matrix metalloproteinase-9 (MMP- 9) -producing cells in the subject and/or inhibit the release of MMP-9 from such cells so as to reduce the likelihood of tumor development or recurrence in the subject, wherein the agent is administered to the subject (a) prior to, (b) during or (c) after the surgery, or any combination of (a) , (b) or (c) .

43. The method of claim 42, wherein the subject is a human .

44. The method of claim 42, wherein the agent is an anti- MMP-9 antibody.

45. The method of claim 42, wherein the agent is an antibody which recognizes an MMP-9-producing cell.

46. The method of claim 45, wherein the MMP-9-producing cell is a monocyte.

47. The method of claim 42, wherein the agent is administered to the subject (a) prior to, (b) during and (c) after the surgery.

48. The method of claim 42, wherein the agent is administered to the subject prior to the surgery.

49. The method of claim 42, wherein the agent is administered to the subject during the surgery.

50. The method of claim 42, wherein the agent is administered to the subject after the surgery.

51. The method of claim 42, wherein the agent is administered to the subject commencing about one month prior to the surgery and continuing until about three weeks after surgery.

52. The method of claim 42, wherein the agent is administered intravenously or orally.

53. The method of claim 42, wherein the method further comprises administering to the subject an amount of a second agent effective to decrease the level of matrix metalloproteinase-9 (MMP-9) in the subject's plasma, wherein the second agent is administered to the subject (a) prior to, (b) during or (c) after, the surgery, or any combination of (a) , (b) or (c) , and does not decrease the level of MMP-9-producing cells in the subject or inhibit the release of MMP-9 from such cells.

54. The method of claim 42, wherein the tumor is a colorectal tumor.