WO1998024417A1 - Endotoxin binding to lipid immobilized hemoglobin - Google Patents

Abstract

The present invention relates to a method of binding endotoxin (or lipopolysaccharide) to lipid immobilized hemoglobin (LIH).

Description

TITLE OF THE INVENTION

ENDOTOXIN BINDING TO LIPID IMMOBILIZED HEMOGLOBIN BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to a method of binding endotoxin to lipid immobilized hemoglobin (LIH) . The method of the present invention can be used both therapeutically to treat or prevent conditions such as sepsis in a patient as well as diagnostically to determine the amount of endotoxin in a biological or environmental sample.

Discussion of the Background

Endotoxin is a bacterial cell wall fragment, sometimes referred to as lipopolysaccharide or LPS. Endotoxin has been shown to be the primary cause of the septic syndrome. Endotoxin accumulates as bacteria translocate from the gut wall in response to hemorrhage or as a result of direct introduction through insult or injury. Endotoxin elicits a number of inflammatory responses which initiate the sepsis syndrome and can be measured in cellular, tissue, and organismal responses. One common response is the production of inflammatory cytokines by phagocytic cells such as macrophages or monocytes. High levels of endotoxin in an animal can cause fever, diarrhea, hemorrhagic shock and other tissue damage. LPS can also induce cellular responses, such as the production of inflammatory agents, such as cytokines. Endotoxins arising from bacteria can also be a contaminating source in food or biomaterials and cause similar reactions. Thus, there is a continuing need for methods for diagnosing endotoxins and decreasing endotoxin levels.

Previously it was suggested that hemoglobin binds endotoxin (Roth et al., 1993, Infection and Immunity 61:3209). Consequently, the administration of hemoglobin based blood substitutes was thought to exacerbate septic shock (Davidson et al., 1988, Critical Care Medicine 16, 606). Thus, blood substitutes were sought with decreased affinity to endotoxin.

Blood substitutes encompass four general classes: free hemoglobin (native tetramer or crosslinked) , perfluorocarbons, hemoglobin or heme moieties anchored to a core particle, and liposome encapsulated hemoglobin (LEH) .

LEH is described, for example, in U.S. Patent Nos. 4,911,929; 4,776,991 and 4,133,874. Fabrication of LEH results in the entrapment of hemoglobin in the interior of the liposome compartment. During the fabrication of this material, hemoglobin adheres to the outer surface of the liposome as evidenced by electron micrographs, and uv-visible spectroscopy (Rabinovici et al. , 1990, Circulatory Shock 31:431) . Further evidence that hemoglobin is bound to the surface is found by treating the liposomes with a protein digestion enzyme (trypsin) which results in enzymatic digestion of surface bound hemoglobin which can be detected by uv-visible spectroscopy. This body of data demonstrates that hemoglobin is surface bound to the liposome. Since surface bound hemoglobin increases the liposome aggregate size, such liposomes are not amenable to 0.2 or 0.45 sterile filtration, an essential feature of a large scale manufacturing process . Much research has focused on diminishing the amount of surface bound hemoglobin. Passivation of the surface with albumin has been shown to reduce the aggregate size, and work is ongoing to demonstrate that the albumin passivation results in reduced surface bound hemoglobin.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide a method for binding endotoxin. Such a method can be used in vitro as a diagnostic method for detecting and reducing the level of endotoxin in a biological or environmental sample. Alternatively, such a method can be used in vivo as a therapeutic to decrease the level of endotoxin.

These and other objects, which will become apparent during the following detailed description, have been achieved by the inventors' discovery that lipid immobilized hemoglobin is effective for binding endotoxin.

The novel aspects of this invention are that 1) the specificity of which endotoxin binds to immobilized hemoglobin and not to other immobilized proteins, 2) the retention of biological activity of the surface immobilized hemoglobin-endotoxin complex, and 3) the immobilization of hemoglobin to a lipid surface which allows endotoxin binding for detecting and reduction.

BRIEF DESCRIPTION OF THE DRAWINGS A more complete appreciation of the invention and many of the attendant advantages thereof will be readily obtained as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

Figure 1 is a cartoon of LEH/surface bound hemoglobin/endotoxin.

Figure 2 shows the radiolabeled endotoxin binding to Liposomes without hemoglobin (top) , liposome encapsulated hemoglobin (middle) , and liposomes which have been bathed in hemoglobin such that hemoglobin is only in contact with the exterior liposomal surface (bottom) . The binding of radiolabeled endotoxin is expressed as % isotopic activity after the radiolabeled endotoxin is incubated with the sample and then the sample centrifuged at 50,000 rpm. The % isotopic activity that remains with the pellet is the 'bound' endotoxin. Note that LEH and liposomes bathed in hemoglobin have significant bound endotoxin, whereas liposomes with no exposure to hemoglobin do not. The binding of endotoxin is mediated by the surface immobilized hemoglobin on the outer surface of the liposomes. Figure 3 illustrates the biological activity of surface bound endotoxin to LEH as measured a first by the production of messenger RNA (mRNA) for the cellular inflammatory agent tumor necrosis factor (TNF-α) , and secondly by the production of TNF-α itself. (A) TNF-α mRNA from RAW 264.7 cells exposed to 0.088 mg/ l LEH (LEH1/LEH2 ) or LEH that had been previously incubated with 1.0 μg/ml LPS at 37 °C for 1-2 hours and then washed of all free LPS (S01/S024) LPS1/LPS24 represent cells challenged with 1 μg/ml LPS for 1 and 24 hours as the positive control. (B) TNF-α production as measured by ELISA from RAW264.7 cells exposed to 1 μg/ml LPS (control) (•) or to 0.088 mg/ml LEH that had been previously incubated with 1.0 μg/ml LPS at 37°C and then washed of all free LPS (o) , ^*p<.05, ^**p<.01. Note that the LEH that has been exposed to LPS and then washed shows biological activity in the ability to elicit TNF-α production. LEH not incubated with endotoxin does not show any biological activity (not shown) .

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS LEH can be prepared using conventional methods such as those disclosed in U.S. Patent Nos. 4,776,991 and 4,133,874;

Farmer & Gaber, Methods in Enzymology, 1986, vol. , pages and Goins et al. , Biotechnology of Blood, 1991, Goldstein

Ed., Butterworth, Stoneham MA, 117-124; each incorporated herein by reference. Briefly, LEH is produced by combining lipids and hemoglobin under a force. Purified hemoglobin can be obtained from commercial sources such as Biopure, Boston MA. Alternatively, hemoglobin can be obtained from red blood cells as described by Tye in U.S. Patent No. 4,529,719; Sim onds et al.,.U.S. Patent No. 4,401,652 and Kothe et al. , U.S. Patent No. 4,526,715 or by reco binant or transgenic methods; each incorporated herein by reference. Hemoglobin in accordance with the invention should be sterile, non-pyrogenic as by rabbit pyrogen test and low endotoxin as determined by the Limulus Amebocyte Lysate (LAL) test (See U.S.P Guidelines; Manning, Chapter 6, Blood Substitutes: Physiological Basis of Efficacy, Winslow et al., ed. , 1995, Birkhauser; Methods in Enzymology, 1987, vol. 189, pl84) .

Lipid constructs in accordance with the present invention can be produced from a variety of lipids. "Lipid construct" as used herein includes multilamellar liposomes, unilamellar liposomes, flat lipid bilayers and onolayers (supported on beads or other solid supports) .

Suitable lipids include phosp atidylcholine with an acyl chain equal to or in excess of 14 carbon atoms (such as distearoyl phosphatidylcholine, hydrogenated soy phosphatidylcholine, sialic acid derivatives thereof) , phosphatidylserine with an acyl chain in excess of 14 carbon atoms, negatively charged lipids (such as phosphatidic acid, dicetyl phosphate, and dimyristoyl-, dipalmitoyl- or distearoyl-phosphatidylglycerol) ; cholesterol; and mixtures thereof. The lipid mixture of the present invention can further contain lipid antioxidants such as vitamin E (α-tocopherol) .

A preferred lipid mixture comprises distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglycerol, and vitamin E in a molar ratio of 10: 9: 0.9: 0.1.

Lipid/hemoglobin mixtures such as LEH can be manufactured either by forming lipid constructs from a mixture of lipids and hemoglobin or by first forming a lipid construct from the lipids and subsequently surface adsorbing the hemoglobin.

In a first embodiment, the lipids can be combined with hemoglobin such that the resulting mixture has a hemoglobin content of at least 20 g%, preferably 20-25 g% , such that the resulting lipid/hemoglobin molar ratio is in the range of 15:1 to 30:1, preferably 20:1.

The lipid/hemoglobin mixture can be suitably formed into liposomes by a number of conventional methods including simple hydration or applying a force such as sonication, hydrodynamic shear, dialysis. Preferably, the resultant multilamellar vesicles can then be homogenized to create large unilamellar vesicles having a particle size of 0.1-0.5, preferably 0.15- 0.2, microns, with a high pressure hydrodynamic shear flow apparatus (Microfluidizer™) . Using the above methods, the amount of hemoglobin in the LEH is preferably 2-12 g%. Alternatively, lipid constructs (or LIH) can be first formed from lipids as described above and then exposed to hemoglobin by bathing the liposomes in solution for 60 minutes at 4°C. Using the above method, the amount of hemoglobin associated with the liposomes is preferably 1-5 mg/ml.

In either method, unencapsulated or unassociated hemoglobin is removed from the lipid constructs using any known separation method such as centrifugation.

Once formed, LEH and LIH in accordance with the present invention can be stored at 4°C for at least 6 months to 1 year. Alternatively, the LEH can be freeze-dried and stored for 6 months or longer.

The method of the present invention comprises contacting a biological or environmental sample in which endotoxin may be present with a solution of LIH and separating any endotoxin from the sample. When the method of the present invention is performed in vitro as a diagnostic, the method further comprises a step of determining the concentration of endotoxin bound to the LIH. When the method is performed therapeutically in vivo , the method may comprise a monitoring step.

For diagnostic applications, the LIH is suitable diluted in a solution to a concentration of from 0.1 to 10%, preferably 1 to 2%.

The biological sample suspected of containing endotoxin can be blood, serum, urine, organ perfusate, pharmaceutical solution, food, or environmental extract. Pharmaceutical solutions include any pharmaceutical agent dissolved in a pharmaceutical carrier (such as saline) . Environmental samples includes soil extracts or waste water effluents or drinking water.

The sample is contacted with (LIH) for a time sufficient to adsorb endotoxin, typically 5 to 180, preferably 60 to 120, minutes .

Endotoxin bound to LIH is removed from the sample by any conventional separation technique such as centrifugation.

Alternatively, LIH can be immobilized onto a carrier such as a ceramic, metal or plastic surface. LIH can either be absorbed onto the surface or attached through a cross-linker such as a lipid, protein or any other bifunctional compound. Methods known in the art for immobilization of hemoglobin onto such surfaces can be used to immobilize the lipid/hemoglobin mixture of the present invention onto said surfaces.

The amount of endotoxin bound to the LIH may be determined by any conventional technique. In particular, LAL assays can be used and are commercially available. The LAL assay is described by Nachum et al., 1982, Laboratory Medicine, 13:112 and Pearson II et al. , 1980, Bioscience 30:461, both incorporated herein by reference. These methods include a gel-clot method, a chromogenic spectroscopic-based LAL, a turbidometric method, a pyrochrome method and a method which consists of contacting the LIH with cells which express TNF-α followed by ELISA quantitation (See Cliff et al, 1995, Art. Cells, Blood Subs . , and Immob . Biotech . , 23, 331-336; incorporated herein by reference).

The amount of endotoxin bound to LIH can be quantitatively determined by comparing the results obtained by exposing the LIH to the biological sample against results obtained by exposing LIH to solutions containing known amounts of endotoxin.

As mentioned above, LIH has previously been used as a blood substitute. The present method entails the administration of LIH for the prevention or treatment of conditions associated with high levels of endotoxin such as sepsis, endotoxemia or food contamination, For these therapeutic purposes, LIH is suitable diluted in a physiological solution such as sterile saline buffered at a pH of about 7-9, preferably 7.4.

The solution of LIH containing a therapeutic amount of LIH is suitably administered to a patient in need thereof either via intravenous injection, intraperitoneal injection or by using an extracorporeal device in which the blood is passed over LIH external to the body.

Suitable patients include those who have from abnormally high levels of endotoxin in their blood to those expected to develop high levels of endotoxin in their blood (for example, patients exposed to actions which increase the risk of hemorrhage) . Blood endotoxin levels are very low (0 endotoxin units/mL) . Clinical symptoms are expected at levels higher than this. Suitable patients include any mammal, particularly humans .

The dosage of LIH will depend on a variety of factors including the extent of sepsis, the age, and weight of the patient. Typically an LIH solution containing 2-12 g% of hemoglobin will be continuously administered to a patient for at least 10 minutes, preferably 0.5 to 4 hours.

It is known that LIH is effectively metabolized by patients. Alternatively, endotoxin bound to LIH could be removed from circulation by dialysis or sedimentation resulting in reduced endotoxin levels.

Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES Example 1 Endotoxin binding to Liposomes with Surface Immobilized Hemoglobin LEH and Liposomes

This experiment was performed in February 1995 and was referred to as LEH/LPS 4 in an ongoing series of experiments designed to elucidate the interaction of LPS with LEH. For this experiment three particular samples were prepared: 1) Liposomes without hemoglobin encapsulated 2) Liposome Encapsulated Hemoglobin (lot ROCAL002) , and 3) Sample 1 bathed in 24 g% Bovine hemoglobin for 90 minutes at 4°C, then washed to remove non-associated protein. Each of these samples were incubated with tritium-labeled LPS for 2 hours at 37 °c with constant shaking. Following centrifugation, separation at 50,000 RPM for 60 minutes, the supernatant (non-liposomal) and pellet (liposomal) were analyzed using liquid scintillation for the tritiated LPS. The pellet was resuspended and centrifuged again (same speed and duration) to verify LPS association. In this example the radioisotope percent associated with the pellet versus the supernate (see attached figure) was as follows: Sample 1 pellet-8% supernate-76%, Sample 2 pellet-47% supernate-55%, and Sample 3 pellet-64% supernate-36%.

Example 2 Cell-based Bioactivity

In this example, the bioactivity of the LPS bound to the liposome was tested. It is well documented that the murine macrophage cell line named RAW 264.7, when exposed to LPS- spiked media in cell culture, produces the cytokine Tumor Necrosis Factor-α (TNF-α) in a time and dose dependent manner (Virca et al., 1989, J. Biol. Chem. 264:21951-21956). The release of this inflammatory mediator is dependent on interaction between the LPS and LPS-binding sites on the macrophage cell surface. This experiment was designed to find out if the LPS bound to the liposomal surface would mimic the response to free LPS by the macrophages and produce TNF-α.

LEH was prepared as described in example 1 and then incubated with non-radioactive LPS at 125 ng/ l in the same manner as in example 2. The LPS-coated LEH was added to the media culture exposed to the macrophages (1 x 10⁶ cells/ml) and compared against cells treated with LPS alone. The amount and kinetic profile of TNF-α produced by macrophages exposed to LPS-coated LEH is similar to that of LPS alone. From this example it appears as though the liposomal-bound LPS is capable of stimulating this macrophage cell line in a manner comparable to that of free LPS. LEH with no associated LPS did not produce a cellular response.

Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

Claims

CLAIMS :

1. A method to decrease the endotoxin concentration in a sample comprising, contacting a sample with a lipid/hemoglobin construct for a time sufficient to bind endotoxin present in said sample to said construct, and removing said construct from said sample.

2. The method of Claim 1, which further comprises the step of determining the amount of endotoxin bound to said lipid/hemoglobin construct.

3. The method of Claim 1, wherein said sample is a biological or environmental sample.

4. The method of Claim 3, wherein said sample is blood, urine, an organ perfusate, a pharmaceutical composition or food extract.

5. The method of Claim 1, wherein said contacting time is 5 to 180 minutes.

6. The method of Claim 1, wherein said lipid/hemoglobin construct is immobilized onto a substrate.

7. The method of Claim 1, wherein said lipid/ hemoglobin construct contains lipid constructs selected from the group consisting of multilamellar liposomes, unilamellar liposomes, flat lipid bilayers and monolayers.

8. The method of Claim 7, wherein lipid/hemoglobin said construct is liposome-encapsulated hemoglobin.