AU599027B2 - Use of an aqueous, stabilised chlorite matrix solution for infectious conditions - Google Patents

Abstract

The solution is administered intravenously and/or locally in infectious conditions caused by parasites, fungi, bacteria, viruses and/or mycoplasmas.

Description

599027

AUSTRALIA

Patentis Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Ciass Application Number: Lodged:5-842 '3 Complel 000 Priority SRelated 0- 0 te Specification Lodged: Accept Ad: Publishcd: EThis documnt contains the amendmtS made und -T Section 49 and is correct Our LODGED AT SU-FrFCE 3 P A P R198 6 Art: 0 00 0 c' 0 0000 o 0 0 00 APPLICANT'S REF.: 4476 B2 OXO CHEMIE GmnbH Name(s) of Applicant(s): Address(es) of Applicant(s):

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as'' a a Actual Inventor(s): MaaBstrasse 24, 6900 Heidelberg, Federal Republic of Germany Dr. Friedrich W. Kuhne Prof. Dr. Erich F. Elstner Dr. Hans-Heinrich Kuhne PHILLIPS, ORMOND3 AND FITZPATRICK Patent and Trade Mark Attc-r u 367 Collins Street Melbourne, Australia, 3000 Address for Service is: Complete Specification for the invention entitled: "USE OF AN AQUEOUS, STABILISED CHLORITE MATRIX SOLUTION FOR INFECTIOUS CONDITIONS" The following statement is a full description of this invention, including thle best method of performing it known to applicant(s): P 19/3/84

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-2- 2 The invention relates to' the use of an aqueous solution of a stabilized chlorite matrix for intravenous zno toj.ii-. administration in infectious conditions caused by parasites,., fungi, bacteria, viruses and/or mycoplasmas. The chlorite matrix used herein the claims and descriptions is described as a negatively charged matrix of chlorite which is chemically stabilized with oxygen or another element of Group VI a and b of the periodic table.

.Stabilized chlorite matrices containing activated oxygen are 1o disclosed in German Patent No. 3,213,389 for which the corresponding US Patent is No. 4,,507,285.

It has been found that, irrespective of their oxygen I, content, stabilized chlorite matrices, which can be activated using bioactivators to give compounds with 4404 S. electron affinity, amplify the "OXIDATIVE BURST" response of phagocytes. It is known from the Literature ('Enhancement of Phagocytosis-Associated Metabolism as a Manifestation of Macrophage Activation' by Richard B Johnston, Dr in Lymphokines, 3:33-56, 1981, Academic Press), that there is a close correlation between the extent of the oxidative response to phagocytosis and the ability to kill microorganisms intracellularly. In vitro, the chemiluminescence associated .with the 'OXIDATIVE BURST' represents a quantiti-:e measure of phagocyte stimulation.

o Measurements in which human granulocytes in whole blood were incubated with non-specifically and specifically opsonized bacteria pathogenic for humans show that addition of o stabilized chlorite matrix solution may result in an increase in the chemiluminescence to 1.7 17 times the 0o control figure, as long as heme-containing bioactivators are dissolved and freely available in the meir'm.

Example 1 Effect of TCDO on -Phagkineiand isa'.monocytogenes Infection The phagocyte stj iaon measured in vitro has been confirmed b gocyte activation (phagokinesis) recorded in vth. c C57 Bl/6 nmous the phagokinoeSis iuedby Sr ,/AR 2a One aspect of the present invention is to provide a method of treating infectious conditions caused by parasites, fungi, bacteria, viruses and mycoplasma which comprises administering intraveneously an effective amount of an aqueous therapeutic solution of a chlorite matrix with a chlorite concentration of' 12 to 72 pmol C102-/ml prepared by reaction of the-following components: sodium chlorite sodium hypochlorite solution chloryl sulfuric acid sodium perborate or So percarbonate and 0 sodium peroxide.

0 00 o The following examples are provided to illustrate the So present invention but are not intended to restrict the scope o 0o of the invention in any way.

Example 1 Effect of Tetrachlorodecaoxide (TCDO) On Phagokinesis and Listeria monocytogenes Infection The tetrachlorodecaoxygen-anion complex (TCDO) I constitutes a new chloro-oxygen compound.

The phagocyte stimulation measured in vitro has been confirmed by phagocyte activation (phagokinesis) recorded in vivo. In the C57 B1/6 mouse the phagokinesis induced by 0975v

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3 administration of a solution of stabilized chlorite matrix (15.5 m mol/1) (single i.p. doses of 0.2 0.5 ml of chlorite matrix per kg of body weight one hour after i.v.

5 administration of 4.5 x 10 Listeria monocytogenes bacteria, corresponding to an infectious lethal dose for of the animals (ILD 80 resulted in a significant increase in the Listeria monocytocenes clearance in the spleen, and in survival of all the animals.

Example 2 Effect ofQ(TCDO) on Humoral and cellular Immune Responses after Candida albicans Infection t An investigation into whether TCDO could influence i .o experimental infections with aerobic and anaerobic S microorganisms was carried out. Experiments were performed I to instigate whether TCDO could modulate humoral or cellular immune responses in vivo which may contribute to host defence reactions against infection.

S O Material and Methods 2.1 Experimental animals iFor all assays, male mice of the inbred line Balb/cABOM (Bomholtgard Ltd, Ry, Denmark) of 20 1 g bodyweight were used.

2.2 TCDO TCDO (WF10, code number of product in development) was available in an isotonic water solution of 15.5 mmol/l.

TCDO was diluted with saline immediately before use. If not 0 otherwise stated, the volume of all intravenous injections S- was 0.5 ml.

2.3 Experimental infection Washed cells of Candida albicans ATCC 10231 of the log growth phase in both containing 1% glucose (E Merck, Darmstadt, FR Germany) were injected intravenously in doses 4 of 6 x 10 Candida cells per 0.25 ml,, corresponding to a dose which was lethal for 75% of the animals (ILD 7 5) as 4 previously determined under identical standardized

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4 conditions For these Candida cells, the mean inhibitory and bactericidal concentrations (MIC and MBC, resp.) of TCDO were found to be above 7.6 mmol/l.

The strictly anaerobically growing strain of Peptostreptococcus intermedius VA 16886, isolated from biopsy material, was used for infections with an anaerobes.

MIC and MBC, in a thioglocycollate medium (Oxoid, Basingstoke, Hampshire, UK) were found to be 0.49 mmol/l and S1.94 mmol/l in broth containing glucose and, in the presence S 10 of additionally 10% foetal calf serum, 0.49 mmol/l and 0.97 mmol/l, respectively.

Germs were cultivated on Schadler anaerobic agar (Oxoid) for 48 h at 37 0 C, rinsed out and washed once with saline concentration suspended in thioglycollate medium Sadjusted to a concentration of 101 cells per ml. Volumes of 0.1 ml were injected intramuscularly (randomized infection) into the hindleg at the same site where 0.05 ml of a 5% solution of lactic acid had been applied the day before. Periods of exposure to air were kept to a barest minimum: infection was carried out by 2 technical assistants within 15 min and bacteria were handled no longer than 30-40 min. Based on pilot experiments under identical conditions, Sthe infectious dose corresponded to an ILD 75 85 4 2.4 Humoral immune response The influence of TCDO on the humoral immune response was evaluated by the Jerne plaque assay technique [13], modified by Cunningham and Szenberg The animals were i immunized by intraperitoneal injections of 1 x 104 sheep red blood cells (SRBC) in 0.5 ml saline. The number of direct plaque forming, immunoglobulin (lg)M producing spleen cells (DPFC) was evaluated on day 3 and that of indirect plaque forming IgG producing spleen cells (IPFC) was assessed on day 7 after immunization and was calculated for 104 spleen cells. Different doses of TCDO were given 1 h before sensitization or in another series of experiments 1 day before evaluation, on days 2 and 6 after sensitization in the case of DPFC and IPFC, respectively.

Cellular immunity The ootpad swelling reaction (FPSR) in mice [15] has been shown to correlate with the cellular immune response 4 For sensitization, doses of 1 x 10 SRBC in 0.5 ml saline were injected intravenously and for challenge 1 x SRBC in 0.04 ml saline into the left hind footpad 4 days later (saline for controls). The difference in the bilateral skin thickness (A mm) before and 24 h after challenge, was taken for evaluation (Oditest 00 T6, I0 Kroeplin, Schluchtern, FR Germany). TCDO was given in single doses either on day 0, 1 h before sensitization, or on day 3, or on day 4, 1 h before the challenge injection.

In separate assays, the influence of repeated TCDO applications was examined.

o Results 2.6 Influence of TCDO on experimental infection with Candida Albicans t In an initial series of experiments, different doses of TCDO were given only once, 1 h after infection. In 2 assays performed on different days, the ILD 75 expected from previous experiments was determined as ILD and 73.3 ILD76 in the control animals. The experiments showed a i 76.7 steep increase in the survival rate up to a dose of 3.1 imol/kg b.w. Higher doses, however, up to 1.8.6 pmol/kg b.w. decreased the survival rate below the control value.

S' 2.7 TCDO treatment of experimental anaerobic infections Experimental infections with Peptostreptococcus intermedius w're treated with single intravenous injections of TCDO 1 h after infection in doses of 3.1 and 12.4 pmol/kg b.w. TCDO. The control mice received saline. Both TCDO doses increased the survival rate in comparison to the saline treated controls.

2.8 Influence of TCDO on humoral immune response A single dose of 3.1 pmol/kg b.w. TCDO given 1 h before sensitization increased the DPFC response significantly. This effect was even more pronounced when 6 TCDO was injected 2 days after sensitization, 1 day before evaluation.

The high dose of 18.6 pmol/kg b.w. had no significant effect on the DPFC response in comparison to controls, irrespective of the time of TCDO administration, either 1 h before or 2 days after sensitization, and in contrast to the fact that this TCDO dose was already inhibitory in experimental Candida infections.

On the other hand, in the case of late TCDO 0o administrations, the higher dose of TCDO had a significantly lower effect in comparison to the lower dose. Similar results were obtained in evaluating the IPFC response.

S Single TCDO doses of 3.1 pmol/kg given 1 h before or 6 days after sensitization, i.e. 1 day before evaluation, resulted in an increased IPFC response.

2.9 Effect on cellular immunity The low single dose of 3.1 pmol/kg b.w. TCDO induced a highly significant FPSR irrespective of the time of administration, given either as a single dose 1 h before or 4 3 days after sensitization. TCDO treatment of the mice 1 h before challenge had no effect.

When TCDO was injected intraperitoneally at the low dose 1 h before sensitization, a slight but significant increase of 5% was observed in FPSR, whereas a high TCDO dose which had shown negative immunomodulation showed no effect in comparison to the controls.

Claims (4)

1. A method of treating infectious conditions caused by parasites, fungi, bacteria, viruses and mycoplasma which comprises administering, intraveneously an effective amount of an aqueous therapeutic solution of a chlorite matrix with a chlorite concentration of 12 to 72 pmol C102 /ml prepared by reaction of the following components: sodium chlorite sodium hypochlorite solution chloryl sulfuric acid sodium perborate or percarbonate and sodium peroxide.

2. A method as claimed in claim 1 wherein the infectious condition is caused by Listeria monocytogenes.

3. A method as claimed in claim 1 wherein the infectious condition is caused by Candida albicans.

4. A method as claimed in claim 1 substantially as hereinbefore described with reference to any one of the S examples. DATED: 22 January, 1990 SOXO CHEMIE GmbH By their Patent Attorneys: PHILLIPS ORMONDE FITZPATRICK AR j I k