FR2586351A1 - PHARMACEUTICAL COMPOSITION BASED ON PEROXODIPHOSPHATE FOR THE INACTIVATION OF BACTERIAL ENDOTOXINS - Google Patents

Abstract

BACTERIAL ENDOTOXINS ARE INHIBITED BY A COMPOUND BASED ON PEROXODIPHOSPHATE, NON-TOXIC, SOLUBLE IN WATER, PHARMACEUTICALLY ACCEPTABLE, IN CONTACT WITH BACTERIAL ENDOTOXIN.

Description

Endotoxins are complex macromolecules composed

  fat, carbohydrate and protein. They are mainly found

  also in the wall of Gram negative microorganisms, and they are usually referred to as "lipopolysaccharides". These macromolecules are toxic to the host and can be fatal. For example, they can cause severe hypotensive shock and also cause various toxic reactions in the body, bone resorption

  understood. In the oral cavity, endotoxins are consi-

  derivatives as an important factor in gum inflammation and localized bone loss, such as

loss of alveolar bones.

  Theoretically, the compounds that release oxygen should inactivate endotoxins. However, due to the speed with which many oxygen-releasing compounds release oxygen, these have little effect

  inhibitor on the development of endotoxins. The ingredients

  which release oxygen more slowly should inhibit

  ber the effect of endotoxins. However, their effectiveness is generally limited, since the necessary conditions

  to the release of oxygen do not correspond to the conditions

reigns in the body.

  As indicated in the patent application in the United States N 726 545, filed on April 24, 1985, mammals, for example, rodents including humans, have in their bodies alkaline phosphatases and phosphatases

  acids. Peroxodiphosphate compounds have the pro-

  please slowly release oxygen. The amount of oxy-

  gene released by these compounds accounts for one-tenth of the

  amount of oxygen released by hydrogen peroxide.

  Only about 50% of the active oxygen of these compounds is released in 20 hours at 25 C, in the presence of phosphatases

  alkaline or acid phosphatases.

  Peroxodiphosphates (PDP) slowly release hydrogen peroxide in the presence of phosphatases, according to the following equation:

O O O

  Il Il phosphatases Il H O X4-O-P-O-O-P-O- -O-O-P-O- O H202 + po4

I I I

O O O-

o 0 0-

  in which X is a non-toxic, pharmaceutical cation

  mentally acceptable, or completes an organic ester fragment.

  The phosphatase responsible for the breakdown of peroxo-

  diphosphate is present in saliva as well as in the

  plasma, intestinal fluids and white blood cells.

  It has been found that bacterial endotoxin also reacts with unaltered PDP. This reaction occurs regardless of the presence of phosphatases, that is, it also takes place outside the body of a warm-blooded animal. However, which is very important, even in the presence of phosphatase, the reaction also takes place when mammals are treated with a PDP according to the

  present invention; It is desirable to establish a poso-

  where the treatment is continued until inacti-

endotoxin removal.

  An object of the present invention is to deactivate endotoxins and thus inhibit their toxic effects, such as

  inflammation, bone resorption and hypotensive shock.

  The description below will show other objects

of the present invention.

  According to some of its objects, the invention relates to a process for the inhibition of lichochypotensive and localized bone resorption, caused by a bacterial endotoxin, which comprises contacting a non-toxic, water-soluble peroxodiphosphate, pharmaceutically acceptable, with endotoxin, to cause inactivation of said

bacterial endotoxin.

  A technique for highlighting inactivation

  endotoxin is to inhibit the induction of production

  tion of a chemotactic factor against poly- leukocytes

  nuclear, hereinafter referred to as "PMN". This factor can be determined using the Boyden chemotaxis method, in

  which rabbit white blood cells are attracted (chemo-

  taxie) by the factor induced by the endotoxin which is produced in their vicinity. In the Boyden method, when incubating a bacterial lipopolysaccharide (endotoxin)

  with serum from a mammal, the reaction occurs

  Next tion: incubation during serum + endotoxin for 1 hour at the chemotactic temfactors gold temperature for PMN ganism The phenomenon of chemotaxis is studied using Boyden chambers, as described by Cates et al., in "Method of the Modified Boyden Chamber, for the measurement of chemotaxis due to PMN "(Modified Boyden Chamber Method for Measuring PMN Chemotaxis, Leucocyte Chemotaxis, Methods, Physiology and Clinical Application, edited by Gallin et

  Quie, Raven Press, New York, 1978, p 67-71). When the endo-

  toxin causes chemotaxis, as in the present invention

  tion, it is possible to quantitatively determine the

  inhibition percent using the Boyden chemotaxis assay. Endotoxins can be introduced into the body of a warm-blooded animal, taking advantage of the presence of these in the wall of Gram negative microorganisms, such as Actinobacillus actinomycetemcomitans (A. a.), Escherichia coli (E. coli), Bacteroides melanenoqenicus

  (B. mel.) And Salmonella typhi (S. typhi).

  The oral endotoxin isolated from A. a. has a toxic action on the alveolar bones. Non-oral endotoxin purified from E. coli can be fatal

for the host.

  Other known techniques for demonstrating the inhibition of the production of endotoxins are carried out using the resorption of a bone culture medium; you can also use an embryo mortality test

of chicken.

  The toxic reaction is effectively inhibited by treatment

  endotoxin in situ in a warm-blooded host with an effective inhibitory amount of a compound based on

  non-toxic peroxodiphosphate, soluble in water, pharma-

  ceutically acceptable. In the body, peroxodiphos-

  phate reacts with endotoxin as a molecule

  intact, while inactivating the peroxoda-based compound

  phosphate. As the endotoxin is inactivated, this shows that

  endotoxin reacts with peroxodiphosphate.

  In general, about 0.1-7% of the peroxide-based compound

  diphosphate in a pharmaceutical vehicle, such as in solution, are effective at a dosage of approximately

  0.2-14 mg per kg of body weight. The effectiveness of inhi-

  bition can be demonstrated by the endotoxin-reducing effect, and is quantitatively determined by

  based on inhibition of chemotaxis against PMN.

  Compounds based on peroxodiphosphate

  ticks, non-toxic, water-soluble, pharmaceutically acceptable, are the alkali metal salts (for example lithium, sodium and potassium), the alkaline earth metal salts (for example magnesium, calcium and strontium) and zinc, tin and ammonium -5quaternary salts, as well as C112 alkyl esters

  adenylyl, guanylyl, cytosylyl and thymylyl.

  Among the inorganic cations, the alkali metals are preferred, the potassium salt in particular. Tetrapotassium peroxodiphosphate is a white crystalline solid, non-hygroscopic, stable, odorless, finely divided, fluid, having a molecular weight of 346.35 and an active oxygen content.

equal to 4.6%.

  Tetrapotassium peroxodiphosphate is soluble in water at 47-51%, at 061 ° C, but insoluble in common solvents such as acetonitrile, alcohols, ethers, ketones,

  dimethylformamide, dimethylsulfoxide and the like. A solu-

  2% aqueous solution has a pH of about 9.6, and a saturated solution of tetrapotassium peroxodiphosphate has a pH of about 10.9. A 10% solution in water did not show a loss of active oxygen at 25 C after 4 months, and at 50 ° C a 10% solution showed a loss of oxygen

active 3% within 6 months.

  Among the organic compounds, those preferred are those

  hydrophobic properties, such as C1_12 alkyl radicals, and those which facilitate the rapid absorption of the peroxodiphosphate fragment by cells, such as

  esters of adenylyl, "guanylyl, cytosylyl and thymylyl.

  Peroxodiphosphate can be administered orally or generally to inhibit excessive secretion of PTH in the body.

  Pharmaceutical vehicles suitable for administration

  oral tration are coated tablets composed of a substance which resists decomposition by gastric acids at the pH prevailing in the stomach (approximately pH 1-3),

  because peroxodiphosphate would be inactivated by these gas acids

  triques. On the other hand, the vehicles in which are incorporated

  filled with compressed granules of the solid substance

  killed by peroxodiphosphoric acid salt, are dissolved by intestinal fluids which have a higher pH (about pH 5.5-10), and do not inactivate peroxodiphosphate, leaving it sensitive to the enzymatic action of phosphatases

  present in humans or other warm-blooded animals.

  A desirable coating solution for tablets is

  of a fatty acid ester, such as n-butyl stearate (normally about 40-50, preferably about 45 parts

  by weight), a wax such as carnauba wax (normally

  about 15-25, preferably about 20 parts by weight), a fatty acid such as stearic acid (normally about 20-30 parts, preferably 25 parts by weight), and an ester of cellulose, such as cellulose acetophthalate (normally 5-15, preferably about 10

  parts by weight) as well as an organic solvent (standard

  approximately 400-900 parts). Other substances of enro-

  desirable bage include shellac and copoly-

  mothers of maleic anhydride and ethylenic compounds, such as polyvinyl methyl ether. Such coatings differ from tablets which are broken down in the oral cavity, in that

  that the constituent substance of the tablets normally contains

  about 80-90 parts by weight of mannitol and about

  30-40 parts by weight of magnesium stearate.

  The peroxodiphosphate granules incorporated in the tablets are obtained. by mixing about 30-50 parts by weight of peroxodiphosphate with about 45-60 parts by weight of a solid polyhydroxylated sugar such as mannitol, and impregnation with about 20-35 parts by weight of a solution of a sugar polyhydroxylated such as sorbitol, sieving to the desired size, mixing with about 20-35 parts by weight of a binder such as magnesium stearate

  and compression of the granules into tablets using a pass-

  tilleuse. The compressed granules are coated by spraying

  tion on these of a foam or a solution of the coating agent, then drying to remove the solvent. Such

  tablets differ from scored tablets, which are charac-

  teristically compressed granules without coating

special protector.

  For the administration of peroxodiphosphate with a prescribed dosage schedule, an effective dose will, in the case of oral administration, from about 0.1 to 6 g

  per kg of body weight per day; in the case of administrative

  In general, such as by intramuscular, intraperitoneal or intravenous injection, the dose ranges from approximately

  0.1 to 2 g per kg of body weight per day.

  Physiologically acceptable pyrogen-free solvents are vehicles suitable for use in the manner recognized in the art for general administration. A phosphate buffered saline at a physiological pH of about 7 to 7.4 is the preferred vehicle for general administration. Such solvents are

  different from the humidifying vehicles used characteristi-

  only in toothpaste. Such a solution is normally prepared by sterilization of deionized distilled water, verification of its non-pyrogenicity by means of the test of horseshoe crab lysate (LAL), described by Tsuji et al. in Pharmaceutical Manufacturing, October 1984, p 35-41, then addition thereto of a phosphate buffer (for example pH approximately 8.5-10), prepared in sterile pyrogen-free water, and of approximately 1- 100 mg of the peroxodiphosphate derivative and sodium chloride at a concentration of approximately 0.5-1.5% by weight. The solution can be placed in

  vials for use, after sterilizing it again

  calf by passing it through a sterilizing 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% can be used.

by weight of calcium chloride.

-8-

  The invention is illustrated with the aid of non-limiting examples.

  below, which show that the peroxodiphosphate (PDP) compound inhibits the chemotaxis caused by the factor produced by endotoxin in serum and inhibits the toxicity of endotoxin to the bones.

Example 1

  PMN is obtained from the peritoneal cavity of adult New Zealand white rabbits 12 hours after 200 ml intraperitoneal injection of a solution

  containing 0.2% glycogen in an isoto- salt solution

  sterile picnic (0.85% NaCl). We collect the cells

  (PMN) from exudate from the peritoneal cavity

  rabbits, and they are purified as described by Taichman

  et al. (Arch. Oral Biol., 21, p 257, 1976). From the endo-

  purified bacterial toxin from E. coli, supplied by Associates of Cape Cod Inc., Woods Hole, Maine, USA, is pretreated with different concentrations of PDP (tetrapotassium salt) for one hour at 37 C. The test is then carried out. laL.chimiotaxie with treated endotoxins and untreated endotoxins, using a Boyden chamber, as described above. The results are

  summarized in Tables 1 and 2.

-9- Treatment

Table 1

  Average chemotaxis value of migrating PMN + CE * Decrease in chemotaxis (%) 1. Control ** 2. Serum *** and 1 ng / ml endotoxin 3. 0.5% PDP and serum *** 4. Endotoxin (1 ng / ml) pretreated with 0.5% PDP and serum ** 5. Endotoxin (0.5 ng / ml) pretreated with 0.5% PDP and serum ** 6. Endotoxin (0, 25 ng / ml) pretreated with 0.5% PDP and serum **

139 4.2

343.0 36.7

142.5 12.0

188.0 + 1-8.4

154.0 2.8

138.5 + 2.8

  -44% compared to (2) -56% compared to (2) -60% compared to (2) * EC = standard deviation ** medium = Earl solution containing 10% bovine serum albumin

*** serum = human serum (normal).

  The results indicated in Table 1 show that,

  as expected, endotoxin triggers a significant release

  aunt of a factor which increases the chemotaxis of PMN (treatment N 2); PDP (0.5%) has no effect on PMN (treatment N 3); and endotoxins pretreated with PDP

  exhibit markedly reduced chemotactic activities

  clouds (treatments N 4, 5 and 6). These results show that

  treatment of endotoxin with PDB inactivates the bio-

logic of the toxin.

-10-

Example 2

  Table 2 gives the results obtained in other tests carried out with the Boyden chamber as in Example 1. The PDP used is in the form of tetrapotassium salt. Treatment

Table 2

  Average value of the chemotaxis of migrating PMN + EC Decrease in chemotaxis (%) 1. Control medium (as in Example 1) 2. Endotoxin (1 ng / ml) and serum * 3. PDP (0.5%) and serum*

4. Endotoxin (1 ng / ml) pre-

  treated with 0.5% PDP and serum *

5. Endotoxin (1 ng / ml) pre-

  treated with 0.25% PDP and serum *

6. Endotoxin (1 ng / ml) pre-

  treated with 0.1% PDP and serum *

136.5 6.3

329.0 139.5

  39.5

  4.9

188.0 9.8

206.5 17.6

231.0 17.6

* serum = see example 1.

  The results shown in the table above show that a minimum effective concentration of PDP also

  low than 0.1% inactivates the biological activity of endotoxin.

-43.0%

-37% -30% -11-

Example 3

  Effects of PDP on Endotoxin Activity in a Bone Culture System The assay is used in which an endotoxin isolated from Acintobacillus actinomycetemcomitans Y4 (AaY4) causes bone resorption in a culture system back

  (Kiley and Holt, Infect. Immun., 30 362-373, 1980), to determine-

  if the PDP inactivates the endotoxin from AaY4, in its action causing bone resorption. A culture of rat embryonic bones is prepared, as described by Raisz, J. Clin. Invest., 44: 103116 (1965), by injection of 45CaC12

  to rats on the eighteenth day of gestation. The ten-

  ninth day, the spleens are sacrificed, then the embryos' radius and ulna are removed, with their cartilaginous ends, and for culture, they are placed

  BGJ medium (Gibco, Buffalo, NY, USA) at 37 C, with 5% CO2.

  The medium is enriched with 5% fetal calf serum.

  heated (57 C for 3 hours). Bones are placed, at a rate of 4 per cup, in boots with 24 cups (Nunc, Gibco)

  containing 0.5 ml of medium per well. We compare the liberation

  tion of 45Ca in culture media from bone incubated in the presence of a test compound, upon release of 45Ca from bone incubated in control media, and the results of resorption are expressed by

this report.

  The endotoxin from AaY4 is supplied by the Dental School of the University of Pennsylvania. The endotoxin of AaY4 is treated with different concentrations of PDP (tetrapotassium salt) at 37 C. The excess PDP is eliminated

  using a dialysis membrane (molecular weight limit

  area: 3,500). This allows non-reactive PDP to be eliminated

  by diffusion, while endotoxin, having a molecular weight

  greater than 3,500, is retained inside the

  poached. The results are summarized in Table 3.

: 1 -12-

Table 3

  Treatment Number 45Ca released 'Test / Rat level% EC control significance 1. Control 6 30.11 1.98

2. 10eg / ml endo-

  AaY4 toxin 6 85.46 4.71 2.87 0.16 97% compared to 1

3. 10 Pg / ml endo-

pre-treated toxin

  with 100 µg PDP 6 78.47 2.9 2.61 0.1 unsigned.

4. 10, pg / ml endo-

  toxin pretreated with 1000 pg of PDP 6 31.98 4.27 1.06 0.14 97% compared to 2 The results show that the endotoxin from AaY4 significantly causes bone resorption

  (comparison of 1 and 2), while pretreatment of the endo-

  toxin with 1000 pg / ml PDP (0.1%) effectively inhibits

  bone resorption activity manifested by endotoxin.

  The above results, obtained in Examples 1-3, illustrate the effects of tetrapotassium PDP and other salts

  PDP-based, non-toxic, water-soluble, pharmaceu-

  tically acceptable, such as other alkali metal salts, alkaline earth metal salts, zinc salt and tin salt, as well as alkyl peroxodiphosphates

  in C1_12 and other organic compounds based on PDP, including

  in particular the adenylyl, guanylyl, cytosylyl and thymylyl esters, as well as the ammonium salts

  quaternary of PDP, in the inhibition of pro- chemotaxis

  voqu6epar the factor generated by the endotoxin-in the

  serum, and inhibition of endotoxin toxicity to

  screw bones, in rats, rabbits and mammals in general.

Claims (1)

CLAIM   Pharmaceutical composition for the treatment of hypotensive shock and localized bone resorption, caused by bacterial endotoxins, characterized in   what it includes as an active ingredient a peroxodi-   non-toxic phosphate, water soluble, pharmaceutical- acceptable.