CA1282004C

CA1282004C - Inactivation of bacterial endotoxins

Info

Publication number: CA1282004C
Application number: CA000516466A
Authority: CA
Inventors: Abdul Gaffar; Edward J. Coleman
Original assignee: Colgate Palmolive Co
Current assignee: Colgate Palmolive Co
Priority date: 1985-08-22
Filing date: 1986-08-21
Publication date: 1991-03-26
Anticipated expiration: 2008-03-26
Also published as: SE468626B; FR2586351B1; GB8620482D0; DE3627759A1; HK593A; DK168513B1; IT8648389A0; FR2586351A1; GB2180451B; DK402086D0; DK402086A; SE8603486L; SE8603486D0; SG108892G; NL8602140A; CH670046A5; GB2180451A; IT1196587B; BE905319A

Abstract

INACTIVATION OF BACTERIAL ENDOTOXINS
ABSTRACT
Bacterial endotoxins are inhibited by a non-toxic, water-soluble, pharmaceutically acceptable peroxy-diphosphate compound in contact with bacterial endotoxin.

Description

~azoo4 62301-1392 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 includiny 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 quicXness 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 commonly assigned Canadian Patent 20 Application No. 485,299, filed June 26, 1985 warm blooded mammals, such as from rodents, up to and including humans have 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 25C in the presence of alkaline phosphatase or acid phosphatase.

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~ Peroxydiphosphate compounds (PDP) release hydrogen il peroxide slowly in the presence of phosphatase enzymes in ¦l accordance with the following equation:

11 11 phosphatases It H O H202~Po4 X~ O~P-O-O-l-O- _~~0-0-~-0-O
wherein X is a non-to~ic pharmaceuticallY acceptable cation . or compl~tes &n organic ester moiety. Phosphata e to break down I
the peroxydiphosphate is present in saliva as well as in plas~aD¦

l intest~.nal fluids and wbite blood cell~.
1, It has been observed that bac~erial endot~xin also reacts with in~act PDP. This reaction occurs independen~ly of ~he l presence of phosphatases; that is, it occurs outside of ehe 1 body of a warm blooded animal to~. HowPver, qu~te importantly, even in the presence of phospha~a~e, ehe reaction al80 occurs when war~ blooded ma~ma~ian animals are trFated with PDP in accordance with the present invention. I~ ig ~e3irable to pro~ide a regimen ~hereby ereat~ent continues unt~l e~doto~ins are inactivated.
It i~ an ob~ect of this inYention to deactivate endo-toxins and thereby lnhlbit their to~lc effects, such a~
inflammation, bone resorption and hypotensivP sbocks.
Other ob~ects of thig invention will be apparent from consideration of the following specification.

~8~0~ 62301-1392 In accordance with certain of its objects this in~en-tion relates to a method for inhibiting hypotensive shock and localized bone resorption caused by bacterial endotoxin which comprises in-troducing a non-toxic water-soluble, pharmaceutical-ly acceptable peroxydiphosphate compound into contact with endotoxin to cause inactivation of said bacterial endotoxin.
The invention provides the use of a non-toxic water-soluble pharmaceutically acceptable peroxydiphosphate compound for inactivation of bacterial endotoxins.
A procedure for evidencing inactivation of endotoxin is by overcoming induction of generation of a factor which is chemotactic to polymorphonuclear leukocytes, hereinafter called "PMN". Such a factor can be assessed in accordance with the Boyden chemotaxis method wherein ~hite 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 ~ith 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 ~or Measuring PMN Chemotaxis" in L ukocyte_Chemotaxis, Methods, Physiology and Clinical Application, edited by Gallin and Quie, Raven Press, N.Y., 1978, pages 67 - 71. When endo-toxin induces chemotaxis as in the present invention, the percentage of inhibition can be quantified using the Boyden chemotaxis test.

~3 Endotoxin material can be introduced into tbe body of a warm bloodPd animal through its presence in the surfaces of gram negative microorganisms, such as Actinobacillus actinomy-cetemcomitens (A.a), Escherichia coli (E. ~oli), Bacteroides melanenogenicus (B. mel) and Salmonella typhi ~S. typhi).
Oral endotoxin isolated fro~ A. a. is toxic to avelolar bone. Non-oral endotoxin purif$ed from E. coli csn prove fatal to the host.
~ , Other known procedur~s for inhibiti~g endoto~in for-mation are doDe using res~rption in a ~one culture mediu~; a chlck embryo lethality test caD al~o be used.
The ~oxic reaction is effectively inhibited bv ~reatin~
endotoxin in situ in a warm blooded host wi~h an inhibiting-effective amount of non-toxic, water-soluble phgr~aceutlcally acceptable peroxvdiphosphate compound. The peroxydiphosphate reacts with the endoto~in in the body as an ntact ~oleculet while ~nacti~ating the pero-xydlpho~phate co~pouud. Since the endotoxin is inactivated, it is appare~t that e~dotoxin react~
with the pero~ydiphosphate.
Generally, about ~ of peroxydlpho~pha~e co~pound in a pharmaceutical carrier, such a~ ln solutlon ~9 effective ln a regimen dosage of about O . 2-14 ~g per kg body weight .
Inhibi~ion effectivenegs cgn be evidenced by reduced e~dotoxin effect and 15 quantified on the bagi~ o f inblblted chemotsxi~
to PMN.

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Typical non-toxic, water-soluble phar~aceutically acceptable pero~y~iphosphate compounds are the alkall metal salts (e.g. lithium, sodium and p~ta~sium)~ alkaline earth metal salts (e.g. magnesiu~, calcium and stronti~m~
and zinc, tin and q~aternary ammonium salts, as well as Cl 12 aIkyl, adenylyl, ~uanylyl, cytosy~yl and thy~ylyl esters.
Alkali metal, particularly potassium salt is preferred from among the inorganic cations. The tetrapotassium pero~ydiphosphate is a stable~ odorless, finely divided, free-flowing, white non-hygroscopic crystalline sol~d ha~lng a molecular weight of 346.35 and an active oxygen content of 4,6%.
Tetrapotassium peroxydiphosphate is 47-51% water-soluble at 0 ~61 C, but insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones, dimethyl forma~ide dimethyl sulfo~ide, and the like. A 2% aqueous ~dl~on has a ~H of about 9.6 and a saturated solution thereof a pU
of about 10.9. A 10% solution in water at 25C sho~ed no active o~ygen los8 after fouT months; and at 50 C a 10%
solution showed an active oxygen 109s of 3% in 6 months.
From among the organic compounds those providing hydrophobic properties such as Cl 12 alkyl radlcal and those which facilitate the rapid uptake of peroxydiphosphate moiety by the cells, such as adenylyl, guanylyl, cytosylyl, and thymylyl, esters are prefe~red.
Peroxydiphosphate compound may be admini~tered orally or systemically to inhibit endotDxins in ~he oral CAVi ty or her p~te of the body,~

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1' Phar~aceuLical carriels suitable for o~al ingestion are coated tablets composed of material which resists breakdown by gastr~c acids in the sto~ach pH
(about 1-3) sincP peroxydiphosphate would be inactivated by such gastric acids. Rather, the carriers, with tableted granules of the peroxydiphosphoric acid salt solid material therein, are ~issol~ed by intestinal ~luids which have a higher pH (about 5.5-10) and d~ not inacti~ate the peroxy-diphosphate~ leaving it subject to en~ymatic action by phosphatase present in humans or other war~ blooded animals.
A desirable tablet coating solution is composed of a fatty acid ester such aR N-butyl stearate(typically about 40-50, preferably about 45 parts by weight), ~ax such as carnuba wax (typically about 15 Z5, preferably about 20 parts by weight), fatty acid such as stearic acid (typicaly about 20-30 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 ~rganic sol~ent (typically about 400-900 parts). Other desirable ~82~
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 con-tains about 80-90 parts by weight o~ manni~ol ana about 30-40 parts by weig~t o-f magnesium stearate.
Tabletted granules of the peroxydiphosphate salt are formed by blending about 30-50 parts by weight of the peroxydi-phosphate salt with about 45-65 parts by weight of a poly-hydroxy ~ugar solid such as mannitol and wetting with about20-35 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 foam of a solution o~ the coating material there-on and drying to remove solvent. Such tablets differ fro~
dental tablets which are typically compressed granules without a special protective coating.
An effective dosage of administration oE peroxydi-phosphate with a prescribed regimen, when administration is by oral ingestion, is about 0.1-2g. per kg of body weight daily, when administration systemic, such as by intramuscular, intra-peritoneal or intravenous injection, the dosage is about 0.1-2g. per kg of body weight daily.

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Physiologically acceptable pyrogen-free solvents are suitable carriers for use in the art-recognized manner for systemic administration. Saline solution buf-fered with phos-phate to a physiological pH of about 7 to 7.4 is t'ne preferred carrier for systemic administration. Such solvents are distinct from water-humectant vehicles typically used in denti-~rices. Such solution is typically prepared by sterilizing deionized distilled water, checking to insure non-pyrogenicity using the Limulus amebocyte lysate (LAL) test described by Tsuji et al in "Pharmaceutical Manufacturing", October, 1984, pages 35-41, and then adding thereto a phosphate buffer (pH
e.g. about 8.5-10) made in pyrogen-free sterile water and about 1-100 mgs. peroxydisphosphate compound derivative and sodium chloride to a concentration o~ about 0.5-1.5~ by weight. The solution can be packed in vials for use after being resteril-ized 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.

~ 04 The following examples illustrate the ability of peroxydiphosphate (PDP) compound to inhibit chemotaxis induced by endoto~in generated factor in serum and to inhibit endotoxin to~icity to bone.

1;28~004 PMN areobtained fro~ the peritoneal cavities of adult New Zealand white rabbits 12 hours af~er intraperitoneal injections of 200 ml of solution contain$ng 0.2% glycogen in sterile isotonic saline (0.85~ NaCl). The cells (P~N) are purified ~rom the exudate obtained from rabbit peritoneal cavity and purified as described by l Taichman et al (Arch. Oral Biol. 21 p. 257, 1976). Bacterial ¦¦ endotoxln pur{fied ~rom E. Coli obtained from Associates ¦~ of Cape Cod Inc. Woods ~ole, Maine, ls pre-treated ¦ with different concentrations of PDP (tetrapo~assium ~alt) a~ 37 for 1 hour. The chemotaxis assay ifi then l run with treated and untrea~ed endotoxins using Boyden ¦ Chamber as described abo~e. The data are su~marized in Tables 1 and 2. ~
TABLE .l Chemotaxis-Mean Number of PMN Percent Reduction T;reatment Mi~ratlng ~ S.D,+ in Chemotaxis 1. Control~+ 139 + 4.2 2. Serum+t+ and 1 nanogram1ml Endotoxin 343.0 + 36.7 3, 0 5~ p+~ and 142.5 + 12.0 4. Endotoxin (lng/ml) pre~reated with 0.5% P+~P and serum 18a.0 + 18.4 - 44 compared to 2 5. Endotoxin (0.5 ng/ml pre-treated wieh 0~ 5~
PDP and seru~++ 154.0 + 2.8 - 56Z compared to 2 6. Endotoxin (0.25 ng/ml) pre-treated with 0.5%
PDP and serum++ 138.5 + 2.8 - 60Z compared to 2 +-S.D. = 9tandard devlation ~+ mediu~ ~ ~a~ solution containing 10% boville ser~ albumi ++/~ serum ~ human fterum (n~r~al) -I;L-The results in Table 1 indicate that endotoxin as expected, induces a great release of a factor which increased chemotaxis of PMN (#2 treatment)g PDP (0.5%) has no effect on PM~ (#3), and end~toxins pre-treated with PDP, have chemotactic activity of ~he toxin significantly reduced (treatments 4, 5 and 6). These data indicate that a tTeatment of endotoxin with PDP, deactivates the biological effect ~of the toxin.

Table 2 ~hows data obtained with further Boyden Chamber Tests as in Example 1 PDP is employed as the tetrapotassium salt.

Chemotaxis-Mean:Number of PMN Perc~nt Reduction Treat=ent Migrating + S.D. in Che~otaxis 1. Co~trol medium (as in Example 1) 136.5 + 6.3 2. Endotoxin 1 ng/ml and serum+ 32900 + 39.5 3. PDP 0.5% and serum+ 139.5 + 4.9 4. Endotoxin (1 ng/ml) pre-treated with 0.5% PDP and serum~ 188.0 + 9.8 -43.0 5. Endotoxin (1 ng/ml) pre-treated with +
0.25% PDP and serum 206.5 + 17.6 -37 6. Endotoxin ~1 ng/ml) pre-trea~ed with 0.1% PDP and serum 231.0 ~ 17.6 -30%
+ serum as in Example 1.
The data in above table show effective concentration of PDP of at least as little as 0.1% de-a~tivates the biological activity of endotoxin.

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Effects of PDP on Endotoxin Activity in Bone Culture System The test in which an endo~oxin isolated from Acintobacillus a~tinomycete~comitans Y4 (AAY4) induces the resorption of bone in a bone culture system (Kiley and Holt, Infect.Im~un. 30:362-373, 1~80) is used to assess whether PDP deactivates the bone resorptive activity of endoto*~ from Y4. Fe~al rat bone culture as described by Raisz, ~. Clin. Invest. 44:1~3-116, 1965, ls prep~red by injecting rats with CaC12 on the 18th day of gestation. The rats are then sacrificed o~ the l9th day~ and radil and ulnae of the embryos, wlth their cartllagenous ends,are remo~ed and placed for culturing in BGJ medium (Gibco, Buffalo, NY) at 37C with 5% C02. The madium is supplemented with 5% heated (57 C for 3 hours) fetal calf serum. Bones are placed 4 to a well in 24 well dishes (Nunc, Gibco) containing 0.5 ml of medium per well. The rele~se of 45Ca into the culture media fro~ bone incubated in the presence of a test a~ent is compared with the release rom bones incubated in control medla, a~d th~ results ~f bone resorption are expressed as a ratio.

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¦ Endotoxin from AAY4 is obtained fro~ the ~niversity of Pennsylvania, School of Dentistry.
AAY4 endotoxin is treated with different concentration of PDP .tetrapotassium salt at 37 C.
The excess PDP is removed by dialysis membrane (3500 mol. w~ maximum). This permits unreactive PDP to diffuse out while the endotoxin having molecular weight greater ~han 3500 $s retained inside the bag.
¦ Table 3 summarizes the dataO

No.of 45Ca released Test/
TreatmPnt ats % + S.D. Control Sig.
l.Control ~ 6 30.11 + 1.98 2.10 ~/ml :
endotoxin : Y4 AA 6 85.46 ~ 4.71 2.87 + 0.16 97~ compared :~: to 1 : 3.10 ~g l ml endotoxin : prs-treated with: 100 not mcg PDP 6 78.47 + 2.9 2.61 + 0.1 significant 4.10 ~g/ml endotoxin pre-treated with 1000 mcg PDP 6 31.98 ~ 4.27 1.06 ~ 0.14 97% compared The data show that endotoxin from Y4 AA significantly induced bone resorption (compare l w~th 2) while a pre-treatment of the endotoxin with lOOOmcg/ml of PDP (0.1%) efeectively inhibits the bone re~orptive activity of the endotoxin.

12f~004 The foregoing results in ExampleP 1-3 are representa-tive of the effects of PDP tetrapolaSS~Um salt and o~her non-toxic water-soluble pharmaceutically acceptable PDP salts such as other alkali ~etal salts, alkal~ne earth metal salts, zinc sal~ and tin salt as well as Cl~l2 alkyl PDP salts and other organic PDP compound~, particularly including the adenylyl, guanylyl, cytosylyl and thymylyl esters and quaternary ammonium PDP salts in inhibiting chemotaxis in-duced by endoto~in generated factor in serum and to inhibit endotoxin toxicity to bone in rats, rabbits and mammals in general.

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Claims

1. The use of a non-toxic water-soluble pharmaceutically acceptable peroxydiphosphate compound for inactivation of bacterial endotoxins.

2. The use according to Claim 1 wherein said peroxydiphos-phate compound is present in an amount of about 0.1 to 7% by weight in a pharmaceutical carrier.

3. The use according to Claim 2 wherein said contact of said peroxydiphosphate compound and said endotoxin is in a warm blooded mammalian animal and said peroxydiphosphate compound is introduced in a regimen dosage of about 0.2-14 mg/kg of body weight of said warm blooded mammalian animal.

4. The use according to Claim 3 wherein said peroxydiphos-phate compound is present in tabletted granules having a coating thereon which is not broken down during passage through the stomach of said warm blooded animal and which coating is dissolv-ed by intestinal fluids having a pH of 5-10.

5. The use according to Claim 3 wherein said peroxydiphos-phate compound is administered to said warm blooded animal in a solution of non-pyrogenic distilled water and sodium chloride buffered with phosphate.

6. The use according to Claim 1 wherein said peroxydiphos-phate compound is present as a salt of alkali metal, zinc, tin or quaternary ammonium or C1-12 alkyl, adenylyl, guanylyl, cytosylyl or thymylyl ester.

7. The use according to Claim 6 wherein said peroxydiphos-phate compound is present as a potassium salt.

8. The use according to Claim 6 wherein said peroxydiphos-phate compound is present as a C1-l2 alkyl ester.

9. The use according to Claim 6 wherein said peroxydiphos-phate compound is present as an adenylyl, guanylyl, cytosylyl or thymylyl ester.

10. A composition for inhibiting hypotensive shock and localized bone resorption caused by bacterial endotoxins, which composition is in ready to use dosage form and contains a non-toxic water-soluble pharmaceutically acceptable peroxydiphos-phate in an amount effective to inactivate bacterial endotoxins, in admixture with a suitable diluent or carrier.

11. A process for preparing a composition in ready to use dosage form, for inhibiting hypotensive shock and localized bone resorption caused by bacterial endotoxins which process is characterized by incorporating as active ingredient in the composition a non-toxic water-soluble pharmaceutically accept-able peroxydiphosphate in an amount effective to inactivate bacterial endotoxins.

12. A composition according to claim 10 wherein said peroxydiphosphate compound is present in an amount of about 0.1 to 7% by weight in said diluent or carrier.

13. A composition according to claim 10 wherein said peroxydiphosphate compound is present as a salt of alkali metal, zinc, tar or quaternary ammonium or C1-12 alkyl, adenylyl, guanylyl, cytosylyl, or thymylyl ester.

14. A composition according to claim 13 wherein said peroxydiphosphate compound is present as a potassium salt.

15. A composition according to claim 13 wherein said peroxydiphosphate compound is present as a C1-12 alkyl ester.

16. A composition according to claim 13 wherein said peroxydiphosphate compound is present as an adenylyl, guanylyl, cytosylyl or thymylyl ester.