Note: Descriptions are shown in the official language in which they were submitted.
~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.
-- 2 --
~7
,~
~Z8ZOO~
!
I
~ 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.
~2~ 0q~
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,~
~ o~
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
62301-1392
~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.
62301-1392
~8~
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
EXAMPLE 1
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.
EXAMPLE 2
Table 2 ~hows data obtained with further Boyden
Chamber Tests as in Example 1 PDP is employed as the
tetrapotassium salt.
TABLE 2
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.
.. ;' !l I
lZ~OOD~
EXAMPLE 3
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.
~'~Z3ZOO~
¦ 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
TABLE 3
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.
--15.
