SE468626B - APPLICATION OF A PEROXIDIFOSPHATE PHOTO SOCIETY FOR INHIBITATION OF HYPOTENSIVE SHOCK AND LOCAL BENRESORPTION - Google Patents

Description

OS O \ IL 'o \ 2 tion: OOOHH phosphatases H H20 X -OPOOPO ------- 9-OOPO ------> HO + P0 4 III 2 2 4 O 0 O- where X is a nontoxic pharmaceutically acceptable cation or terminates an organic ester moiety. Phosphatase for the degradation of peroxide diphosphate 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, i.e. it also occurs outside the body of a warm-blooded animal. However, which is important, this reaction also occurs in the presence of phosphatase, when warm-blooded mammals are treated with PDP in accordance with the present invention. It is desirable to provide a regimen with which treatment is continued until the endotoxins are inactivated.

An object of the present invention is to inactivate endotoxins and thereby inhibit their toxic effects, such as inflammation, bone resorption and hypotensive shocks. Other objects of the present invention will become apparent from the following description.

In accordance with certain embodiments of the invention, it relates to a method of inhibiting hypotensive shock and local bone resorption caused by bacterial endotoxin, and comprises binding a non-toxic water-soluble pharmaceutically acceptable peroxyphosphate compound in contact with endotoxin to effect inactivation of said bacterium. 1/4:.

Ch CO CH PJ (JN A procedure for detecting inactivation of endotoxin is by overcoming the induction of the generation of a factor that is chemotactic to polymorphonuclear leukocytes, hereinafter referred to as "PMN". Boyden's chemotaxis method, according to which white blood cells from a rabbit are attracted (chemotaxis) by endotoxin-induced factor generated in the area In Boyden's method, when a bacterial endotoxin-lipopolysaccharide is incubated with a serum from a mammal, the following occurs: incubated Serum and endotoxin ----- -achemotactic factors for PMN at body temperature for 1 hour The chemotaxis phenomenon is studied using Boyden's chamber as described by Cates et al, "Modified Boyden Chamber Method for Measuring PMN Chemotaxis" in Leukocyte Chemotaxis, Methods, Physiology and Clinical Application, published by Gallin and Quie, Raven Press, New York, 1978, pages 67 - 71. When endotoxin induces chemotaxis as according to the present invention. According to the invention, the percentage of inhibition can be quantified using Boyden's chemotaxis test.

Endotoxin material can be introduced into the body of a warm-blooded animal by its presence on the surfaces of gram-negative microorganisms, such as Actinobacillus actinomycetemcomitens (Aa), Escherichia coli (E.coli), Bacteroides melanenogenicus (B. mel) and Salmonella typhi (S. typhi).

Oral endotoxin isolated from A.a. is toxic to alveolar bones. Non-oral endotoxin purified from E. coli may prove fatal to the host.

Other known procedures for inhibiting endotoxin formation are performed using resorption in a bone culture medium, and a chicken embryo lethality test may also be performed. used.

The toxic reaction is effectively inhibited by treating endotoxin in situ in a warm-blooded host with an effective inhibitory amount of a non-toxic water-soluble pharmaceutically acceptable peroxide phosphate compound. The peroxyphosphate reacts with the endotoxin in the body as an intact molecule, inactivating the peroxyphosphate compound. Since the endotoxin is inactivated, it is obvious that the endotoxin reacts with the peroxide diphosphate.

In general, approx. 0.1 - 7% peroxide diphosphate compound in a pharmaceutical carrier, as in solution, effective in a prescribed dose of approx. 0.2 - 14 mg per kg body weight. The inhibition efficacy can be ascertained by reduced endotoxin effect and quantified on the basis of inhibited chemotaxis to PMN.

Typical non-toxic water-soluble pharmaceutically acceptable peroxide phosphate compounds are the alkali metal salts (eg lithium, sodium and potassium), alkaline earth metal salts (eg magnesium, calcium and strontium) and zinc, tin and quaternary ammonium salts, as well as Cl C12 alkyl, adenylyl, guanylyl, cytosylyl and thymylyl esters. Alkali metal, especially potassium salt, is preferred among the inorganic cations. The tetrachotassium peroxide diphosphate is a stable, odorless, finely divided, free-flowing, white, non-hygroscopic crystalline solid with a molecular weight of 346.35 and an active oxygen content of 4.6%.

F1 Tetra potassium peroxide diphosphate is 47-51% water soluble at 0-61 ° C, but insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethylformamide, dimethylsulfoxide and the like. A 2% aqueous solution has a pH of approx. 9.6 and a saturated solution thereof a pH of 468 626 approx. 10.9. A 10% solution in water at 25 ° C showed no active oxygen loss after 4 months and at 50 ° C a%% solution showed a loss of active oxygen of 3% for 6 months.

Among the organic compounds, those which exhibit hydrophobic properties such as C 1 -C 12 alkyl radical and those which facilitate rapid uptake of the peroxide diphosphate moiety by the cells, such as adenylyl, guanylyl, cytosylyl and thymylyl esters are preferred.

The peroxydiphosphate compound can be administered orally or systemically to inhibit endotoxins in the oral cavity or other parts of the body.

Pharmaceutical carriers suitable for oral administration are coated tablets composed of materials which resist the degradation of gastric acids at the pH prevailing in the stomach (ca. 1 - 3), as peroxide diphosphate would be inactivated by such gastric acids. Instead, the carriers containing tableted granules of the peroxydiphosphoric acid salt in the form of solids dissolve the intestinal fluids, which have a higher pH (about 5.5 - 10) and do not inactivate the peroxide phosphate but leave it to be exposed to the enzymatic action of phosphatase which is present in humans or other warm-blooded animals. A suitable tablet coating solution is composed of a fatty acid ester such as N-butyl stearate (typically about 40-50 and preferably about 45 parts by weight), wax such as carnuba wax (typically about 15-25 and preferably about 20 parts by weight), fatty acid such as stearic acid (typically about 20-30 and preferably 25 parts by weight) and cellulose esters such as cellulose acetate phthalate (typically about 5-15 and preferably about 10 parts by weight) and organic solvent (typically about 400-900 parts). Other suitable coating materials include shellac and copolymers of maleic anhydride and ethylene compounds such as polyvinyl methyl ether. Dy- w 468 626 equal coatings are distinct from tablets which decompose in the oral cavity in which the tablet material typically contains approx. 80 - 90 parts by weight of mannitol and approx. 30 - 40 parts by weight of magnesium stearate.

Tableted granules of the peroxydiphosphate salt are formed by mixing approx. 30 - 50 parts by weight of the peroxyphosphate salt with approx. 45 - 65 parts by weight of a solid polyhydroxy sugar such as mannitol and wetting with approx. 20 to 35 parts by weight of a solution of a polyhydroxy sugar compound such as sorbitol, sifting to the right size, mixing with approx. 20 to 35 parts by weight of a binder such as magnesium stearate and compressing the granules into tablets with a tablet forming machine.

The tableted granules are coated by spraying a foam 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 any special protective coating.

An effective dose of peroxide diphosphate at a prescribed course, when administered by oral administration, is approx. 0.1 - 6 grams per kg body weight daily and when the administration is systemic such as by intramuscular, intraperitoneal or intravenous injection, the dosage is approx. 0.1 - 2 grams per kg body weight daily.

Physiologically acceptable pyrogen-free solvents are suitable carriers for use in conventional systemic administration. Saline solution buffered with phosphate to a physiological pH of approx. 7 - 7.4 is the preferred carrier for systemic administration. Such solvents differ from water-humectant vehicles typically used in dentifrices. Such a solution is typically prepared by sterilizing deionized distilled water, checking to ensure pyrogen freedom using Limulus amebocyte lysis certificate (LAL) described by Tsuji et al in 1 "Pharmaceutical Manufacturing", October 1984, pages 41, and then adding a phosphate buffer (pH eg 8.5 - 10) prepared in pyrogen-free sterile water and approx. 1 - 100 mg peroxide diphosphate derivatives and sodium chloride to a concentration of approx. 0.5 - 1.5% by weight. The solution can be packaged in test tubes for use after re-sterilization by passage through a microporous filter. Alternatively, other solutions such as Ringer's solution containing 0.86% by weight of sodium chloride, 0.03% by weight of potassium chloride and 0.033% by weight of calcium chloride may be used.

The following examples illustrate the ability of the peroxide diphosphate compound (PDP) to inhibit chemotoxic induced by endotoxin-generated factor in serum and to inhibit endotoxin toxicity to bone.

Example 1 PMN is obtained from the peritoneal cavities of adult white New Zealand rabbits 12 hours after intraperitoneal injections of 200 ml of solution containing 0.2% glycogen in sterile isotonic saline (0.85% NaCl). The cells (PMN) are purified from the exudate obtained from the rabbit's peritoneal cavity and purified as described by Taichman et al (Arch. Oral Biol. 21 p. 257, 1976). Bacterial endotoxin purified from E. coli obtained from Associates of Cape Cod Incorporated, Woods Hole, Maine, is pretreated with various concentrations of PDP (tetrachotassium salt) at 37 ° C for 1 hour. The chemotaxis test is then performed with treated and untreated endotoxins using Boyden's chamber as described above. The data obtained are summarized in Tables 1 and 2. 468 626.

Table 1 Chemotaxis - average number of mig-% reduction Treatment of PMN + SD in chemotaxis 1. Control011 ++ 139 1 4.2 2. Serum +++ and 1 ng / m1 endotoxin 343.0 in 37.6 3. 0.5% PDP and ++ 142.5 in 12.0 4. Endotoxin (1 ng / ml) pretreated with 0.5% PDP 0Ch + Serum serum ++ 100.0 in 18.4 - 44% compared to 2. Endotoxin (0.5 ng / ml) pretreated with 0.5% PDP and serum ++ 154.0 in 2.8 - 56% compared to 2 6. Endotoxin (0.25 ng / ml) pretreated with 0, 5% PDP and serum ++ 138.5 3 2.8 - 60% compared to 2 + SD = standard deviation ++ medium = Earl's solution containing 10% bovine serum albumin +++ serum = human serum (normal) The results in Table 1 indicate that endotoxin, as expected, induces a strong release of a factor that increased chemotaxis of PMN (treatment No. 2), PDP (0.5%) had no effect on PMN (No. 3) and endotoxins pretreated with PDP significantly reduced the chemotactic activity of the toxin (treatments Nos. 4, 5 and 6). These data indicate that CN treatment with endotoxin with PDP inactivates the biological effect of the toxin.

Example 82 Table 2 shows data obtained by means of additional Boyden Chamber tests as in Example 1. PDP is used in the form of the tetra potassium salt.

Table 2 Chemotaxis - average number of mig-% reduction Treatment concerning PMN + S.D. in chemotaxis 1. Control medium (as in Example 1) 136.5 in 6.3 2. Endotoxin 1 ng / ml and serum + 329.0 in 39.5 3. PDP 0.5% and serum + 139.5 3 4.9 Endotoxin (1 ng / ml) pretreated with 0.58 PDP and serum * 188.0 in 9.8 -43.08. Endotoxin (1 ng / ml) pretreated with 0.25% PDP and serum * 206.5 in 17.6 -378 6. Endotoxin (1 ng / ml) pretreated with 0.18 PDP and serum * 231.0 in 17, 6 -30% + serum according to Example 1.

The data in the table above show that an effective concentration of PDP of at least as little as 0.1% deactivates the biological activity of the endotoxin.

OK 03 Ch FJ C \ Example 3 Effects of PDP on endotoxin activity in bone culture system The test in which an endotoxin isolated from Acintobacillus actinomycetemcomitans Y4 (AAY4) induces the resorption of bone in a bone culture system (Kiley and Holt, Infect. Immun.: 362-373, 1980 ) was used to determine whether PDP inactivates the bone resorption activity of endotoxin from Y4. Rat fetal bone culture as described by Raisz, J. Clin.

Invest. 44: 103-116, 1965, was prepared by injecting rats with 45 CaCl 2 on the eighteenth day from conception. The rats are then sacrificed on the nineteenth day and the radii and ulnae from embryos with their cartilage ends are removed and placed for cultivation in BGJ medium (Gibco, Buffalo, New York) at 37 ° C with 5% CO 2. The medium is supplemented with 5% warm (57 ° C for 3 hours) fetal calf serum. The legs are placed four and four in a compartment in 24-compartment plates (Nunc, Gibco) containing 0.5 ml of medium per compartment. Release of av zav 45Ca to the culture medium from bone incubated in the presence of a testagen is compared with the release from bone incubated in control medium, and the results for 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 tetrachotassium salt at 37 ° C. Excess PDP is removed through dialysis membranes (3500 molecular weight maximum). This allows non-reactive PDP to diffuse out while retaining endotoxin having a molecular weight in excess of 3500 retained on the inside of the bag. The data obtained are summarized in Table 3. [> O \ CO (H IJ Ch ll Table 3 45Ca Released Test / + S.D.

Number Treatment rats% control Significance H- 1. Control 6 30.11 1.98 --- --- 2. lolpq / ml endotoxin 6 H- ss, 4e 4.71 2, s7io, 16 97% compared to l 3 10 / ng / ml endotoxin pretreated with 100 mcg PDP 6 H-7s, 47 2.9 2.61io, 1 not significant 4. lojug / ml endotoxin pretreated with 1000 mcg PDP 6 1, o6io, 14 31 , 98 in 4.27 97% compared to 2 Data show that endotoxin from Y4 AA significantly induced bone resorption (compare 1 with 2), while a pretreatment of the endotoxin with 1000 mcg / ml PDP (0.1%) effectively inhibits the bone resorptive activity of endotoxin.

The results of Examples 1-3 are representative of the effects of PDP tetracali metal 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 C 1 -C 12 alkyl PDP. salts and other organic PDP compounds, in particular including adenylyl, guanylyl, cytosylyl and thymylyl esters and quaternary ammonium PDP salts in inhibiting endotoxin-induced chemotaxis factor in serum and inhibiting endotoxin toxicity to bone in rats, rabbits and mammals in general.

Claims (6)

10 15 20 25 J; C'J \ CO O \ I J CA A3 P a t e n t k r a v Use of a non-toxic, water-soluble, pharmaceutically acceptable peroxide phosphate compound in the form of an alkali metal, zinc, tin or quaternary ammonium salt or a C 1 -C 12 alkyl, adenylyl, guanylyl, cytosylyl or thymylyl ester for the manufacture of a medicament for inhibiting hypotensive shock and local bone resorption caused by bacterial endotoxins. Use according to claim 1, characterized in that the peroxydiphosphate compound is present in an amount of about 0.1 - 7% in a pharmaceutical carrier. Use according to any one of claims 1 and 2, characterized in that the peroxydiphosphate compound is present in tableted granules with a coating thereon, which does not degrade during passage through the stomach of warm-blooded mammals and which is dissolved by intestinal fluids. with a pH of 5 - 10. Use according to any one of claims 1 - 3, known as potassium salt. of the presence of the peroxide diphosphate compound Use according to any one of claims 1 - 3, characterized as C1-C12-alkyl ester. of the presence of the peroxide diphosphate compound Use according to any one of claims 1 to 3, characterized as adenylyl, guanylyl, cytosylyl or thymylyl ester. of the presence of the peroxide diphosphate compound