DE10318009B4 - Means for inactivating infectious prions - Google Patents

Abstract

Use of a disinfectant for inactivating infectious prions on surfaces, instruments and in liquids, wherein the disinfectant is a synergistically effective mixture of one or more hydroxybenzoic acids (A) and one or more phenols (B), in combination with alkyl sulfonates, alkyl sulfates and / or alkylaryl sulfonic acid and / or alkylaryl sulfonates and / or alkylaryl ether sulfates having 1-3 EO groups and / or alkyl ether sulfates having 1-3 EO groups, their sodium, potassium and ammonium salts, with primary or branched chains of length C ₈ -C ₁₈ as anionic surfactants Contains (C)
wherein the hydroxybenzoic acids are selected from 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3, 5-Dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or mixtures thereof, wherein the phenols are selected from phenol, 2-iso-propyl-5-methylphenol, 2 Methylphenol, 3-methylphenol, 4-methylphenol, hexylresorcinol, 2-phenylphenol, 3-methyl-4-chlorophenol, 3,5-dimethyl-4-chlorophenol, 2-benzyl-4-chlorophenol, mono-, di-, and trihalogenated phenols or mixtures thereof, and
wherein the weight ratio of the hydroxybenzoic acids (A) to the phenols (B) is between 10:90 and 90:10.

Description

A hitherto unknown threat - since the appearance of BSE and proven portability this animal disease to humans - the new variant of Creutzfeldt-Jacob disease (vCJK). It has recently become known that it has been possible to that the classic variant of CJK is also the result of an infection can be with the BSE agent. The causative agent of transmissible spongiform encephalopathies (TSEs = CJK, vCJK, Kuru, etc.) applies to inactivation as a special challenge.

Cause of the TSE diseases are misfolded glycoproteins, which were designated by their discoverer S. Prusiner as prion (s) (proteinaceus infectious only). For the pathological variant of the prion exists a physiological counterpart, which is also called prion isoform and abbreviated as PrP ^c (c = cell). For the pathological variant, the terms PrP ^{Sc, BSE, CJD are} short in the literature.

The altered chemical and physical properties of the misfolded prions are used in diagnostic assays to differentiate between infectious and physiological proteins. The infectious form of the prions shows partial resistance to proteases due to the secondary structure. The prion isoform PrP ^c (c = cell), however, is completely digested by the enzyme.

Out explained the beta-sheet structure u. a. also the resistance against high temperatures, UV rays, acids and disinfectants as well as the insolubility in water.

The Ausdünnbarkeit Infectiousness Misfolded prions leaves the degree of infectivity be titratable in the animal model.

at The surgical treatment of CJD suspected cases has so far been used Device (surgical instruments, Endoscopes, etc.) should only be disposed of to reduce the risk of iatrogenic transmission by avoiding surgical instruments on humans.

The Problems in the inactivation of prions and possibilities for cleaning and Decontamination of medical devices is described in the communication "The Variant Creutzfeldt-Jakob Disease (vCJK) in the Bundesgesundheitsblatt 2002: 45 on pages 376-394 discussed.

With the occurrence of CJK and vCJD, as a human BSE infection, The situation has changed in Europe and, more recently, in the USA and Japan changed sustainably. Instead of fewer CJK cases is also due to epidemiological extrapolations of an unknown, much larger number infected persons who are asymptomatic for many years be, d. H. which can not be classified as vCJK suspected cases yet as a carrier the disease this, in the case of treatment with chirurgical Instruments or endoscopes, can continue to spread.

A similar Problem arises in the production of food animal material. To interrupt BSE infection chains - their Paths only incomplete are known - appears the use of an agent to inactivate infectious prions for the entire range of food production - from livestock husbandry to towards the production of meat and sausage products - urgently needed. The indication for a such means are given for all 1 areas with infectious Material can be contaminated, these include Structural facilities (areas, soils) as well as tools, instruments or liquids.

At present, only alkalis (eg caustic soda, potash lye) and hypochlorite compounds (chlorine bleach, chlorine elimination) are available as recognized effective inactivation agents of PrP ^res . Both classes of compounds cause hydrolysis or denaturation of the prion protein in a sufficiently high concentration.

A Use of these chemicals for permanent disinfection of surgical Instruments or endoscopes are prohibited because of their aggressive properties to expect that the instruments will soon become useless would.

This applies for the same reason also for Area of food production from animal material.

The DE 43 17 083 A1 discloses a disinfectant comprising a combination of a phenol and a hydroxybenzoic acid (salicylic acid) may contain. The disclosed disinfectant is used to kill parasites.

from that there was a need for compounds or classes of compounds to seek, on the one hand, in terms of their corrosive properties (Metal corrosion, stress corrosion cracking, etc.) are manageable and on the other hand a sufficient inactivating effect against pathogenic Prions have.

at looking for disinfectants with material compatible Properties became surprising found that mixtures of phenols and hydroxybenzoic acids infectious prions to be able to denature in such a way that their infectivity will be annulled.

The present invention is the use of a disinfectant for inactivating infectious prions on surfaces, instruments and in liquids, wherein the disinfectant is a synergistically effective mixture of one or more hydroxybenzoic acids (A) and one or more phenols (B), in conjunction with alkylsulfonates , Alkyl sulfates and / or alkylaryl sulfonic acid and / or alkylaryl sulfonates and / or alkylaryl ether sulfates having 1-3 EO groups and / or alkyl ether sulfates having 1-3 EO groups whose sodium, potassium and ammonium salts, with primary or branched chains of length C ₈ -C ₁₈ as anionic surfactants (C).

The hydroxybenzoic are selected from 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 2,3,4-dihydroxybenzoic acid. Trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or Mixtures thereof.

The Phenols are selected from phenol, 2-iso-propyl-5-methylphenol, 2-methylphenol, 3-methylphenol, 4-methylphenol, Hexylresorcinol, 2-phenylphenol, 3-methyl-4-chlorophenol, 3,5-dimethyl-4-chlorophenol, 2-benzyl-4-chlorophenol, mono-, di- and trihalogenated phenols or mixtures thereof.

The weight ratio the hydroxybenzoic acids (A) to the phenols (B) is between 10:90 and 90:10.

preferred embodiments are in the subclaims specified.

following listed Examples and tables serve to illustrate the present invention and proof of synergism.

According to FC Kull and PC Eisman, Applied Microbiology 9, 538-41 (1946), synergism is proved to be established when the result F <1 is obtained by applying the following formula. F = QA / Qa + QB / Qb (Eq.1) where the characters mean: F <1 synergism F = 1 Additive effectiveness F> 1 antagonism Qa = Amount of A alone to the endpoint Qb = Amount of B alone to the endpoint QA = Amount of A in the mixture with B QB = Amount of B in the mixture with A

at the investigation of synergists as a single substance could not always find an endpoint because of any concentration increase because of Methodology and / or physical properties of the respective substances was not feasible. The highest concentration used in the test was in these cases assumed as the concentration to the endpoint and to the calculation used in equation (1).

To demonstrate the effectiveness and the synergistic effect by the Ge invention Mix Formulation Examples 1-6 were used. Examples 1 and 4 describe formulations used according to the invention.

Examples

example 1 Parts by weight Alkyl (C10-C13) sulfate Na salt 10.00 4-chloro-3-methylphenol 15.00 2-hydroxy benzoic acid 10.00 2-propyl 25,00 Deionized water 40,00 Example 2 Alkyl (C10-C13) sulfate Na salt 10.00 2-hydroxy benzoic acid 10.00 2-propyl 25,00 Deionized water 55,00 Example 3 Alkyl (C8-C14) sulfonate Na salt 10.00 4-chloro-3-methylphenol 15.00 2-Propylakohol 25,00 Deionized water 50,00 Example 4 Alkyl (C10-C13) sulfate Na salt 10.00 phenol 20.00 4-hydroxybenzoic acid 10.00 2-propyl 25,00 Deionized water 35,00 Example 5 Alkyl (C10-C13) sulfate Na salt 10.00 4-hydroxybenzoic acid 10.00 2-propyl 25,00 Deionized water 55,00 Example 6 Alkyl (C10-C13) sulfate Na salt 10.00 phenol 20.00 2-propyl 25,00 Deionized water 45,00

In vitro testing

The in vitro test method known as Western Blot makes it possible to qualitatively determine the inactivation of prions via the denaturation / destruction of PrP ^res .

All Substances or substance mixtures were first examined by blotting. Could not in this way degradation of the infectious brain homogenate by protease K, the formulation was rejected as ineffective.

mixtures that led to that after exposure to the infectious material a proteolytic Degradation by the enzyme again possible was classified as potentially effective and a quantitative one Investigation in the animal model supplied.

starting material for the Test were 100 μl - approaches of a 5% brain homogenate. The infection was scrapie strain 263 K used.

From a hamster brain (with about 1 g weight) results in about 1 mg of brain tissue in 20 .mu.l approach with an average infectivity of 10 ⁶ -10 ⁷ infectious units (LD ₅₀ ). As a control, the brain is used by healthy hamsters.

Everyone Test batch contained:

• 20 μl brain homogenate
• 30 μl H ₂ O
• 50 μl Test substance (various conc.)

Everyone Control batch contained:

• 20 μl brain homogenate
• 80 μl H ₂ O

To 30 minutes Incubation of the batches at the desired Temperature of 37 ° C on a shaker, These were used to neutralize (inactivate) the acidic test substances adjusted to pH 7.0. The control batch was the same Neutralization buffer, the neutralization buffer for the control batch however, was previously adjusted to pH 7.0. To neutralize the acids averaged 2.5 mM-1000 mM Tris base used. The volume of all adjusted to pH 7.0 approaches gave 150 μl.

2 μl each batch (test batch or control batch) were then admixed with 18 μl H ₂ O and 2 μl proteinase K stock solution (1 mg / ml) (Boehringer Mannheim).

Of the enzymatic degradation by proteinase K was carried out for 1 hour at 37 ° C.

As a control served approaches to which instead of 2 ul proteinase K 2 ul H ₂ O was added and which were also incubated for 1 hour at 37 ° C.

Finally, each batch was mixed with 5 μl of protein solubilization buffer. 20 μl of each batch was used for SDS gel electrophoresis. Gels are blotted onto PVDF membranes ¹ (0.45 microns).

Analysis of the blot with specific antibodies:

The Blotfolie was used to avoid non-specific binding on the PVDF membrane treated with milk protein (Blocking the binding sites) and subjected to various rinses, afterwards with the primary monoclonal anti-prion protein antibody 3F4 incubated. After another Pretreatments and rinses was then with the secondary Antibody-AP goat conjugated anti mouse IgG - incubated.

To rinse again was incubated with a luminescent buffer (a light emission is thereby generated at the locations of the membrane where the Prion proteins are located). After removal of excess solution, the membrane was placed in a Exposure box spent and developed on x-ray film.

The Evaluation of the blot leaves unequivocally detect if any effect by agent activity on the infectious material took place. Nevertheless, this procedure gives no reliable information about the Extent of inactivation and possible remaining residual infectivity.

The in vitro method allows - according to international Scales - only a qualitative statement of effectiveness and only then as validated when using the quantitative in vivo method (animal model) an affirmative Result can be achieved.

In vivo testing (animal model)

to execution of the experiments in the animal model hamster were neutralized with 20 .mu.l of the Samples and controls are inoculated intracerebrally (solutions such as in the blot method). For Each sample was used to number more than 8 hamsters to arrive at a statistically reliable statement.

animals where instead of an inactivating substance uses only water on average, died 85 days after inoculation at scrapie.

By Application of the mixtures according to the invention is a delay reach the disease onset of animals of 135 days and more. Infection dose and incubation time correlate with each other, therefore including appropriate titration experiments for the incubation period an associated titer reduction will be indicated graph (1). A delay from Z. B. 85 days to 125 days corresponds to a titer reduction of more than 4 logarithmic units or a reduction of the infectivity by 99.99%.

Grafik (1) log(ILD) = –0,1007 × Id + 15,115 log(ILD) = infektiöse Einheiten Id = Inkubationszeit in Tagen (Gl. 2) Regressions-Statistik: Bestimmtheitsmaß: 0,9835 Standardfehler: 0,2710 Schnittpunkt: 15,1155; Standardfehler 0,72668 Variable: 0,1007; Standardfehler 0,00650 The infectivity of the brain homogenate (number of infectious units / particle) was determined by dilution to the end point. The averaged results from the dilution series are semilogarithmically in linear relation to the incubation times. From these investigations the graphic representation (1) and the regression function (equation 2), which was used to calculate the results of the investigation in Tables 1-3, resulted.

Graphic (1) log (ILD) = -0,1007 × I d + 15,115 log (ILD) = infectious units I d = Incubation time in days (equation 2) Regression statistics: of determination: .9835 Standard Error: .2710 Intersection: 15.1155; Standard error 0.72668 Variable: 0.1007; Standard error 0.00650

The averaged results of Example Formulations 1-9 are presented in Tables 1-3. Unless otherwise indicated, the numerical values given are the infectious titre (log LD ₅₀ ) of the reduced infectious dose, the difference of which from the starting dose gives the reduction factor. Table 1 Concentration [%] / log (infectivity) samples log (inf. dose) 0.5% 1.0% 2.0% 5.0% example 1 6.67 2.02 Example 2 6.67 6.45 6.23 5.55 5.25 Example 3 6.67 6.22 5.88 5.45 4.34 Table 2 Concentration [%] / log (infectivity) samples log (inf. dose) 0.5% 1.0% 2.0% 5.0% Example 4 6.71 3.83 2.43 Example 5 6.71 6.61 6.52 6.35 5.95 Example 6 6.71 6.63 6.52 5.89 5.61

The Outcome is considered to be sufficiently effective if the infection titer was reduced by 4 log levels, i. H. if a 99.99% reduction Infectiousness is reached.

Out Table 1 follows that with the acid component from Example 2 and the component from Example 3 is not sufficient Effectiveness could be achieved. Formulation example 1, on the other hand contained both components and showed already at 0.5% concentration sufficient inactivation of infectious prions.

Substituting these results in the formula of Kull and Eisman (equation 1), one obtains F = 0.5 × 15/5 × 15 + 0.5 × 10/5 × 10 = 0.20

Of the Numerical value 0.20 thus provides clear evidence of the existence a synergistic effect.

For the table 2 results for F = 0.20 as a numerical value.

As a result, is the efficacy against pathogenic prions and the synergistic Effect of the mixtures according to the invention proved by the results presented.

Claims (9)

Use of a disinfectant for inactivating infectious prions on surfaces, instruments and in liquids, wherein the disinfectant is a synergistically effective mixture of one or more hydroxybenzoic acids (A) and one or more phenols (B), in combination with alkyl sulfonates, alkyl sulfates and / or alkylaryl sulfonic acid and / or alkylaryl sulphonates and / or alkylarylethersul containing 1-3 EO groups and / or alkyl ether sulfates having 1-3 EO groups whose sodium, potassium and ammonium salts, with primary or branched chains of length C ₈ -C ₁₈ as anionic surfactants (C), wherein the Hydroxybenzoic acids are selected from 2-hydroxybenzoic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid, 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5- Dihydroxybenzoic acid, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid or mixtures thereof, wherein the phenols are selected from phenol, 2-iso-propyl-5-methylphenol, 2-methylphenol , 3-methylphenol, 4-methylphenol, hexylresorcinol, 2-phenylphenol, 3-methyl-4-chlorophenol, 3,5-dimethyl-4-chlorophenol, 2-benzyl-4-chlorophenol, mono-, di- and trihalogenated Phenols or mixtures thereof, and wherein the weight ratio of the hydroxybenzoic acids (A) to the phenols (B) is between 10:90 and 90:10. Use according to claim 1, characterized in that the weight ratio of hydroxybenzoic acids (A) to the phenols (B) is between 20:80 and 80:20. Use according to claim 1 or 2, characterized in that the halogenated phenols selected are from 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2-bromophenol, 3-bromophenol, 4-bromophenol, 2-iodophenol, 4-iodophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol and 2,4,6-trichlorophenol. Use according to any one of claims 1 to 3, characterized in that the disinfectant further comprises nonionic surfactants of the alkylpolyethylene glycol ether type having from 3 to 11 EO groups, hydrotroping agents selected from butylmonoglycol sulfate, cumene sulphonate, toluenesulphonate, xylenesulphonate, as sodium, potassium or ammonium salts individually or as a mixture and aliphatic alcohols and / or glycols of chain length C ₂ to C ₁₂ as a solvent individually or as a mixture and aliphatic carboxylic and / or hydroxycarboxylic acids of chain length C ₁ to C ₆ individually or as a mixture as pH regulators and sequestering agents. Use according to one the claims 1 to 4, characterized in that the weight ratio of Alkyl sulfonates or sulfates and / or alkylaryl sulfates and / or Ether sulfates and their salts (C) with the acids and phenols (A + B) in relationship C: (B + A) = 1:99 and 99: 1. Use according to one the claims 1 to 5, characterized in that the weight ratio of Hydrotroping agents and their salts, individually or in admixture between 0.1 and 70 wt .-% based on the total weight of the disinfectant concentrate lies. Use according to one the claims 1 to 6, characterized in that the weight ratio of Alcohols individually or mixed with each other between 1 and 90% by weight based on the total weight of the disinfectant concentrate lies. Use according to one the claims 1 to 7, characterized in that between 0.1-30% by weight one or more sequestrants of the aminoacetic acid type or phosphonic and their respective derivatives are included. Use according to one the claims 1 to 8 in aqueous, diluted Solutions.