DE10318009A1 - Disinfectant for inactivating infectious prions on surfaces and instruments and in liquids comprises a mixture of hydroxybenzoic acids and phenols - Google Patents

Abstract

Disinfectant for inactivating infectious prions on surfaces and instruments and in liquids comprises a mixture of hydroxybenzoic acids and phenols and selected anionic and nonionic surfactants, hydrotropes, solvents and pH regulators. Disinfectant for inactivating infectious prions on surfaces and instruments and in liquids comprises a mixture of hydroxybenzoic acids, e.g. 2-, 3- and/or 4-hydroxybenzoic acid, 2,3-, 2,4-, 2,5-, 2,6-, 3,4- and/or 3,5-dihydroxybenzoic acid and/or 2,3,4-, 2,4,6- and/or 3,4,5-trihydroxybenzoic acid, and phenol or substituted phenols, e.g. 2-isopropyl-5-methylphenol, 2-, 3- or 4-methylphenol, hexylresorcinol, 2-phenylphenol, 3-methyl-4-chlorophenol, 3,5-dimethyl-4-chlorophenol, 2-benzyl-4-chlorophenol and/or mono-, di- and trihalophenols; anionic surfactants selected from sodium, potassium and ammonium alkylsulfonates, alkyl sulfates, alkylarylsulfonates, alkylaryl ether sulfates with 1-3 ethylene oxide (EO) units and alkyl ether sulfates with 1-3 EO units, all with carbon chain lengths of 8-18; nonionic surfactants selected from alkyl polyethylene glycol ethers with 3-11 EO units; hydrotropes selected from sodium, potassium and ammonium butyl monoglycol sulfate, cumenesulfonate, toluenesulfonate and xylenesulfonate; solvents selected aliphatic alcohols and glycols with carbon chain lengths of 2-12; and pH regulators selected from aliphatic carboxylic and hydroxycarboxylic acids with carbon chain lengths of 1-6. ACTIVITY : Antibacterial; Neuroprotective. MECHANISM OF ACTION : None given.

Description

A hitherto unknown threat - since the appearance of BSE and proven portability this animal disease on humans - the new variant of Creutzfeldt-Jacob disease (vCJK). It was recently announced that it was possible to prove that the classic variant of CJD is the result of an infection with the BSE pathogen. The causative agent of transmissible spongiform encephalophatias (TSEs = CJK, vCJK, Kuru etc.) is considered to be inactivation a special challenge.

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

The changed chemical and physical properties of the incorrectly folded prions are used in diagnostic examination procedures to differentiate between infectious and physiological proteins. The infectious form of the prions shows a partial resistance to proteases due to the secondary structure. The prion isoform PrP ^c (c = cell) is completely digested by the enzyme.

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

The Ausdünnbarkeit infectivity misfolded prions the degree of infectivity become titratable in the animal model.

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

With the occurrence of CJK and vCJK as a BSE infection in humans, the situation has changed in Europe and more recently in the USA and Japan changed permanently. Instead of fewer CJK cases is also based on epidemiological projections of an unknown, incomparably larger number infected people who are asymptomatic for many years will be d. H. which cannot be classified as suspected vCJD, nevertheless as a carrier the disease this, in the case of treatment with surgical Instruments or endoscopes.

A similar Problems arise in the production of food from 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 area of food production - from livestock to towards the production of meat and sausage products - urgently needed. The indication for a such means arises for all areas with infectious Material may be contaminated these include structural facilities (areas, soils) as well as tools, instruments or liquids.

Currently, only recognized alkalis (e.g. sodium hydroxide solution; potassium hydroxide solution) and hypochlorite compounds (chlorine bleaching solution; chlorine releasers) are available as recognized inactivating agents of PrP ^res . In sufficiently high concentrations, both classes of compounds cause hydrolysis or denaturation of the protein portion in the prion.

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

This applies to the same reason for Field of food production from animal material.

Hence the need to search for compounds or classes of compounds that on the one hand, they can be controlled with regard to their corrosive properties (metal corrosion, stress corrosion cracking, etc.) and on the other hand they have a sufficiently inactivating effect against pathogenic prions.

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

following listed Examples and tables serve to explain the present invention and evidence of synergism.

According to F.C. Kull and P.C. Eisman, Applied Microbiology 9,538-41 (1946) applies a synergism as proven if by using the calculation formula below the result F <1 comes about.

F = QA / Qa + QB / Qb (Eq. 1) where the characters mean:
F <1 synergism
F = 1 Additive effectiveness
F> 1 antagonism
Qa = set of A alone at the end point
Qb = amount of B alone at the end point
QA = amount of A mixed with B
QB = amount of B mixed with A

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

Formulation examples 1-6 were used to demonstrate the effectiveness and the synergistic effect of the mixtures 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 Parts by weight Alkyl (C10-C13) sulfate Na salt 10.00 2-hydroxy benzoic acid 10.00 2-propyl 25,00 Deionized water 55,00 Example 3 Parts by weight Alkyl (C8-C14) sulfonate Na salt 10.00 4-chloro-3-methylphenol 15.00 2-Propylakohol 25,00 Deionized water 50,00 Example 4 Parts by weight 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 Parts by weight Alkyl (C10-C13) sulfate Na salt 10.00 4-hydroxybenzoic acid 10.00 2-propyl 25,00 Deionized water 55,00 Example 6 Parts by weight 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 using the blot method. Could not break down the infectious brain homogenate by protease in this way K are found, the formulation was rejected as ineffective.

mixtures which resulted in that after acting on the infectious material a proteolytic Degradation by the enzyme possible again were classified as potentially effective and quantitative Investigation carried out in the animal model.

starting material for the Test were 100 μl batches of one 5% brain homogenate. Scrapie strain 263 became infected K used.

A hamster brain (weighing approx. 1 g) produces approx. 1 mg brain tissue in a 20 μl mixture with an average infectivity of 10 ⁶ –10 ⁷ infectious units (LD ₅₀ ). The brain of healthy hamsters is used as a control.

Each test approach contained:
20 ul brain homogenate
30 ul H ₂ O
50 μl test substance (various conc.)

Each control approach included:
20 ul brain homogenate
80 ul H ₂ O

To 30 minutes Incubate the approaches at the one you want Temperature of 37 ° C on a shaker, these were used to neutralize (inactivate) the acidic test substances adjusted to pH 7.0. The control approach was the same Neutralization buffer offset, the neutralization buffer for the control approach however, was previously adjusted to pH 7.0. To neutralize the acids were an average of 2.5 mM-1000 mM Tris base used. The volume of all set to pH 7.0 approaches gave 150 μl.

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

The Enzymatic degradation by proteinase K took place at 37 ° C. for 1 hour.

As a control, batches were used to which 2 μl H ₂ O was added instead of 2 μl proteinase K and which were also incubated at 37 ° C. for 1 hour.

Finally, 5 μl protein solubilization buffer was added to each batch. 20 ul of each batch was used for SDS gel electrophoresis. The gels are blotted on PVDF ¹ membranes (0.45 μm).

Analysis of the blot with specific antibodies:

The Blot film was used to avoid unspecific binding on the PVDF membrane treated with milk protein (Blocking of the binding sites) and various flushing processes, after that with the primary monoclonal anti-prion protein antibody 3F4 incubated. After another Pretreatment and rinsing was done then with the secondary Antibody - AP conjugated goat anti mouse IgG - incubated.

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

The Evaluation of the blot leaves undoubtedly recognize whether an effect due to agent activity on the infectious material has taken place. However, this procedure does not provide reliable information about the The extent of inactivation and the possible remaining infectivity.

The enables in vitro method - according to international Standards - only a qualitative statement of effectiveness and is only then considered 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, hamsters were neutralized with 20 μl Inoculated samples and controls intracerebrally (solutions such as with the blot method). For each sample was used to number more than 8 hamsters to arrive at a statistically reliable statement.

animals where only water is used instead of an inactivating substance statistically died 85 days after inoculation of scrapie.

By Use of the mixtures according to the invention is a delay the disease onset of the animals can be reached for 135 days and more. Infection dose and incubation time correlate with each other, therefore including corresponding titration experiments for the incubation period an associated titer reduction can be given graphic (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 in infectivity by 99.99%.

Grafik (1) log(ILD) = –0,1007 × Id + 15,115 (Gl. 2)log(ILD) = infektiöse Einheiten
I_d = Inkubationszeit in Tagen Regressions-Statistik:

The infectivity of the brain homogenates (number of infectious units / particles) was determined by dilution to the end point. The averaged results from the dilution series are semi-logarithmically linear in relation to the incubation times. The result of these examinations is the graphical representation graphic (1) and the regression function (equation 2), which are used to calculate the examiners results from Tables 1–3.

Graphic (1) log (ILD) = -0.1007 × I d + 15.115 (Eq. 2) log (ILD) = infectious units
I _d = incubation time in days of regression statistics:

The averaged results of Example Formulations 1-9 are shown in Tables 1-3. Unless otherwise stated, the numerical values given mean the infection titer (log LD ₅₀ ) of the reduced infectious dose, the difference from the starting dose giving the reduction factor.

Table 1

Table 2

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

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

If you put these results in the formula of Kull and Eisman (Eq. 1), you get
F = 0.5 × 15/5 × 15 + 0.5 × 10/5 × 10 = 0.20

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

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

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

Claims (8)

Disinfectant for inactivating infectious prions on surfaces, instruments and in liquids based on a synergistically effective mixture of mono-, di- and trihydroxybenzoic acids and phenols, the anionic and non-ionic surfactants as wetting agents, hydrotroping agents, primary and secondary alcohols as solvents and aliphatic carboxylic acids and hydroxycarboxylic acids - Can contain as pH regulators and sequestering agents, characterized in that a) they have synergistically effective combinations of aromatic monohydroxycarboxylic acids such as the 2-; 3-; 4-hydroxybenzoic acids, the dihydroxybenzoic acids, 2,3-; 2.4; 2.5; 2,6; 3,4; 3,5-dihydroxybenzoic acid, or the trihydroxybenzoic acids, 2,3,4-trihydroxybenzoic acid, 2,4,6-trihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, individually or mixed, and phenol or substituted phenols such as 2-isopropyl 5-methylphenol, 2-; 3- or 4-methylphenol, hexylresorcinol, 2-phenylphenol, 3-methyl-4-chlorophenol, 3,5-dimethyl-4-chlorophenol, 2-benzyl-4-chlorophenol, mono-, di- and trihalogenated Phenols such as 2-; 3-; 4-chlorophenol, 2-; 3-; 4-bromophenol, 2-; 4-iodophenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol individually or mixed, in combination with alkyl sulfonates, alkyl sulfates and / or alkylarylsulfonic acid and / or alkylarylsulfonates and / or alkylaryl ether sulfates with 1 -3 EO groups and / or alkyl ether sulfates with 1-3 EO groups containing their sodium, potassium and ammonium salts with primary or branched chains of length C8-C18 as anionic surfactants, and nonionic surfactants of the alkylpolyethylene glycol ether type with 3- 11 EO groups can contain. b) butyl monoglycol sulfate, cumene sulfonate, toluenesulfonate, xylene sulfonate, as sodium, potassium or ammonium salt individually or as a mixture as hydrotroping agents and aliphatic alcohols and / or glycols of chain length C2-C12 as solvents or as a mixture and aliphatic carbon and / or May contain hydroxycarboxylic acids of chain length C1 to C6 individually or as a mixture as pH regulators. Disinfectant according to claim 1, characterized in that the weight ratio of hydroxybenzoic acids (A) to the phenols (B) can be between 1:99 and 99: 1. Disinfectant according to claim 1 and 2, characterized characterized in that the weight ratio of the alkyl sulfonates or sulfates and / or alkylarylsulfates and / or ether sulfates and their Salts (C) with the acids and phenols (A + B) in the ratio C: (B + A) = 1:99 and 99: 1. Disinfectant according to claim 1, characterized in that the weight ratio the hydrotroping agent and its salts, individually or in a mixture with each other between 0.1 and 70% by weight based on the total weight of the disinfectant concentrate. Disinfectant according to claim 1, characterized in that the weight ratio of the alcohols individually or in a mixture with one another between 1 and 90% by weight based on the total weight of the disinfectant concentrate can lie. Disinfectant according to claim 1, characterized in that between 0.1-30% by weight of one or more sequestering agents of the aminoacetic acid or phosphonic acid type and their respective de may contain derivatives. Use of the disinfectant according to one of the Expectations 1 to 6 for inactivating prions on surfaces, instruments and liquids in the field of medicine and all technical facilities for food production from animal finishing. Use of the disinfectant according to one of the Expectations 1 to 6 in aqueous, diluted Solutions.