Title:  Method of treating depressed reticuloendothelial system function

United States Patent:  6,686,332

Issued:  February 3, 2004

Inventors:  van Leeuwan; Paul A. M. (Amsterdam, NL); Boermeester; Marja A. (Amsterdam, NL)

Assignee:  Xoma Corporation (Berkeley, CA)

Appl. No.:  689097

Filed:  October 12, 2000

Abstract

The present invention provides methods of treating adverse physiological effects associated with depressed reticuloendothelial system function comprising administering to a subject suffering from depressed reticuloendothelial system function an effective amount of a BPI protein product.

SUMMARY OF THE INVENTION

The present invention provides novel methods for the treatment of adverse effects associated with depressed reticuloendothelial system function and specifically treatment of adverse physiological effects associated with impaired liver function resulting e.g., from physical, biological and chemical insult to the liver. Conditions associated with impaired RES function include conditions which directly affect the liver including conditions associated with lowered blood flow to the liver via the portal vein or hepatic artery. Such conditions include but are not limited to, liver cirrhosis, liver transplantation, bile duct obstruction and depressed blood flow from the splenic bed.

More specifically, the invention provides methods for treating conditions associated with depressed reticuloendothelial system function comprising administering to a subject an amount of a BPI protein product effective to alleviate adverse physiological effects resulting from impaired capacity of the RES to clear and inactivate bacteria, bacterial particulates and endotoxin from circulation in the blood. The invention thus provides methods for treatment of endotoxin related sepsis-like conditions associated with impaired liver function resulting from physical (including surgical), chemical and biological (including bacterial and viral) insults to the liver. BPI administration according to the invention is particularly advantageous in the context of pre- and/or post-treatment of subjects undergoing liver surgery. Such methods are particularly preferred where the liver surgery comprises liver transplant or liver resection (hepatectomy) wherein transitory or permanent loss of RES function by Kupffer cells of the liver gives rise to adverse hemodynamic changes, leukocytosis and metabolic acidosis. Benefits resulting from treatment according to the invention include reduction in inflammatory response to liver resection and enhanced regenerative capacity of the remnant liver.

The invention further provides the use of a BPI protein product in the manufacture of a medicament for treatment of adverse physiological effects associated with depressed reticuloendothelial system function, including uses wherein the depressed reticuloendothelial function comprises diminished function of Kupffer cells of the liver such as when the diminished Kupffer cell function results from physical, chemical or biological insult to the liver. The methods of using BPI protein products in the manufacture of such medicaments include those wherein the BPI protein product is rBPI₂₃, rBPI₂₁, rBPI, rBPI₄₂ dimer and peptides as set out in SEQ ID NOS:3 through 224. The BPI protein products may also be used in the manufacture of such medicaments in conjunction with a pharmaceutically-acceptable diluent, adjuvant or carrier.

DETAILED DESCRIPTION OF THE INVENTION

The invention is based on the discovery that administration of BPI protein products attenuates the adverse effects associated with depressed reticuloendothelial system function, particularly effects associated with impaired liver function. The dysfunction or partial resection of the hepatic phagocytic system, i.e. the Kupffer cells, results in reduced clearance of circulating potentially pathogenic particles. In addition, host injury increases intestinal permeability, thus promoting translocation of bacteria or their products (endotoxins) from the gut into the portal or lymphatic circulation. BPI protein products are shown herein to reduce the hemodynamic and metabolic alterations and the inflammatory responses that occur after partial hepatectomy, and also to improve the regenerative response of the liver as measured by liver cell proliferation.

Specifically contemplated by the invention is the treatment of adverse physiological effects resulting from physical, chemical and biological insult to the liver by administering BPI protein products to subjects exposed to such insults. Physical insult to the liver is exemplified by partial or total hepatectomy, such as accompanies transplantation, and trauma. Chemical insult is exemplified by results of exposure to hepatotoxic substances such as chloroform, glucosamine, carbon tetrachloride and ethanol. Biological insult is exemplified by infectious and non-infectious diseases such as viral hepatitis and chronic inflammatory hepatitis. The BPI protein products are preferably administered systemically, such as intravenously, intraperitoneally, or by intramuscular or subcutaneous injection.

As used herein, "BPI protein product" includes naturally and recombinantly produced BPI protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; and biologically active polypeptide analogs of BPI protein or fragments or variants thereof, including cysteine-substituted analogs. The BPI protein products administered according to this invention may be generated and/or isolated by any means known in the art. U.S. Pat. No. 5,198,541, the disclosure of which is incorporated herein by reference, discloses recombinant genes encoding and methods for expression of BPI proteins including recombinant BPI holoprotein, referred to as rBPI or rBPI₅₀ and recombinant fragments of BPI. Co-owned, copending U.S. patent application Ser. No. 07/885,501 and a continuation-in-part thereof. U.S. patent application Ser. No. 08/072,063 filed May 19, 1993 and corresponding PCT Application No. 93/04752 filed May 19, 1993, which are all incorporated herein by reference, disclose novel methods for the purification of recombinant BPI protein products expressed in and secreted from genetically transformed mammalian host cells in culture and discloses how one may produce large quantities of recombinant BPI products suitable for incorporation into stable, homogeneous pharmaceutical preparations.

Biologically active fragments of BPI (BPI fragments) include biologically active molecules that have the same or similar amino acid sequence as a natural human BPI holoprotein, except that the fragment molecule lacks amino-terminal amino acids, internal amino acids, and/or carboxy-terminal amino acids of the holoprotein. Nonlimiting examples of such fragments include a N-terminal fragment of natural human BPI of approximately 25 kD, described in Ooi et al., J. Exp. Med., 174:649 (1991), and the recombinant expression product of DNA encoding N-terminal amino acids from 1 to about 193 or 199 of natural human BPI, described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992), and referred to as rBPI₂₃. In that publication, an expression vector was used as a source of DNA encoding a recombinant expression product (rBPI₂₃) having the 31-residue signal sequence and the first 199 amino acids of the N-terminus of the mature human BPI, as set out in FIG. 1 of Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (specified by GAG) rather than lysine (specified by AAG). Recombinant holoprotein (rBPI) has also been produced having the sequence (SEQ ID NOS: 1 and 2) set out in FIG. 1 of Gray et al., supra, with the exceptions noted for rBPI₂₃ and with the exception that residue 417 is alanine (specified by GCT) rather than valine (specified by GTT). Other examples include dimeric forms of BPI fragments, as described in co-owned and co-pending U.S. patent application Ser. No. 08/212,132, filed Mar. 11, 1994, and corresponding PCT Application No. US95/03125 filed Mar. 13, 1995, the disclosures of which are incorporated herein by reference. Preferred dimeric products include dimeric BPI protein products wherein the monomers are amino-terminal BPI fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of BPI holoprotein. A particularly preferred dimeric product is the dimeric form of the BPI fragment having N-terminal residues 1 through 193, designated rBPI₄₂ dimer.

Biologically active variants of BPI (BPI variants) include but are not limited to recombinant hybrid fusion proteins, comprising BPI holoprotein or biologically active fragment thereof and at least a portion of at least one other polypeptide, and dimeric forms of BPI variants. Examples of such hybrid fusion proteins and dimeric forms are described by Theofan et al. in co-owned, copending U.S. patent application Ser. No. 07/885,911, and a continuation-in-part application thereof. U.S. patent application Ser. No. 08/064,693 filed May 19, 1993 and corresponding PCT Application No. US93/04754 filed May 19, 1993, which are all incorporated herein by reference and include hybrid fusion proteins comprising, at the amino-terminal end, a BPI protein or a biologically active fragment thereof and, at the carboxy-terminal end, at least one constant domain of an immunoglobulin heavy chain or allelic variant thereof.

Biologically active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more amino acid residues have been replaced by a different amino acid. For example, co-owned, copending U.S. patent application Ser. No. 08/013,801 filed Feb. 2, 1993 and corresponding PCT Application No. US94/01235 filed Feb. 2, 1994, the disclosures of which are incorporated herein by reference, discloses polypeptide analogs of BPI and BPI fragments wherein a cysteine residue is replaced by a different amino acid. A preferred BPI protein product described by this application is the expression product of DNA encoding from amino acid 1 to approximately 193 or 199 of the N-terminal amino acids of BPI holoprotein, but wherein the cysteine at residue number 132 is substituted with alanine and is designated rBPI_21.DELTA.cys or rBPI₂₁. Other examples include dimeric forms of BPI analogs; e.g. co-owned and co-pending U.S. patent application Ser. No. 08/212,132 filed Mar. 11, 1994, and corresponding PCT Application No. US95/03125 filed Mar. 13, 1995, the disclosures of which are incorporated herein by reference.

Other BPI protein products useful according to the methods of the invention are peptides derived from or based on BPI produced by recombinant or synthetic means (BPI-derived peptides), such as those described in co-owned and copending PCT Application No. US94/10427 filed Sep. 15, 1994 which corresponds to U.S. patent application Ser. No. 08/306,473 now U.S. Pat. No. 5,652,332 filed Sep. 15, 1994, and PCT Application No. US94/02465 filed Mar. 11, 1994, which corresponds to U.S. patent application Ser. No. 08/209,762, filed Mar. 11, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/183,222, filed Jan. 14, 1994, which is a continuation-in-part of U.S. patent application Ser. No. 08/093,202 filed Jul. 15, 1993 (for which the corresponding international application is PCT Application No. US94/02401 filed Mar. 11, 1994), which is a continuation-in-part of U.S. patent application Ser. No. 08/030,644 filed Mar. 12, 1993, the disclosures of all of which are incorporated herein by reference. Illustrative endotoxin binding and neutralizing peptides include those set out in SEQ ID NOS:3 through 224.

Presently preferred BPI protein products include recombinantly-produced N-terminal fragments of BPI, especially those having a molecular weight of approximately between 21 to 25 kD such as rBPI₂₃ or rBPI₂₁, or dimeric forms of these N-terminal fragments (e.g., rBPI₄₂ dimer). Additionally, preferred BPI protein products include rBPI and BPI-derived peptides.

The administration of BPI protein products is preferably accomplished with a pharmaceutical composition comprising a BPI protein product and a pharmaceutically acceptable diluent, adjuvant, or carrier. The BPI protein product may be administered without or in conjunction with known surfactants, other chemotherapeutic agents or additional known anti-microbial agents. A preferred pharmaceutical composition containing BPI protein products (e.g., rBPI, rBPI₂₃) comprises the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM NaCl, pH 5.0) comprising 0.1% by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, N.J.) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington. Del.). Another preferred pharmaceutical composition containing BPI protein products (e.g., rBPI₂₁) comprises the BPI protein product at a concentration of 2 mg/ml in 5 mM citrate, 150 mM NaCl, 0.2% poloxamer 188 and 0.002% polysorbate 80. Such preferred combinations are described in co-owned, co-pending PCT Application No. US94/01239 filed Feb. 2, 1994, which corresponds to U.S. patent application Ser. No. 08/190,869 filed Feb. 2, 1994 and U.S. patent application Ser. No. 08/012,360 filed Feb. 2, 1993, the disclosures of all of which are incorporated herein by reference.

The following illustrative example of practice of methods of the invention involves prophylactic administration of BPI protein products to alleviate postoperative complications attending liver resection.

EXAMPLE 1

Effect of BPI Protein Product on Hemodynamic and Metabolic Parameters of Rats Subjected to Liver Resection

In this example, the effect of a BPI protein product (rBPI₂₃) administered by a continuous intravenous infusion was determined on rats subjected to a 70% liver resection according to the general methods described by van Leeuwen et al., Surgery 110: 169-175 (1991). Specifically, male Wistar rats (230-250 g) received a 70% liver resection or a sham operation under light ether anesthesia, and were treated with either rBPI₂₃ (8 resected rats, 7 sham rats) or thaumatin a "control" protein having similar molecular weight and isoelectric point (8 resected rats, 8 sham rats). Specifically, the rats were treated with a first loading dose of either rBPI₂₃ or thaumatin at 1 mg/kg followed 20 minutes later by a second loading dose of rBPI₂₃ or thaumatin followed immediately by a continuous intravenous infusion of either rBPI₂₃ or thaumatin at 0.2 mg/kg/hr. Various physiological parameters were measured 4 hours after the resection or sham operation along with IL-6 levels which were determined using the B9 bioassay according to the methods of Helle et al., Eur. J. Immunol. 18:1535-1540 (1988). An alternative assay for IL-6 is described in Helle et al., J. Immunol. Meth. 138:47-56 (1991). The results of these assays are shown in Tables 1, 2 and 3 below.

Control resected rats demonstrated a significantly decreased mean arterial pressure and heart rate compared to control, sham-operated animals. These variables dramatically increased with BPI treatment in the resected rats. Blood pH was significantly decreased in the resected control group (p<0.05), whereas the leukocyte count and hematocrit were significantly increased compared to levels of control, sham-operated animals (p<0.005 and p<0.05, respectively). In the BPI treated resected rats, these parameters were restored to near sham levels.

Levels of IL-6, an important inflammatory mediator, were profoundly elevated in the resected control group compared to the sham control group and the sham BPI treated group. In contrast, the IL-6 levels of the BPI treated resected group were significantly reduced from those of the resected control group as shown in Table 3.

These results show that the early postoperative course following partial hepatectomy is characterized by substantial hemodynamic and metabolic changes. Perioperative infusion of rBPI₂₃ in rats prevented early postoperative hypotension and bradycardia, metabolic acidosis as well as leucocytosis, and also reduced IL-6 levels. These data show that systemic endotoxemia and/or bacteremia, possibly of gut origin, is a major cause of liver surgery postoperative hemodynamic and metabolic derangements including leukocytosis and metabolic acidosis and that administration of BPI protein products can prevent those conditions.

EXAMPLE 2

Effects of BPI Protein Product on Liver Cell Proliferation and Metabolism of Rats Subjected to Liver Resection

The effects of administration of BPI protein product (rBPI₂₃) were determined on liver cell proliferation and liver metabolism of rats subjected to liver resection using procedures essentially as in Example 1. Male Wistar rats (230-250 g, Harlan CPB, Zeist, The Netherlands) were allowed to acclimatize to the laboratory environment for five days with free access to water and rat chow (Hope Farms, Woerden, The Netherlands). The animals were housed under standard environmental conditions with a 12-hr light/dark cycle. Chow was withdrawn on the evening before surgery. Surgery was performed between 9:00 and 11:00 am to avoid chronobiological variations.

The rats were randomized into different groups that underwent either a two-thirds partial hepatectomy (PHX) or a sham operation and were treated peri-operatively with either 0.9% saline or rBPI₂₃. This resulted in the following three groups: (1) rats subjected to a sham operation and treated with saline (n=8); (2) rats subjected to partial hepatectomy and treated with saline (n=8); and (3) rats subjected to partial hepatectomy and treated with rBPI₂₃ (n=8). In addition, two groups of five animals each were used to assess the effects of rBPI₂₃ on sham-operated rats and the effects of a control protein thaumatin (an iso-electric, iso-kd protein) on partially hepatectomized rats.

Prior to the start of the treatment and surgical procedures, the animals were anaesthetized with ether and placed in a supine position. First, a loading dose of the drug or placebo was given via the tail vein: 1 mL 0.9% sodium chloride or 1 mg/kg rBPI₂₃ in 0.5 mL 0.9% saline. Then, a PE-50 catheter (Fisher Scientific, Springfield, N.Y.) was placed via the right jugular vein into the superior caval vein and subcutaneously tunnelled into the interscapular region. Through a spring wire (Instech Laboratories Inc., Plymouth Meeting, Pa.) this intravenous line was connected to a swivel (Instech Labs Inc.) and a micro-infusion pump (Harvard Apparatus, Boston, Mass.). Once the connection was made, a second loading dose of the drug or placebo comprising either 1 mL 0.9% saline or 2 mg/kg rBPI₂₃ in 1 mL 0.9% saline was injected slowly into the intravenous line. Immediately afterwards, a continuous infusion was started of 0.9% saline or 0.2 mg/kg/hr rBPI₂₃ at an infusion rate of 500 .mu.L per hr.

The rats subsequently underwent either a two-third partial hepatectomy, according to the method of Higgins and Anderson Arch. Pathol., 12:186-202 (1931), or a sham operation. Resection of the median and left lateral lobes of the liver was performed with a single vicryl ligature that was carefully placed around the lobes using cotton wool sticks to prevent bleeding from the liver bed. Sham animals underwent a midline laparotomy and gentle manipulation and exteriorization of the median and left lateral lobes, without actual resection. The incision was closed in two layers by vicryl sutures. Within 20 minutes the animals regained consciousness and moved freely while continuing to receive their intravenous infusion. The animals received no food or oral fluids during the study. At 24 hours after surgery, each animal was reanesthetized using Ketamine HCl (50 mg/kg intraperitoneally) and the abdomen was reopened to remove the remnants of the liver. Liver samples up to 0.5 cm³ were frozen immediately in liquid nitrogen or used in conventional histological studies.

Liver enzymes were evaluated as follows. Cryostat sections of constant 8 .mu.m thickness were cut at -25 C. on a motor-driven cryostat (Bright, Huntingdon, UK), placed on clean glass slides and stored at -20^oC. until use. Sections were allowed to dry for at least 5 min at 37^oC. before incubation. Incubations were performed at 37^oC. according to methods described in detail by Van Noorden and Frederiks, "Enzyme Histochemistry: a Laboratory Manual of Current Methods". Oxford, Oxford University Press (1992). Alkaline phosphatase (EC 3.1.3.1) activity was demonstrated using a quantitative indoxyl-tetrazolium salt method. The incubation medium contained 18% (w/v) polyvinyl alcohol (PVA; weight average M. 70,000-1000,000; Sigma, St. Louis, Mo.) in Tris-HCl buffer (pH 9.0), 0.8 mM 5-bromo-4-chloro-3-indolyl phosphate (Boehringer, Mannheim, Germany) as substrate, 0.45 mM 1-methoxy phenazine methosulfate (1-mPMS, Serva, Heidelberg, Germany), 10 mM MgCl₂, 5 mM sodium azide and 5 mM tetranitro blue tetrazolium salt (tetranitro BT; Serva, Heidelberg, Germany). Incubation lasted for 15 minutes and control incubations were performed in the presence of substrate and 10 mM tetramizole (Sigma). Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) activity was demonstrated using a quantitative tetrazolium salt method. The incubation medium consisted of 100 mM phosphate buffer (pH 7.45) containing 18% (w/v) PVA, 10 mM glucose-6-phosphate (Serva) as substrate, 0.8 mM NADP⁺ (Boehringer), 5 mM sodium azide, 0.45 mM 1-mPMS and 5 mM tetranitro BT. Sections were incubated for 10 minutes. Control incubations were performed in the absence of substrate and co-enzyme. Phosphogluconate dehydrogenase (EC 1.1.1.44) activity was demonstrated using a quantitative tetrazolium salt method. The medium was 100 mM phosphate buffer (pH 7.45) containing 18% (w/v) PVA. 8 mM 6-phosphogluconic acid (BDH Chemicals Ltd, Poole, Dorset, UK), 0.8 mM NADP⁺, 5 mM sodium azide, 0.45 mM 1-mPMS and 5 mM tetranitro BT. Incubation was performed for 10 min and control media lacked substrate. Afterwards, all sections were rinsed in 100 mM phosphate buffer (pH 5.3) at 60^oC. to stop the reaction immediately and to remove all of the viscous medium from the sections. Sections were embedded in glycerin-gelatin.

The lipid content of the liver was assessed as follows. Sections were air-dried, treated briefly in 70% ethanol and incubated 30 minutes in a saturated Sudan Black B solution Merck, Darmstadt, Germany; 300 mg/100 mL 70% (v/v) ethanol). Afterwards, sections were rinsed twice in 70% ethanol, and once in 50% ethanol and distilled water before mounting in glycerin-gelatin. In order to measure total amounts of DNA and protein per unit tissue volume, sections were stained with the quantitative combined Feulgen-Naphthol Yellow S (NYS) staining method.

Cytophotometrical analysis of final reaction products was performed as described by Van Noorden and Frederiks, supra, with a Vickers M85a scanning and integrating cytophotometer (Vickers Instruments, York, England). Per rat, 10 readings were made in periporal and pericentral zones in each of 2 sections, both for test and control reactions. The relative integrated absorbance values were converted into mean integrated absorbance (MIA) by reference to a calibration curve. For specific absorbance due to enzyme activity, MIA values obtained in control reactions were subtracted from MIA values obtained in test reactions. For calculation of enzyme activity, MIA values were computed into .mu.moles of substrate converted per minute per cm³ liver tissue by using the molar extinction coefficient of 19,000 for tetranitro BT-formazan.

Absorbance generated by dehydrogenase activity was measured at 557 nm using a 6.3x planachromatic objective (numerical aperture 0.20), a bandwidth setting of 65, a csanning spot with an effective diameter of 3.2 .mu.m and a mask with a diameter of 63 .mu.m. The area scanned per measurement was thus 3117 .mu.m². Formazan generated by alkaline phosphatase activity was measured at 557 nm using a 16x objective (numerical aperture 0.45), a bandwidth setting of 65, scanning spot with diameter 1.25 .mu.m and a mask with diameter 50 .mu.m. The total area scanned per measurement was 1963 .mu.m².

Sudan Black B stained sections were scanned at 595 nm using the same setting as for alkaline phosphatase activity measurements. Faulgen-NYS stained sections were analyzed cytophotometrically at 560 nm (Feulgen) and 430 nm (NYS) with a 6.3x objective (numerical aperture 0.20), bandwidth 65, a 3.2 .mu.m scanning spot and a mask with a diameter of 159 .mu.m and 95 .mu.m respectively. The total area scanned for Feulgen stain was 19.856 .mu.m² and for NYS, 7088 .mu.m².

For the demonstration of proliferating cell nuclear antigen PCNA), a modified streptavidin-biotin-diamino benzidine (DAB) method was used. All incubations were carried out at room temperature in a moist chamber, and all sections were rinsed in 0.01 M phosphate buffered saline (pH 7.4) between each step. Cryostat sections were dried overnight and fixed (for 2 minutes at room temperature) in 4% phosphate buffered formaldehyde (Merck), followed by upgraded and downgraded ethanol series. Sections were pre-incubated for 20 minutes with 10% normal goat serum, decanted and incubated for 60 minutes with a 1:100 dilution of mouse MAb PC 10 (Dakopatts, DAKO a/s, Glostrup, Denmark) directed against PCNA. Endogenous peroxidase activity was blocked using 0.3% (v/v) H₂ O2 and 0.1% (w/v) sodium azide for 15 min. Subsequently, sections were incubated with biotinylated rabbit-anti-mouse Ig at a 1:200 dilution, containing 10% human AB serum for 30 min, followed by an incubation with StreptABComplex (DAKO), prepared 30 minutes in advance using 0.5% (v/v) streptavidin, 0.5% (v/v) biotinylated horseradish peroxidase (HRP) and 10% (v/v) human AB serum. To detect peroxidase activity, sections were incubated for 10 minutes with 0.5 mg/mL DAB and 0.3% (v/v) H₂ O₂ in 50 mM Tris-HCl buffer (pH 7.6) and finally counterstained with haematoxylin. The PCNA index for periportal and pericentral areas was determined by analysis of the percentage of PCNA-positive liver cells out of 300 liver cells in both periportal and pericentral zones. For each rat, mean values of measurements were calculated for both periportal and pericentral zones. Results are expressed as means+standard error of the means per group of animals. Statistical analysis was performed by the non-parametric Mann-Whitney U Test. A p-value of less than 0.05 (two-tailed) was considered significant.

PCNA expression is displayed in FIG. 1. In sham-operated animals, cell proliferation was virtually absent. In partially hepatectomized animals, there was a high rate of cell proliferation 24 hours post-surgery, particularly in the periportal zones. Treatment with rBPI₂₃ significantly increased liver cell proliferation, as indicated by PCNA expression, in both hepatocytes and sinusoidal cells in both zones of liver lobules.

Lipogenesis and lipid accumulation in the liver is correlated to liver damage and reduced regenerative capacity. Partial hepatectomy induced a 5-fold lipid accumulation in liver, compared to the sham-operated animals (p<0.001). Treatment with rBPI₂₃, treatment significantly reduced lipid content by 20-30% (p<0.05). Lipid content was always higher periportally than pericentrally.

Treatment with rBPI₂₃ had no effect on alkaline phosphatase, glucose-6-phosphate dehydrogenase or phosphogluconate dehydrogenase activity. After partial hepatectomy, alkaline phosphatase (AP) activity in bile canalicular membranes was significantly increased both periportally (p<0.005) and pericentrally (p<0.001) compared to sham-operated animals. Predominant AP activity in partially hepatectomized rats was in pericentral zones, compared to periportal zones in sham-operated rats. In addition, AP activity was observed at the sinusoidal membranes of hepatocytes in hepatectomized rats, but not in sham-operated animals. Glucose-6-phosphate dehydrogenase (G6PD) activity was significantly decreased at 24 hr after partial hepatectomy compared with sham-operated rats (p<0.001 periportally; p<0.01 pericentrally). In addition, the higher periportal G6PD activity observed in sham-operated animals had disappeared. Phosphogluconate dehydrogenase (PGDH) activity, which was always higher pericentrally than periportally, was markedly decreased in partially hepatectomized rats compared with sham-operated animals.

Cytophotometric measurements of enzyme activity or lipid content did not need correction because the ratios of total DNA over total protein were similar in all periportal and pericentral zones of all animals. Partially hepatectomized rats that received thaumatin showed no significant difference from partially hepatectomized rats that received saline. Moreover, infusion of rBPI₂₃ or thaumatin compared to infusion of saline had no significant effects on the measured parameters in sham-operated animals (data not shown).

These results show that rBPI₂₃ treatment stimulated liver cell proliferation and reduced lipid accumulation after partial hepatectomy.

EXAMPLE 3

Effects of BPI Protein Product on the Local Inflammatory Response of Rats Subjected to Liver Resection

The effects of a BPI protein product, rBPI₂₃, were determined on the local inflammatory response of rats subjected to liver resection according to the procedure described in Example 2. In particular, effects on infiltration of immune cells and expression of major histocompatibility complex (MHC) class II antigens of macrophages (a macrophage activation marker) in the remnant liver were studied using immunohistochemical techniques.

The rats were divided into three groups: (1) rats that underwent a sham operation with saline treatment (n=8); (2) rats that underwent partial hepatectomy with saline treatment (n=8); and (3) rats that underwent partial hepatectomy with rBPI₂₃ treatment (n=8). Before catheter insertion, the rats were given a loading dose of 0.5 mL 0.9% saline or 1 mg/kg rBPI₂₃ in 0.5 mL buffer solution (containing 20 mM sodium citrate, 150 mM sodium chloride; pH 5) via the tail vein. After catheterization, the rats were administered a second loading dose of either 1 mL 0.9% saline or 2 mg/kg rBPI₂₃ in 1 mL (0.5 mL buffer solution plus 0.5 mL 0.9% (w/v) NaCl). Immediately thereafter the rats were administered 0.9% saline or 0.2 mg/kg/hr rBPI₂₃ by continuous infusion at a rate of 500 .mu.L per hr, which 'was continued for a 24-hour period. After 24 hours, liver samples were obtained as described in Example 2.

Alkaline phosphatase (AP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), bilirubin and glucose serum levels were assessed by automated laboratory analysis. Ammonia levels were assayed by a standard enzymatic method. Sections of paraffin-embedded liver samples were subjected to conventional histology. All sections were read in a blinded fashion. Polymorphonuclear neutrophils (PMNs) were stained with chloracetate-esterase. Mast cells were stained using a Toluidine Blue O staining procedure. Proliferating cell nuclear antigen was also measured using the procedures described in Example 2.

Immunohistology was performed on the liver samples as follows. Incubations were carried out at room temperature in a moist chamber, and all sections were rinsed in 0.01 M phosphate buffered saline (PBS), pH 7.4, between each step, unless otherwise specified. Cryostat sections were air-dried for 30 minutes, fixed in acetone (for 7 minutes at 4^oC.) and air-dried again (30 minutes) before incubation for 60 minutes with monoclonal antibodies (MAb) ED 1, ED 2 or OX 3 (Serotec, Hilversum, Netherlands) diluted 1:500 in PBS containing 0.2% (w/v) bovine serum albumin (BSA). MAb ED 1 recognizes a cytoplasmic antigen in monocytes and the various types of macrophage populations. MAb ED 2 recognizes membrane antigens of resident macrophages. MAb OX 3 is directed against a polymorphic determinant of the human MHC class II antigen (rat Ia antigen). Sections were then incubated for 30 minutes with rabbit-anti-mouse IgG peroxidase (Dakopatts, Copenhagen, Denmark) diluted 1:200 in PBS/BSA containing 1% (v/v) normal rat serum to reduce non-specific staining. Afterwards, sections were stained for peroxidase activity for 10 minutes using, 1 mM 3-amino-9-ethylcarbazole (AEC, Sigma, St. Louis, Mo., USA) dissolved in 5% (v/v) dimethyl formamide (DMF) and 0.01% (v/v) H₂ H₂ in 50 mM acetate buffer (pH 4.9). Finally, after rinsing in distilled water, sections were counterstained with hematoxylin for 1 minute and rinsed thoroughly before mounting in glycerol jelly.

To assess the composition of the hepatic mononuclear phagocytic cell pools, sections stained with mAb ED 1 (a marker for monocytes and all macrophage populations) were compared to consecutive sections stained with ED 2 (a marker for resident macrophages). After sham operation, the majority of these macrophages were both ED 1-positive (ED 1+) and ED 2-positive (ED 2+) and, thus, were predominantly Kupffer cells. After partial hepatectomy, an increase in the number of ED 1+ cells was observed compared to sham-operated rats. In partially hepatectomized rats, far more macrophages were ED 1+ than ED 2+, particularly in periportal areas, indicating an increase in the number of non-resident macrophages and monocytes. Livers of partially hepatectomized rats treated with rBPI₂₃ had fewer ED 1+ cells compared to saline-treated rats.

Expression of MHC class II antigen (an indicator of antigen presentation) was evaluated by immunohistochemical staining using mAb OX 3. Following partial hepatectomy, an increase in OX 3+ cells, predominantly in periportal areas, was observed compared to the sham-operated group. This increase in OX 3+ cells was not observed in livers from partially hepatectomized animals treated with rBPI₂₃.

Conventional histological examination showed that, at 24 hours after partial hepatectomy, increased numbers of PMNs were found predominantly in periportal areas. Infrequent aggregates of PMNs in close contact with small patches of necrosis were also found. In partially hepatectomized animals treated with rBPI₂₃, fewer PMNs were found periportally and virtually no PMNs were found in other areas, and no aggregates of PMNs or necrosis were observed. In all groups of animals only few mast cells were found, mainly adjacent to larger vessels.

PCNA values (means+standard error) are shown in Table 4 below. Liver cells of sham-operated rats expressed virtually no PCNA. At 24 hours after partial hepatectomy, a larger number of cells express PCNA, predominantly in periportal zones. Treatment of partially hepatectomized rats with rBPI₂₃ led to a significant increase in liver cell proliferation both periportally (p<0.01) and pericentrally (p<0.01).

Serum levels of measured parameters known to be related to liver function are depicted in Table 5. After 24 hours, circulating levels of alkaline phosphatase, AST, ALT and ammonia were significantly increased following partial hepatectomy compared to sham operated rats (p<0.0005, p<0.0005, p=0.0005 and p=0.01, respectively). Bilirubin levels were only slightly elevated following partial hepatectomy, though significantly higher than in the sham operated group (p=0.001). In addition, glucose levels were significantly lower then those found in sham animals (p<0.001). In the rBPI₂₃ treated partially hepatectomized animals, AST and ALT levels were significantly reduced (p<0.05 vs. untreated) but still elevated compared to sham-operated rats. Ammonia levels were also lower in animals treated with rBPI₂₃ (p=0.1) and were close to those in the sham-operated rats. Other measured parameters of liver function (AP, bilirubin and glucose) were not affected by treatment with rBPI₂₃.

These results show that, following partial hepatectomy, there is a local inflammatory response. This inflammatory response is characterized by a profound influx of mononuclear phagocytes and a moderate infiltration of PMNs and coincides with increased serum levels of markers of liver dysfunction (AST, ALT, ammonia), implying damage of liver parenchyma by these reactions. There is also a higher proportion of macrophages expressing Ia antigens, which are indicative of activation and possibly antigen-presentation. Treatment with rBPI₂₃ reduced hepatic inflammation and partially prevented liver failure. In addition, liver cell proliferation liver regeneration (e.g., as assessed by PCNA expression) was significantly enhanced by rBPI₂₃ treatment.

Claim 1 of 4 Claims

What is claimed is:

1. A method of treating a human subject subjected to liver surgery comprising administering to said subject an amount of bactericidal permeability-increasing protein (BPI) protein product effective to reduce liver failure.

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