WO1994018989A1 - Use of heparin to inhibit interleukin-8 - Google Patents

Abstract

A method and medicament for the inhibition of the cytokine interleukin-8 comprising administering a treatment effective amount of the sulfated polysaccharide heparin to a human in need thereof. The medicament preferably is administered by aerosolization. In specific embodiments of the invention, the ratio of heparin to interleukin-8 is greater than about 0.1 and less than about 5.0. Preferably, the heparin medicament includes a physiologically acceptable carrier which may be selected from the group consisting of physiologically buffered saline, isotonic saline and normal saline.

Description

USE OF HEPARIN TO INHIBIT INTERLEUKIN-8

BACKGROUND OF THE INVENTION The present invention relates to medical treatment of humans and closely related primates and more specifically to methods and medicaments for blocking the effects of interleukin-8, a cytokine causing neutrophil influx into inflamed and injured tissues in various disease states. Accumulation of activated polymorphonuclear leukocytes (PMNs) in tissues is a major pathophysiologic event mediating inflammation and tissue injury in a diverse variety of diseases, including ischemia- reperfusion injury (J.L. Romson, et al. Circulation (1983) 67:1016-1023), adult respiratory distress syndrome (J.E. Rinaldo, et al. New England Journal of Medicine (1982) 306:900-909), cystic fibrosis (N.G. McElvaney, et al. Journal of Clinical Investigation (1992) 90:1296-1301) and psoriasis (H. Pinkus, et al. Journal of Investigative Dermatology (1966) 46:109-115). Once present, PMNs enhance tissue injury by release of oxidants such as hypochlorous acid and cationic connective tissue proteases such as collagenase, elastase and cathepsin G. An important stimulus responsible for attracting PMNs into injured organs has been recently discovered to be a basic 8-10 kilodalton human protein called interleukin-8 (IL-8), one of the most potent chemotaxins for human PMNs (J.J. Oppenheim, el al. Annual Review of Immunology (1991) 9:617-648). IL-8 is produced by a variety of human cell types in response to injury, including monocytes, macrophages, t-lymphocytes, fibroblasts, endothelium, keratinocytes, hepatocytes, chonedrocytes and tracheal epithelium, and is a universally important local signal attracting PMNs in a variety of disease processes. IL-8 secreted by cultured cells after stimulation by anoxia and hyperoxia mediates PMN influx into tissues after ischemia- reperfusion and hyperoxic injury (A. P. Metinko, et al. Journal of Clinical Investigation (1992) 90:791-798). As a consequence of PMN migration into injured tissues such as infarcted myocardium (an example of ischemia- reperfusion) and ventilated lung exposed to high oxygen (an example of hyperoxic injury), the tissues undergo a degree of injury much worse than they would from the primary disease process alone. IL-8 is elevated in bronchoalveolar lavage fluid of patients with adult respiratory distress syndrome (ARDS), and causes neutrophil recruitment and mortality in this disease (E.J. Miller, et al. American Review of Respiratory Diseases (1992) 146:427-432). Regardless of the primary cause of lung injury in ARDS (sepsis, pneumonia, aspiration, toxic gas inhalation, or massive trauma), the degree of lung injury is greatly worsened when IL-8 attracts PMNs into the lung and the PMNs subsequently release their toxic products. IL-8 is also markedly increased in respiratory secretions of patients with cystic fibrosis (CF) and is thought to be a major stimulus producing airway inflammation and consequent airway disease in CF patients (N.G. McElvaney et al. Journal of Clinical Investigation (1992) 90:1296-1301). In CF, IL-8 is thought to be a primary stimulant to the development of lung disease. While the cause of initial inflammation in the CF airway is unknown, this inflammation causes airway cells to make and release IL-8, which in turn attracts activated PMNs into the airway. The PMNs release toxic oxygen radicals and proteases which enhance the degree of injury and stimulate production of even more I -8, initiating an IL-8 dependent downward spiral of inflammatory airway destruction that ultimately results in progressive obstructive lung disease. Finally, IL-8 is present in psoriatic scales (J.M. Schroeder, et al. Journal of Immunology (1987) 139:3474-3483) and is a potent growth stimulus for keratinocytes (G. Krueger, et al. Journal of Investigative Dermatology (1990) 94:545), suggesting a role for IL-8 in mediating psoriasis and other leukocyte- mediated skin disorders. SUMMARY OF THE INVENTION As explained below, this invention describes how heparin and heparin-like compounds are useful in blocking the biologic activity of IL-8 for attracting PMNs into tissues. Heparin is a naturally occurring heterogeneous group of straight-chain anionic mucopolysaccharides present in mast cells of a variety of tissues such as liver, lung, large arteries and intestine. It is a polymer of repeating units of the sugars (1) alpha- - iduronic acid 2-sulfate, (2) 2-deoχy-2-sulfamino-alpha-D- glucose 6-sulfate, (3) beta-D-glucuronic acid, (4) 2- acetamido-2-deoχy-alpha-D-glucose and (5) alpha-L-iduronic acid. These sugars are present in decreasing amounts, usually in the order (2)>(1)>(4)>(3)>(5). The polymer is formed by alternating-1 ,4, linkages between C1 and C4 of adjacent sugars across an interposed oxygen, and is strongly acidic because of its content of covalently linked sulfate and carboxylic acid groups. Representative subunits of the structure of heparin are shown in FIGURE 1. Heparin is commonly provided as the sodium salt as indicated in U.S. Patent No 3,062,716 (Montaudraud) and as "Entry 4543" in The Merck Index. 672 (10th edn. 1983), although the salts of calcium, potassium, barium, lithium, ammonium and other cations are also known. The major commercial utility of heparin is as an anticoagulant. Heparin functions as an anticoagulant by virtue of the presence of a repeating pentasaccharide structure, which binds to the serum protein antithrombin III. The attachment of heparin to antithrombin III greatly enhances the natural anticoagulant activity of antithrombin III against thrombin and against factor Xa in the coagulation cascade. This, in turn, effects anticoagulation of the blood. However, other uses of heparin not necessarily dependent on anticoagulation are described, including inhibition of human tumor metastases and inhibition of smooth muscle growth. The aforementioned biologic effects of IL-8 and its important role in mediating a number of diseases suggest that any means of blocking the biologic actions of IL-8 would be extremely useful in treatment of medical conditions to which this cytokine contributes. However, there has been no method described to date of blocking I - 8 activity.

It is an object of the present invention to provide a method for inhibiting IL-8 activity in humans and closely related primates that also make IL-8 as a major chemotactic stimulus for neutrophils.

It is a further object of the present invention to provide a method for inhibiting IL-8 activity by aerosol treatment with the therapeutic agent. It is an advantage of the present invention that the therapeutic agent is produced from a toxicologically characterized compound with low toxicologic potential when administered by inhalation of an aerosol.

Consideration of the specification, including the several figures and examples to follow will enable one skilled in the art to determine additional objects and advantages of the invention.

The present invention provides a medicament for the inhibition of IL-8 in humans and closely related primates comprising a treatment effective amount of the sulfated polysaccharide heparin or heparin-like polymers, their components and derivatives. In preferred embodiments of the invention, the medicament is administered by aerosolization. In other embodiments of the invention, the effective molar ratio of heparin to IL-8 is chosen to be greater than about 0.1 and less than about 5.0. Preferably, the medicament includes a physiologically acceptable carrier which may be selected from the group consisting of physiologically buffered saline, isotonic saline and normal saline. The pH of the heparin and carrier mixture is preferably equal to or greater than 6.5 but less than 7.4. BRIEF DESCRIPTION OF THE DRAWINGS Reference to the following detailed description may help to better explain the invention in conjunction with the drawings in which: FIGURE 1 shows the proposed structure of representative subunits of heparin.

FIGURE 2 shows a graph of the inhibitory effect of increasing doses of heparin against the chemotactic activity of human IL-8 for human PMNs; FIGURE 3A-C show chest x-rays of a human patient with adult respiratory distress syndrome before heparin therapy, immediately after therapeutic response and much later when heparin therapy had been stopped and his lung disease had worsened; and FIGURE 4 shows a graph of the Alveolar-arterial gradient for oxygen ([A-a]0₂) in the patient of FIGURE 3 with adult respiratory distress syndrome treated with aerosolized heparin.

DETAILED DESCRIPTION OF THE INVENTION

The active receptor binding site of the IL-8 molecule resides on the alpha-helix at the carboxyterminal region of the molecule. Heparin inhibits IL-8 because the heparin binding region of IL-8 also resides on the carboxyterminal region (J.J. Oppenheim, et al. Annual

Review of Immunology (1991) 9:617-648). Once attached to IL-8 in this region, the long polysaccharide heparin polymer is placed in close proximity to create steric hindrance that effectively impairs binding of PMN and other cell surface receptors at the nearby receptor binding site. It would be expected that modifications and derivatives of heparin and other heparin-like sulfated polysaccharides would also have similar IL-8 blocking activity. When used clinically to block IL-8 activity and prevent chemotaxis of PMNs, heparin would not necessarily be expected to affect the cause of the primary disease process causing tissue injury. However, in blocking IL-8 biologic activity, heparin would favorably affect overall disease outcome by decreasing the signal for influx of activated inflammatory PMNs into injured tissues, thereby decreasing the overall picture of injury.

The toxicity of heparin is well understood. Its major side effect is anticoagulation of the blood, for which it is widely used therapeutically by intravenous administration. It is not absorbed into the systemic circulation orally and reaches appreciable blood levels to produce anticoagulation only after aerosolization of doses above 8 mg/kg (L.B. Jaques, et al. Lancet (1976) 2:1157- 1161). Thus, one can administer IL-8 blocking doses of heparin by intrapulmonary aerosolization without causing significant undesirable systemic anticoagulation. To decrease the risk of toxicity even further one can modify heparin in a number of ways, including selective and partial desulfation, chemical over-sulfation, acetylation free hydroxyl groups with acetic, succinic and other carboxylic acid anhydrides, and esterification of the carboxylate groups of heparin. All of these methods have been reported to decrease the anticoagulant activity of heparin but would not necessarily affect the ability of heparin to inhibit IL-8. Used in this manner, heparin would be most useful for the treatment of respiratory diseases such as chronic bronchitis, adult respiratory distress syndrome (ARDS) and cystic fibrosis by administration to the respiratory tree directly as an aerosol. Heparin prepared from porcine intestinal mucosa is preferred over that from bovine lung because of the risk of thrombocytopenia from the latter form of commercially available heparin. A convenient commercial source of porcine intestinal mucosal heparin is Scientific Protein Laboratories of Waunakee, WI. A dose of from about 25 to 100 mg of heparin dissolved in 3 ml of sterile isotonic saline is aerosolized into the lung about every 4 to 12 hours using any common clinically available aerosol device (such as the DeVilbiss or Acorn nebulizers) attached to a positive pressure source (either compressor or compressed air or oxygen) to generate aerosols of particles less than 10 microns mass median diameter. Ideally, preservative free heparin should be used to avoid airway reactivity frequently associated with commonly used antibacterial and preservative ingredients. To this end, the heparin can be packaged in unit dose vials designed for a single dose administration and containing 50 mg heparin in 1 ml of isotonic sterile saline or 100 mg heparin in 2 ml of isotonic sterile saline. These vials can then be autoclaved to insure sterility. The pH of the final product should be adjusted from 6.5 to 7.4 (preferably 7.0) for compatibility with the airway and to prevent bronchospasm and direct injury from administration of solutions that are acid or alkaline with respect to the airway environment. The vials should be of Type 1 borosilicate glass, a material with which heparin and other sulfated polysaccharides are compatible. The vials should also have a "flip-tear" rubber seal for ease of opening. The final product can contain a flavoring such as peppermint to improve patient acceptance of the product.

In order to facilitate a further understanding of the invention, the following examples primarily illustrate certain more specific details thereof.

Example I demonstrates the potent activity of heparin as an inhibitor of IL-8 mediated chemotaxis of human PMNs. Example II shows the therapeutic benefit of using aerosolized heparin to block IL-8 mediated chemotaxis of PMNs, thereby suppressing lung inflammation in acute lung injury.

EXAMPLE I Inhibition of IL-8-induced Human PMN Chemotaxis bv Heparin The inhibition of IL-8 by heparin was measured by studying the ability of heparin to inhibit IL-8 induced chemotaxis of human PMNs. Chemotaxis was performed using a 48 well microchemotaxis chamber. Chemotaxis is assessed by quantitating movement of PMNs through a filter in response to a stimulus on the opposite side. Chemoattractant solutions IL-8 (1 microgram) and N-formyl- methionyl-leucyl-phenylalanine (FMLP, 10"⁸ M) were loaded into wells of the bottom plate, then covered by a 5 micrometer Millipore filter that was sealed by a silicone gasket and the top plate. Human PMNs harvested in standard fashion by dextran sedimentation and Ficoll- Hypaque centrifugation were suspended in buffer (135 mM NaC1 , 4.5 mM KC1 , 1.3 mM mgC1₂, 1.5 mM CaC1₂, 10 mM Hepes, 6 mM glucose, 1% human serum albumin) and loaded into the top wells (1.0 x 10^"5 PMNs/well). The chamber was incubated at 37 degrees C for 2 hours, and filters were stained with hematoxylin and eosin. The number of cells which had completely migrated through to the bottom side of the filter was counted in 10 random fields at 45x magnification using a 5x5 mm optical grid. PMN migration with each chemoattractant was normalized to the positive FMLP control. The effect of heparin on chemotaxis of human PMNs is shown in FIGURE 2. In these studies heparin was added to bottom wells with IL-8 or FMLP, but not to top wells with PMNs where it might have a direct membrane depressant effect on PMN chemotaxis unrelated to cytokine inhibition (Y. Matzner, et al. Thrombosis and Haemostasis (1984) 52:134-137). FIGURE 2 shows that both FMLP and IL- 8 are potent stimuli for PMN chemotaxis in this system (second pair of bars from left). Heparin 100 micrograms has no effect on random migration (third pair of bars from left). However in doses ranging from 0.1 to 100 micrograms, heparin markedly inhibits PMN chemotaxis from IL-8 (fourth through seventh pairs of bars from left). In contrast, heparin has no effect on chemotaxis from FMLP, demonstrating that the inhibition is specific for IL-8 and not a nonspecific inhibition of PMN chemotaxis in general. Because heparin is a polydisperse mixture of sulfated polysaccharides of varying chain lengths and degrees of sulfation, individual component parts and derivatives of heparin would also be expected to inhibit IL-8 activity.

EXAMPLE II Successful Treatment of Acute Lung Injury with Heparin To demonstrate the efficacy of inhibiting IL-8 activity therapeutically in man, we present the case of a 70 year old man who developed adult respiratory distress syndrome (ARDS) after massive aspiration of gastric contents. The patient's initial chest x-ray is shown in FIGURE 3A. Despite being placed on a mechanical ventilator and treatment with systemic antibiotics, aerosolized beta agonists and intravenous aminophylline, the patient remained seriously hypoxemic and had widespread lung inflammation. After informed consent was obtained from family, aerosolized heparin was initiated at a dose of 5,000 units (33 mg, or 0.33 mg/kg) every 4 hours to block IL-8 activity, prevent influx of PMNs into the lung and thereby hopefully save the patient's life. The heparin was administered by dissolving it in 3 ml of sterile 0.9% NaC1 and aerosolizing it into the lung using the nebulizer present in the ventilator circuit. The dose of heparin was then increased to 7,500 units (50 mg, or 0.5 mg/kg) every 4 hours the following day. Therapeutic effect was measured by the Alveolar-arterial oxygen gradient ([A-a]0₂), the difference between partial pressures of oxygen in the lung gas exchange unit (alveolus) and the arterial blood. Rising (A-a)0₂ gradients indicate a widening difference between the partial pressures of inhaled oxygen and oxygen in arterial blood caused by worsening gas exchange from diffuse lung injury, and falling gradients indicate improvement in gas exchange and lung injury.

FIGURE 4 shows that the (a-a)0₂ gradient fell dramatically and progressively with the initiation of aerosolized heparin therapy. In parallel with the improvement in gas exchange, there was a marked improvement in lung inflammation on the patient's chest x- x-ray (FIGURE 3B). Sixty hours after aerosolized heparin was begun, the treating physician was requested by hospital administration to stop aerosolized heparin because the treatment was considered unconventional. Because of the prolonged (up to 6 days) lung residence time of intrapulmonary heparin (L.B. Jaques, et al. Lancet (1976) 2:1157-1161; J. Mahadoo, et al. Artery (1980) 7:438-447), the therapeutic effect of heparin aerosol persisted for several more days, during which (A-a)0₂ fell 270% from a pre-treatment level of 606 to a post-treatment nadir of 223 mm Hg. However, after heparin therapy was stopped the patient's lung disease gradually worsened, his lung inflammation returned visibly on x-ray (FIGURE 3C), his (A-a)0₂ gradient increased progressively back to 606 mm Hg (FIGURE 4) and he died of respiratory failure.

No complications occurred from aerosolized heparin therapy in this patient. The activated partial thromboplastin time, a measure of systemic anticoagulation from heparin, remained at 35 to 37 seconds before, during and after heparin aerosolization. The platelet count remained normal. There was no evidence of intrapulmonary hemorrhage or blood in tracheal secretions. These observations suggest that the effect of aerosolized heparin in the doses used was localized primarily to the lung, without systemic absorption sufficient to cause anticoagulation or complications thereof.

The appended claims set forth various novel and useful features of the invention.

Claims

WHAT IS CLAIMED IS:

Claim 1. A medicament for the inhibition of the cytokine interleukin-8 comprising a treatment effective amount of the sulfated polysaccharide heparin, its components and derivatives, and heparin-like polysaccharides, their components and derivatives.

Claim 2. The medicament of Claim 1 wherein said medicament is administered by aerosolization.

Claim 3. The medicament of Claim 1 wherein the ratio of heparin to interleukin-8 is greater than about 0.1 and less than about 5.0.

Claim 4. The medicament of Claim 1 including a physiologically acceptable carrier.

Claim 5. The medicament of Claim 1 wherein said carrier is selected from the group consisting of physiologically buffered saline, isotonic saline and normal saline.

Claim 6. The medicament of Claim 1 wherein said medicament and carrier have a pH of greater than or equal to 6.5 and less than or equal to 7.4.

Claim 7. A method for inhibiting interleukin-8 in humans comprising administering a treatment effective amount of heparin and heparin-like polysaccharide of claim 1 to a human.

Claim 8. The method of Claim 7 wherein said treatment effective amount is administered by aerosolization.

Claim 9. The method of Claim 7 wherein the ratio of heparin to interleukin-8 is greater than about .1 and less than about 5.0.

Claim 10. The method of Claim 7 including mixing said heparin with a physiologically acceptable carrier.

Claim 11. The method of Claim 7 wherein said carrier is selected from the group consisting of physiologically buffered saline, isotonic saline and normal saline.

Claim 12. The method of Claim 7 wherein said medicament and carrier have a pH of greater than or equal to 6.5 and less than or equal to 7.4.