GB2180451A - Inactivation of bacterial endotoxins - Google Patents

Abstract

A non-toxic, water-soluble, pharmaceutically acceptable peroxydiphosphate compound is used to inhibit hypotensive shock and localised bone resorption caused by bacterial endotoxins. Preferred compounds are alkali metal, zinc, tin or quaternary ammonium salts or C1-12 alkyl, adenylyl, guanylyl, cytosylyl or thymylyl esters.

Description

SPECIFICATION Inactivation of Bacterial Endotoxins The present invention relates to a method for inhibiting hypotensive shock and localised bone resorption caused by bacterial endotoxin which comprises introducing a non-toxic water-soluble, pharmaceutically acceptable peroxydiphosphate compound into contact with endotoxin to cause inactivation of the said bacterial endotoxin.

Endotoxins are complex macromolecules containing lipid, carbohydrate and protein. They are mainly found in the surface of gram negative organisms and are usually referred to as lipopolysaccharides. These macromolecules are toxic to the host and can be fatal. For instance, they can cause severe hypotensive shocks, and also elicit a variety of toxic reactions in the body including bone resorption. In the mouth, endotoxins have been implicated as a major factor in the inflammation of gum tissues and in localized bone loss such as alveolar bone loss.

Theoretically, compounds which release oxygen could inactivate endotoxins. However, due to the quickness with which many oxygen-evolving compounds release oxygen, they generally have little effect in controlling endotoxin growth. Those compounds which release oxygen more slowly could control endotoxin effect. However, their effectiveness is generally limited in that the conditions of oxygen-release do not correspond to the conditions prevailing in the body.

As described in GB Patent Application No. 8515105, filed 14th June, 1985, warm blooded mammals, such as from rodents, up to and including humans and alkaline phosphatase or acid phosphatase in their bodies. Peroxydiphosphate compounds possess the property of slow release of oxygen. The amount of oxygen which they release is one-tenth the amount released by hydrogen peroxide. Only about 50% of their active oxygen is released in 20 hours at 25"C in the presence of alkaline phosphatase or acid phosphatase.

Peroxidiphosphate compounds (PDP) release hydrogen peroxide slowly in the presence of phosphatase enzymes in accordance with the following equation:

wherein X represents a non-toxic pharmaceutically acceptable cation or completes an organic ester moiety.

Phosphatase to break down the peroxydiphosphate is present in saliva as well as in plasma, intestinal fluids and white blood cells.

It has been observed that bacterial endotoxin also reacts with intact PDP. This reaction occurs independently of the presence of phosphatases; that is, it occurs outside of the body of a warm blooded animal, too. However, quite importantly, even in the presence of phosphatase, the reaction also occurs when warm blooded mammalian animals are treated with PDP in accordance with the present invention. It is desirable to provide a regimen whereby treatment continues until endotoxins are inactivated.

A procedure for evidencing inactivation of endotoxin is by overcoming induction of generation of a factor which is chemotactic to polymorphonuclear ieukocytes, hereinafter called "PNM". Such a factor can be assessed in accordance with the Boyden chemotaxis method wherein white blood cells of a rabbit are attracted (chemotaxis) by endotoxin induced factor generated in the area. In the Boyden method, when a bacterial endotoxin lipopolysaccharide is incubated with a serum from a mammalian, what occurs is.

Incubated at Serum and endotoxin

chemotactic factors for PMN body temperature for 1 hr.

The chemotaxis phenomenon is studied using Boyden chambers as described by Cates et al, "Modified Boyden Chamber Method for Measuring PMN Chemotaxis" in Leukocyte Chemotaxis, Methods, Physiology and ClinicalApplication, edited by Gallin and Quie, Raven Press, N.Y., 1978, pages 67-71. When endotoxin induces chemotaxis as in the present invention, the percentage of inhibition can be quantified using the Boyden chemotaxis text.

Endotoxin material can be introduced into the body of a warm blooded animal through its presence in the surfaces of gram negative microorganisms, such asActinobaccilus actinomycetemcomitens (A.a.), Escherichia coli (E. coil), Bacteroides melanenogenicus (B. mel) and Salmonella typhi (S. typhi).

Oral endotoxin isolated from A.a. is toxic to avelolar bone. Non-oral endotoxin purified from E. coli can prove fatal to the host Other known procedures for inhibiting endotoxin formation are done using resorption in a bone culture medium; a chick embryo lethality test can also be used.

The toxic reaction is effectively inhibited by treating endotoxin in situ in a warm blood host with an inhibiting-effective amount of non-toxic, water-soluble pharmaceutically acceptable peroxy-diphosphate compound. The peroxydiphosphate reacts with the endotoxin in the body as an intact molecule, while inactivating the peroxydiphosphate compound. Since the endotoxin is inactivated, it is apparent that endotoxin reacts with the peroxydiphosphate.

Generally, about 0.17% of peroxydiphosphate compound in a pharmaceutical carrier, such as in solution is effective in a regimen dosage of about 0.2-14 mg per kg body weight. Inhibition effectiveness can be evidenced by reduced endotoxin effect and is quantified on the basis of inhibited chemotaxis to PMN.

Typical non-toxic, water-soluble pharmaceutically acceptable peroxydiphosphate compounds are the alkali metal salts (e.g. lithium, sodium and potassium), alkaline earth metal salts (e.g. magnesium, calcium and strontium) and zinc, tin and quaternary ammonium salts, as well as C112 alkyl, adenylyl, guanylyl, cytosylyl and thymylyl esters. Alkali metal, particularly potassium salt is preferred from among the inorganic cations. The tetrapotassium peroxydiphosphate is a stable, odourless, finely divided, freeflowing, white non-hygroscopic crystalline solid having a molecular weight of 346.35 and an active oxygen content of 4.6%.

Tetrapotassium peroxydiphosphate is 47-51% water-soluble at 00--610C, but insolubie in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethyl formamide, dimethyl sulphoxide, and the like. A 2% aqueous solution has a pH of about 9.6 and a saturated solution thereof a pH of about 10.9. A 10% solution in water at 25"C showed no active oyxgen loss after four months; and at 50"C a 10% solution showed an active oxygen loss of 3% in 6 months.

From among the organic compounds those providing hydrophobic properties such as C1 -12 alkyl radical and those which facilitate the rapid uptake of peroxidiphosphate moiety by the cells, such as adenylyl, guanylyl, cytosylyl, and thymylyl esters are preferrred.

Peroxydiphosphate compound may be administered orally or systemically to inhibit endotoxins in the oral cavity or other parts of the body.

Pharmaceutical carriers suitable for oral ingestion are coated tablets composed of material which resists breakdown by gastric acids in the stomach pH (about 1-3) since peroxydiphosphate would be inactivated by such gastric acids. Rather, the carriers, with tabletted granules of the peroxydiphosphoric acid salt solid material therein, are dissolved by intestinal fluids which have a higher pH (about 5.510) and do not inactivate the peroxydiphosphate, leaving it subject to enzymatic action by phosphatase present in humans or other warm blooded animals.A desirable tablet coating solution is composed of a fatty acid ester such as N-butyl stearate (typically about450, preferably about 45 parts by weight), wax such as carnauba wax (typically about 1 25, preferably about 20 parts by weight), fatty acid such as stearic acid (typically about 2030 parts, preferably 25 parts by weight) and cellulose ester, such as cellulose acetate phthalate (typically about 5--15, preferably about 10 parts by weight) and organic solvent (typically about 400900 parts. Other desirable coating materials include shellac and copolymers of maleic anhydride and ethylenic compounds such as polyvinyl methyl ether.Such coatings are distinct from tablets which are broken down in the oral cavity in which the tablet material typically contains about 8090 parts by weight of mannitoi and about 30 < 0 parts by weight of magnesium stearate.

Tabletted granules of the peroxydiphosphate salt are formed by blending about 3050 parts by weight of the peroxydiphosphate salt with about 4565 parts by weight of a polyhydroxy sugar solid such as mannitol and wetting with about 2035 parts by weight of a polyhydroxy sugar compound solution such as sorbitol, screening to size, blending with about 2035 parts by weight of a binding agent such as magnesium stearate and compressing the granules into tablets with a tablet compressing machine. The tabletted granules are coated by spraying a form of a solution of the coating material thereon and drying to remove solvent. Such tablets differ from dental tablets which are typically compressed granules without a special protective coating.

An effective dosage of administration of peroxydiphosphate with a prescribed regimen, when administration is by oral ingestion, is about 0.1-6 mg per kg of body weight daily; when administration is systemic, such as by intramuscular, intraperitoneal or intravenous injection, the dosage is about 0.1-2 mg per kg of body weight daily.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in the art-recognised manner for systemic administration. Saline solution buffered with phosphate to a physiological pH of about 7 to 7.4 is the preferred carrier for systemic administration. Such solvents are distinct from water-humectant vehicles typically used in dentifrices.Such solution is typically prepared by sterilising deionised distilled water, checking to ensure non-pyrogenicity using the Limulus amebocyte lysate (LAL) test described by Tsuji et al in "Pharmaceutical Manufacturing", October, 1984, pages 3541, and then adding thereto a phosphate buffer (pH e.g. about 8.510) made in pyrogen free sterile water and about 1-100 mg peroxydiphosphate compound derivative and sodium chloride to a concentration of about 0.51.5% by weight. The solution can be packed in viais for use after being resterilised by passing through a micropore filter. As alternatives, other solutions such as Ringer's solution containing 0.86% by weight sodium chloride, 0.03% by weight potassium chloride and 0.033% by weight calcium chloride may be used.

The invention may be put into practice in various ways and a number of specific embodiments will be described to illustrate the invention with reference to the accompanying examples which iliustrate the ability of peroxidiphosphate (PDP) compound to inhibit chemotaxis induced by endotoxin generated factor in serum and to inhibit endotoxin toxicity to bone.

EXAMPLES 1Ato 1F PMN are obtained from the peritoneal cavities of adult New Zealand white rabbits 12 hours after intraperitoneal injections of 200 ml of solution containing 0.2% glycogen in sterile isotonic saline (0.85% NaCI). The cells (PMN) are purified from the exudate obtained from the rabbit peritoneal cavity and purified as described byTaichman et al. (Arch. Oral Biol. 21 page 257, 1976). Bacterial endotoxin purified from E. Coli obtained from Associates of Cape Cod Inc. Woods Hole, Maine, is pre-treated with different concentrations of PDP (tetrapotassium salt) at 37"C for 1 hour. The chemotaxis assay is then run with treated and untreated endotoxins using Boyden Chambers as described above. The data are summarised in Tables 1 and 2.

TABLE 1 Chemotaxis mean No. of % Reduction Example Treatment PMN migrating in chemotaxis 1A Control (Serum A2) 139+4.21 1B Serum B3 and 1 343.0*36.7 nanogram/ml endotoxin 1C 0.5% PDPand 142.5*12.0 serum3 1D Endotoxin (1 ng/ml) 188.0+18.4 -44% compared to 1B pretreated with 0.5% PDP and serum2 1E Endotoxin 154.0*2.8 -56% compared to 1B (0.5 ng/ml pre treated with 0.5% PDP and serum A2 1F Endotoxin 138.5*2.8 -60% compared to 1B (0.25 ng/ml) pre treated with 0.5% PDP and serum A2 Notes on Table 1 1 This is the standard deviation (S.D.) 2 Serum A=Earl's solution containing 10% bovine serum albumin 3 Serum B=human serum (normal).

The results in Table 1 indicate that endotoxin as expected, induces a great release of a factor which increased chemotaxis of PMN (Example 1 B treatment); PDP (0.5%) has no effect on PMN (Example 1C); and endotoxins pretreated with PDP, have the chemotactic activity of the toxin significantly reduced (Examples 1D, E and 1 F). These data indicate that a treatment of endotoxin with PDP, deactivates the biological effect of the toxin.

EXAMPLES 2Ato 2F Table 2 shows data obtained with further Boyden Chamber Tests as in Examples lAto 1 F. PDP is employed as the tetrapotassium salt TABLE 2 Chemotaxis mean No. of PMN % Reduction Example Treatment migrating in chemotaxis 2A Control medium 136.5~6.3 (as in Ex. 1A) 2B Endotoxin 1 ng/ml 329.0*39.5 and serum3 2C PDP0.5% and 139.5*4.9 serum 2D Endotoxin (1 ng/ml) 188.0+9.8 -43.0% pretreated with 0.5% PDP and serum1 2E Endotoxin (1 ng/ml) 206.5*17.6 -37% pretreated with 0.25% PDP and serum3 2F Endotoxin (1 ng/ml) 231.0+17.6 -30% pretreated with 0.1% PDP and serum3 Notes on Table 2 1as in Table 1 3 as in Table 1.

The data in the above Table 2 shows that PDP is effective at a concentration at least as low as 0.1% to de-activate the biological activity of endotoxin.

EXAMPLE 3 This shows the effects of PDP on endotoxin activity in a bone culture system.

The test is one in which an endotoxin isolated from Actinobacillus actinomycetemcomitans Y4 (AAY4) induces the resorption of bone in a bone culture system (Kiley and Holt, Infect Immun. 30:362-373, 1980).

The test is used to assess whether PDP deactivates the bone resorptive activity of endotoxin from Y4. Foetal rat bone culture as described by Raisz, J. Clin. Invest. 44:103--116, 1965, is prepared by injecting rats with 45CaCI2 on the 18th day of gestation. The rats are then sacrificed on the 19th day, and radii and ulnae of the embryos, with their cartilagenous ends, are removed and placed for culturing in BGJ medium (Gibco, Buffalo, NY) at 37"C with 5% CO2. The medium is supplemented with 5% heated (57"C for 3 hours) foetal calf serum. Bones are placed four to a well in twenty four well dishes (Nunc, Gibco) containing 0.5 ml of medium per well.The release of 45Ca into the culture media from bone incubated in the presence of a test agent is compared with the release from bones incubated in control media, and the results of bone resorption are expressed as a ratio.

Endotoxin from AAY4 is obtained from the University of Pennsylvania, School of Dentistry. AAY4 endotoxin is treated with different concentrations of PDP tetrapotassium salt at 37"C. The excess PDP is removed by dialysis membrane (3500 mol. wt. maximum). This permits unreactive PDP to diffuse out while the endotoxin having molecular weight greater than 3500 is retained inside the bag. Table 3 summarises the data.

TABLE 3 No. of Testy Example Treatment Rats % 45Ca released Control Significance 3A Control 6 30.11+1.981 3B 10 ug/mI endotoxin 6 85.46+4.71 2.87*0.16 98% compared to 3A Y4AA 3C 101lg/mlendotoxin 6 78.47+2.9 2.61+0.1 notsignificantly pretreated with different from 3B 100 mcg PDP 3D 10 Cig/ml endotoxin 6 31.98+4.27 1.06+0.14 97% compared to 3B prated with 1000 mcg PDP Notes on Table 3 1 Table 1 The data show that endotoxin from Y4AA significantly induced bone resorption (compare 3A to 3B) while a pretreatment of the endotoxin with 100 mcg/ml of PDP (0.1 %) effectively inhibits the bone resorptive activity of the endotoxin.

The foregoing results in Examples 1-3 are representative of the effects of PDP tetrapotassium salt and other non-toxic water-soluble pharmaceutically acceptable PDP salts such as other alkali metal salts, alkaline earth metal salts, zinc salt and tin salt as well as C1~,2 alkyl PDP salts and other organic PDP compounds, particularly including the adenylyl, guanylyl, cytosylyl and thymylyl esters and quaternary ammonium PDP salts in inhibiting chemotaxis induced by endotoxin generated factor in serum and to inhibit endotoxin toxicitytobone in rats, rabbits and mammals in general.

Claims (12)

1. A non-toxic water-soluble, pharmaceutically acceptable peroxydisphosphate compound for use in inhibiting hypotensive shock and localised bone resorption caused by bacterial endotoxins.

2. Peroxydiphosphate compounds in the form of a salt of alkalimetal, zinc, tin or quaternary ammonium or C1 -12 alkyl, adenylyl, guanylyl, cytosylyl or thymylyl ester, for use in inhibiting hypotensive shock and localised borie resorption caused by bacterial endotoxins.

3. A composition comprising peroxydiphosphate compound present in an amount of about 0.17% in a pharmaceutical carrier.

4. A composition as claimed in Claim 3, in which the said peroxydisphosphate compound is present in tabletted granules having a coating thereon which is not broken down during passage through the stomach of a warm blood animal and which coating is dissolved by intestinal fluids having a pH of 510.

5. A composition as claimed in Claim 3, in which the said peroxydiphosphate compound is in a solution of non-pyrogenic distilled water and sodium chloride buffered with phosphate.

6. A composition as claimed in Claim 3,4 or 5 in which the peroxydiphosphate compound is present as a potassium salt.

7. A composition as claimed in Claim 3,4 or 5 in which the said peroxydisphosphate compound is present as a C,-,2 ester.

8. A composition as claimed in Claim 3,4 or 5 in which the said peroxydiphosphate compound is present as an adenylyl, guanylyl, cytosylyl or thymylyl ester.

9. The use of a non-toxic water-soluble, pharmaceutically acceptable peroxydiphosphate compound in the preparation of a medicament for use in inhibiting hypotensive shock and localised bone resorption caused by bacterial endotoxins.

10. The use of a non-toxic water-soluble pharmaceutically acceptable peroxydiphosphate compound in the preparation of a medicament for inactivation of bacterial endotoxins.

11. A method for inhibiting hypotensive shock and localised bone resorption caused by bacterial endotoxins, which comprises introducing a non-toxic water-soluble pharmaceutically peroxydiphosphate compound into contact with endotoxin thereby causing inactivation of said bacterial endotoxin.

12. A method as claimed in Claim 11 in which the said contact of the said peroxydiphosphate compound and the said endotoxin is in a warm blooded mammalian animal and the said peroxydiphosphate compound is introduced in a regimen dosage of about 0.2-14 mg/kg of body weight of the said warm blood mammalian animal.