EA013519B1

EA013519B1 - Antibacterial or pediculicidal preparation comprising allicin

Info

Publication number: EA013519B1
Application number: EA200700663A
Authority: EA
Inventors: Норман Джон Беннетт; Питер Дэвид Джослинг
Original assignee: Стоун Айлэнд Холдингз Лтд.
Priority date: 2001-09-21
Filing date: 2002-09-23
Publication date: 2010-06-30
Also published as: EA009169B1; ZA200403002B; GB0122793D0; EA200700663A1; EA200400451A1; EP1435928A1; AU2002334074B2; WO2003024437A1

Abstract

The present invention relates to allicin. In particular, it relates to antibacterial and pediculicidal preparations comprising allicin or an allicin metabolite and a pharmaceutically acceptable excipient. There are also synergistic effects of allicin in preparations further comprising at least one further antibacterial or pediculicidal.

Description

The present invention relates to allicin.

Allicin, a sulfur compound having the formula

It is a major active compound characterized by numerous therapeutic properties, which are described for garlic (LSHit κηΐίνίι.ιιη). In its natural state, garlic does not contain allicin, but its precursor, alliin [(+) 8-allyl-L-cysteine sulfoxide]. Alliin is converted to allicin as a result of the action of the enzyme allinase or alli-lyase, also a component of garlic. Alliin and allinase get together when chopped garlic cloves. The following equation represents the synthesis scheme.

ABOUT

C | allinaaa / noo

----— ~ allicin alliin pyruvic acid O + 2. ,, - 1 + 2.

= 4

II Oh

However, allinase is rapidly and irreversibly inactivated by the product of its reaction, allicin, and is also inactivated under acidic conditions, such as in the stomach. Therefore, in practice, the output of allicin from chives is significantly lower than the theoretical maximum. Indeed, the output is usually about 0.3-0.5%.

AO 97/39115 describes a continuous method for the synthesis of allicin, which involves preparing a column containing allinase immobilized on a solid carrier, passing the solution of alliin through the column and obtaining a solution of allicin in the effluent.

In addition, the authors of this application received allicin in a spray-dried form and purchased it in capsules from Achscht 1n1Atyopa1 Ytyyeb οί Na1G Noshe, MPIagu Voab, Vous. Tank! 8i88eh, ΝΥ31 7п, IpPeb Kshdbot, under the trade name allimaks (ABB1MAX).

The present invention is based on studies of the new therapeutic properties of allicin.

In the most general aspect, the present invention relates to an antibacterial or pediculicidal preparation containing allicin or allicin metabolite and a pharmaceutically acceptable excipient.

Preferably, the preparation contains at least one other antibacterial or pediculidal agent.

More preferably, another agent is selected from (1) penicillins, including ampicillin, piperacillin, carbenicillin, amoxicillin, methicillin and penicillin C; () aminoglycosidases, including gentamicin, tobramycin, streptomycin and amikacin; (w) tetracyclines; (ίν) macrolides, including erythromycin; (ν) cephalosporins and cefamycins, including cefuroxime, cefamandole and moxalactam; and (νί) fusidic acid, rifampicin, novobiocin, vancomycin, ciprofloxacin, chloramphenicol and metronidazole.

Accordingly, the allicin metabolite is at least one of the number ΌΑΌ8 (diallyl disulfide), ΌΑΤ8 (diallyl trisulfide), adzoena, allitridium, or vinyldithiine.

Preferably, for oral administration or administration in the form of a suppository, vaginal suppository, or nasal preparation, a pharmaceutically acceptable excipient is a solid composition to which allicin or its metabolite is added. More preferably, if desired, the solid composition contains a volume-enhancing agent such as lactose, microcrystalline cellulose or dicalcium phosphate; a thickener such as gum or starch; a disintegrating agent such as starch sodium glycolate or cross-linked povidone; release agent, such as magnesium stearate; emulsifier; surfactant and sweeteners, flavors and colorants. Most preferably, allicin is bound by a spray-drying method, and the solid composition contains modified starch, such as maltodextrin, gum arabic, silicon dioxide, and an emulsifier, such as magnesium stearate.

Accordingly, for topical administration, a pharmaceutically acceptable excipient is included in a cream or soap. Alternatively, the excipient may be a lotion, ointment, toothpaste, mouthwash or a hair preparation such as shampoo, styling gel or conditioner. If appropriate, such preparations may include combinations of the following substances: surfactants, flavoring agents, dyes, stabilizers, antioxidants, emulsifiers, thickeners, waxes, glycerols, fats, suspending agents, defloculatory substances and antioxidants, all of which can may or may not be hypoallergenic. Preferably, the cream excipient contains white semi-solid paraffin, an emulsifier, such as stearate, preferably magnesium stearate, glycerin, water, yellow semi-solid paraffin, and a stabilizer, such as potassium citrate. Most preferably, the excipient cream is an aqueous cream, preferably an aqueous cream BP. Accordingly, the filler-soap contains sulfuric acid ester, cocamide and cocobetaine. Possibly, the excipient, in addition, may include flavors and colors.

- 1 013519

Preferably, for oral, parenteral or topical use, the ratio of allicin to excipient is one that provides allicin concentrations between 1 and 2000 ppm, preferably between 50 and 1000 ppm, more preferably between 250 and 500 ppm.

FIG. Figure 1 shows the average sizes of the zones for allicin fluid relative to clinical isolators 81a1yosossik aiteik, which showed resistance, intermediate resistance and sensitivity to mupirocin;

in fig. 2 - the average size of the zones for allicin cream relative to clinical isolators 81aryuososik aiteik, which showed resistance, intermediate resistance and sensitivity to mupirocin;

in fig. 3 — minimal inhibitory (M1C) and minimal bactericidal (MBC) concentrations for allicin (μg / ml) relative to clinical isolators 81131103;

in fig. 4 - inhibition zones formed by allicin against MK8L103;

in fig. 5 shows the reaction of degree 1 with the Oxford strain and streptomycin;

in fig. 6 - reaction of degree 2 with the Oxford strain and vancomycin;

in fig. 7 - reaction of degree 3 MK8L102.

The above and other aspects of the present invention are further described in more detail with reference to the following examples only as illustrative example.

1. The antimicrobial properties of single allicin.

Determination of the minimum inhibitory concentration (M1C) by the method of tube dilution.

Using an aseptic method, 1 ml of double concentration nutrient medium was poured into each of the 11 Kahn tubes (Kyap). To the first tube, tube 1, (labeled control) was added 1 ml of sterile deionized water. To the second tube, tube 2, 1 ml of allicin (2000 ppm in aqueous solution) was added, mixed using a mechanical mixer, and 1 ml was transferred from tube 2 to tube 3. The process was repeated sequentially until tube 11.

Therefore, a series of double dilutions between 1/2 and 1/1024 was obtained. 4 μl of culture grown overnight in a nutrient medium was used to inoculate each tube. The contents of the tubes were mixed and incubated at 37 ° C for 24 hours. Observed for turbidity in tubes and the lowest concentration without turbidity was recorded as M1C.

Determination of the minimum bactericidal concentration (MBC).

From each of the M1C-series tubes, in which no turbidity was detected, 100 μl were taken and scattered on the surface of the nutrient agar plate using a sterile glass spatula. The plates were incubated at 37 ° C for 24 hours and the appearance of bacterial colonies was monitored. The lowest concentration at which no viable bacteria was observed was recorded as MBC.

Determination of M1C using multipoint inoculum.

Allicin stock solution 1/4 v / v prepared by adding 5 ml of allicin (2000 ppm in an aqueous solution) to 15 ml of sterile water. 10 ml of the prepared solution was taken, added to 10 ml of double-molten, cooled nutrient agar, mixed, and used to prepare a 1/8 v / v dilution cup. The remaining 10 ml of allicin (1/4) was diluted with 10 ml of sterile water to obtain a solution with a dilution of 1/8 v / v. 10 ml of a 1/8 solution was used to prepare 1 cup diluted 1/16 v / v. Serial dilution was continued until no 1/1024 v / v dilution of allicin cups was obtained. The plates were allowed to equilibrate and dried at 44 ° C for 15 minutes.

The tested microorganisms (Table 1) were cultivated in nutrient broth at 37 ° C for 18 hours. 0.3 μl of undiluted cultures were seeded onto the surface of the prepared dried cups using a multipoint inoкулятор liter. The plates were incubated at 37 ° C for 24 hours and monitored for growth. The total number of viable organisms was determined according to the Miles-Misra method (M11ecM1kga).

- 2 013519

Table 1

Grand positive bacteria Gram-negative bacteria Yeast Vas111iz 8iŠH1z Aegolupaz lubgoria SapsIy a! Ysapz EpTegozosiz ESSEHGSYA SOI £ aes11z YSTS 10418 Yztegga Рцец Цотопз аегидхпоза toposudedepez ΜβΤΐιίάίΙΙίη 5a1topa11a EurYttsg1shp Ee1z1apT Sarrow aigeis (ΜΚΞΆ) 31ARW ΥθΧΞίηίβ AYEUS ISST 6571 endogram: 1s

The minimum inhibitory concentrations established by the method of tube dilution.

Of the microorganisms tested, Rkeibotopak aegidtock was the least sensitive to allicin, and SapFba a1Sapk was the most sensitive (Table 3). The values of M1C and MBC for allicin were calculated and are presented in Table. 2. The obtained sample of allicin was analyzed at a concentration of 2000 hours.

Minimum inhibitory concentrations established using a multi-point inoculant.

M1C was determined using a multipoint inoculator (Table 3). M1C values were calculated using allicin at a concentration of 2000 ppm, and the results were in good agreement with the results obtained by an in vitro dilution method.

The antimicrobial activity of allicin was demonstrated against eleven microbial species. The relative sensitivities of the microorganisms differed, the Rkeibotopak aegidschock was the least sensitive, and SapFba a1Sapk was the most sensitive.

Staphylococcus is one of the most important types of bacteria that cause disease in humans. They are normal inhabitants of the upper respiratory tract, skin, vagina and intestines. They are representatives of a group called pyogenic cocci (pyogenic). Staphylococcus is easily transmitted from asymptomatic carriers (without signs of disease) or from subjects with the disease through skin contact, aerosols, or from inanimate objects. Staphylococcus can cause a pathological process in almost every organ and tissue of the body.

- 3 013519

table 2

M1C and MVS, op tube dilution 5 * ISTS 6571 03/04/01 06/04/01 AND 04/18/01 Concentration (parts per million) M1C 1/128 1/64 1/64 2 3 MVS 1/4 1/8 1/4 375 The total number of viable (CFU / ml) 1.43x10 “Σ'ЗхТС 1.43x10 “

E. coI ΝΟΤΟ 10418 03/04/01 06/04/01 ' 04/18/01 Concentration (parts million) on M1C 1/128 1/64 1/32 28 MVS 1/32 1/16 1/16 94 The total number of viable (CFU / ml) 1, 13x10 ' 6.75x10 " 1,05χ10 ^1υ

E. £ aesAz 03/04/01 06/04/01 04/18/01 Concentration (parts million) on M1C 1/16 1/32 1/32 75 MVS 1/4 1/2 1/4 600 The total number of viable (CFU / ml) 1.80x10 “ 1, 63x10 “ 7.75x10 '

A, luMgorY1a 03/04/01 06/04/01 04/18/01 Concentration (parts million) on Mts 1/64 1/16 1/256 49 MVS 1/32 1/16 1/32 75 The total number of viable (CFU / ml) 2,55x10 " 4.25x10 “ 2.33x10 "

Z. Bur 03/04/01 06/04/01 04/18/01 Concentration (parts million) on Mts 1/64 1/64 1/32 31 MVS 1/16 1/16 1/8 150 The total number of viable (CFU / ml) 8.90x10 “ 6,00x10 “ 1.80x10 “

- 4 013519

Υ. épbegosošus 03/04/01 06/04/01 04/18/01 Concentration (parts million) on M1C 1/512 1/64 1/128 15 mws 1/64 1/64 1/64 28 The total number of viable (CFU / ml) 9, 50x10 ' 1,70h10 ^e ^{1 and} 2,18h10

B. shopwarming 03/04/01 06/04/01 04/18/01 Concentration (parts million) on M1C 1/64 1/128 1/64 23 mws 1/8 1/4 1/16 262 The total number of viable (CFU / ml) 2.30x10 " 7.75x10 ' 1,15x10 ^th

MKZA 03/04/01 06/04/01 Concentration (parts million) on M1C 1/64 1/64 28 mws 1/4 1/2 675 The total number of viable (CFU / ml) 9,00x10 ' ”9.75x10 '

R. aegidposa 03/04/01 06/04/01 04/18/01 Concentration (parts million) on M1C 1/8 1/8 1/8 225 mws 1/8 1/4 1/4 375 The total number of viable (CFU / ml) 5.25x10 " G, 25x10 ⁹ 3.75x10 "

S. a1b1capz 03/04/01 06/04/01 04/18/01 Concentration (parts million) on M1C 1/512 > 1/512 1/512 > 3.5 mws 1/128 1/32 1/64 33 The total number of viable (CFU / ml) 3, 40x10 " 3.05x10 “ 3, WLO ^1,

- 5 013519

Table 3

M1C, determined using a multipoint inoculator

3.AiFellIfA 6571 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/128 1/128 1/128 14 M1C (Series 2) <1/40 1/128 1/128 1/128 The total number of viable (CFU / ml) 1.43x10 “ 2,3x10 “ 1, 43Х10 ⁸

E. So11 MHTS 10418 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) MTS (Series 1) <1/40 1/128 1/128 1/128 1 l M1C (Series 2) <1/40 1/128 1/128 1/128 1 h The total number of viable (CFU / ml) 1,13x10 ^th 6.75x10 ^e 1, O5x1O ¹⁰

E. £ aesa115 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/32 1/32 1/32 56 M1C (Series 2) <1/40 1/32 1/32 1/32 The total number of viable (CFU / ml) 1.80x10 “ 1, 63x10 ^minutes 7.75x10 ⁷

A. Iubgorpia 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/128 1/256 1/128 12 M1C {Series 2) <1/40 1/128 1/256 1/128 The total number of viable (CFU / ml) 2.55x10 ^e 4.25x10 “ 2.33x10 °

- 6 013519

8. £ Ur! T1tigl 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/64 1/64 1/64 28 M1C (Series 2) <1/40 1/64 1/64 1/64 The total number of viable (CFU / ml) 8, βΟχίδ ⁵ 6,00x10 “ 1.80x10 '

Υ. epbegosoNbtsa 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) MHS (Series 1) <1/40 1/128 1/256 1/128 12 M1C (Series 2) <1/40 1/128 1/256 1/128 The total number of viable (CFU / ml) 9.50x10 ' 1.10x10 ' 2.18 x ¹⁰

s topsubodeduz 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/64 1/128 1/64 21 M1C (Series 2) <1/40 1/128 1/128 1/64 The total number of viable (CFU / ml) 2.30x10® 7.75x10 ' 1,15x10 ^th

Meza 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1} <1/40 1/64 1/128 1/64 26 M1C (Series 2) <1/40 1/64 1/64 1/64 The total number of viable (CFU / ml) 9,00x10 ' 9.75x10 ' 7.50x10 '

- 7 013519

R. aegidhoza 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) 1/15 > 1/8 1/8 > 1/8 > 197 M1C (Series 2) 1/15 > 1/8 1/8 > 1/8 The total number of viable (CFU / ml) 5.25x10 ⁸ 1,25x10 ' 3.75x10 '

S. a1Sypz 03/04/01 06/04/01 12/04/01 04/18/01 Concentration (parts per million) M1C (Series 1) <1/40 1/256 1/512 1/256 > 5 MTS (Series 2) <1/40 1/512 1/512 1/256 The total number of viable (CFU / ml) '3,40х10 ^b 3.05x10 ° 3,00x10 °

B.5bt0115 03/04/01 06/04/01 12/04/01 04/18/01 Concepting (parts per million) M1C (Series 1) <1/40 1/256 1/512 1/64 > 2 M1C (Series 2) <1/40 1/512 1/64 1/64

Methicillin (or its related antibiotics) is one of the main drugs used for the treatment of infectious diseases caused by paralysis. Methicillin-resistant 81yearusssi5ageic (ICBA) is a major nosocomial problem (infections caused by strains acquired in the hospital). Most of these strains are resistant to a wide variety of antibiotics (including some of the most recent). Some strains are also resistant to agents, such as mupirocin, currently used to combat asymptomatic carriage and colony formation in hospitals. In some intensive care units, 10–20% of patients may be infected with ICBA.

Using diffusion tests determine the sensitivity of isolates to antimicrobial agents, measuring the zone of inhibition around the cumulative indicator of antimicrobial agents. However, these tests are the most common tests used for testing for antimicrobial resistance. Inhibition zones of at least 6 mm, which are smaller than those of a known control strain, indicate bacterial sensitivity to the antimicrobial agent. The dimensions of the zones of 12 mm or less usually indicate resistance, there is also an intermediate resistant group between the indicated levels.

Using the above criteria, in the following examples, when comparing the sizes of zones with dimensions of antibiotic-sensitive control zones, clinical isolates were classified as (1) resistant to mupirocin, (and) intermediately resistant, and () sensitive to mupirocin.

Bacterial strains.

clinical isolates and one control strain 8 (ar11 aigeik (Oxford strain, ΝΟΤΟ 6571) were tested in test phase 1. All strains were provided by the royal hospitals (Roua1 Loioui and 8ΐ Va111151) in London and were identified as having multiple resistance to antibiotics.Subsequently, seventeen of these strains plus control were selected for phase 2 tests.

Phase 1. Diffusion analysis.

Initially mentioned tests were carried out to test the effectiveness of creams. However, the results of the study of creams were compared with the results of testing garlic and allicin liquid using standard agar well methods. Resistance to mupirocin was confirmed using a diffusion test with a standard paper disk. Penalty B (d). Müller-Ginton agar plates (MIELEG-Nash1op) were seeded (lawn layer) at a standardized concentration of 8 (ap11 aigeic. The cups were left to air dry. Round holes of standard size were cut out in an agar culture medium and filled with the same amount (100 μl) of cream or The antibacterial activity was determined by measuring the zones of inhibition formed around each well.

- 8 013519

Sensitivity to mupirocin.

Oxford 81ary. Aigeik formed a zone of 35 mm. 5 strains were identified as fully sensitive (zone sizes from 33 to 45 mm), 12 zones showed intermediate sensitivity (zone sizes between 12 and 23 mm) and 13 strains were resistant (no zone of inhibition).

Allicin solution activity.

An aqueous solution of allicin liquid has been shown to be highly active against all 81ary strains. Aigeik tested up to levels of 250 ppm, (Fig. 1).

Allicin cream activity.

When a conventional water cream was formulated, it was found that allicin was highly active against all strains at concentrations of 500 ppm. The indicated concentrations correspond to suitable levels for use as a topical agent (Fig. 2).

Phase 2. Minimum inhibitory concentrations (M1C) and minimum bactericidal concentrations (MBC) for allicin in relation to §1aryy1osossi8 aigeik.

Seventeen clinical isolates (selected from the group of phase 1 strains) and one control strain (Oxford strain, ICTS 6571) 81ary were tested. aigeik. Each strain was cultivated overnight at 37 ° C in Muller-Ginton broth (χοίά Ü1. CM405). The cultures were diluted with the mentioned broth to obtain a concentration of BT 7 CFU / ml, and a tenfold final concentration was used.

ml of aqueous solution of allicin (at a concentration of 5000 ppm) was added to 8 ml of MullerGinton broth (the broth was concentrated to avoid the effect of dilution of the medium) and diluted twice to obtain a concentration range between 1000 ppm (μg / ml) and hours / million (µg / ml) plus 0 hours / million (µg / ml) in the negative control. 1 ml of each mixture was taken from each medium and 1 ml of bacterial concentrate was added, reaching a final concentration of approximately 65 CFU / ml.

These broth cultures were incubated overnight at 37 ° C. The next day, the broths were examined for growth (turbid cultures). The lowest concentration that did not show any growth (transparent) was considered as M1C.

The culture containing M1C and all the above concentrations were subcultured on nutrient agar plates (Oxo bieniu, CM3) to determine the MFR. 0.1 ml of each culture was collected and cultured. The culture with the highest concentration at which growth was observed (turbidity) in the M1C study was also subcultured as a positive control. The plates were incubated overnight at 37 ° C. The highest concentration at which growth was observed (visible bacterial colonies) was considered as MBC.

In order to prepare a cream or solution for clinical use against §1aryupport, it is important to establish optimal concentrations of the antimicrobial agent that is active against the tested strains. The results are compared in Table. 4 below.

The studied control strain (Oxford 81ary. Aigeik) showed M1C 32 µg / ml and MBC 256 µg / ml. M1C§ for 17 tested clinical isolates were either 16 or 32 µg / ml, MBC§ were either 128 or 256 µg / ml. Most clinical isolates had M1C § 16 µg / ml and MBC § 128 µg / ml, see table. five.

Table 5

MTS 16 MTS 32 TOTAL MVS 128 13 2 15 MVS 256 2 0 2 TOTAL 15 2 17

88% of clinical isolates had a µA 16 µg / ml and 88% of clinical isolates had an MBC С 128 µg / ml, see FIG. 3. Of the 17 strains tested, 3 were sensitive to mupirocin (as indicated by a disk diffusion test), 8 showed intermediate sensitivity and 6 were resistant. All 6 strains resistant to mupirocin had a µA 16 µg / ml, 4 strains had a MBC 128 128 µg / ml and 2 had a MBC 256 256 µg / ml.

- 9 013519

Table 4

LPSZ (ALLIMAKS)

Breeding (parts (+> “Growth (-) - no growth (oh) - Oxford control 8 (arb.1cho per million) 1000 500 250 125 62.5 31.25 15.6 7.3 3.7 1.8 0.9 0 §1арЬ.М> КС 825 (oh) <-) (-) (-) " 0 0 006) (-) 0 (-) (-X +) (+ X +) (+) (+) (+ X +) (+ X +) (+ X +) Oh 32 867 0000 00 (-) (-) 0 (-) 0 (-) (-) () (+ X +) (+) (+) (+ X +) (+ X +) (+) (+) 567 sixteen 868 (-) (-) (-) (-) (-) (-) (-) (-) 00 (-) 0 00 (+ X +) (+ X +) (+ X +) (+) (+) (+ X +) 868 sixteen 869 (-) (-) () (-) (-) () (-) (-) 00 (-) 0 () (·) (-X +) (+) (+) (+ X +) (+) (+) (+ X +) 869 sixteen 870 (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) () (+) (+ X +) (+ X +) (+ X +) (+) (+) 870 sixteen 872 (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (+) (+ X +) (+ X +) (+ X +) (+) (+) (+ X +) 572 32 873 0 0 (-) (-) (-) (-) 00 (-) (-) (-) (-) (-) (-) (-X +) (+) (+) (+ X +) (+) (+) (+) (+) §73 sixteen 874 (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) (-) 00 (-X +) (+ X +) (+ X +) (+) (+) (+ X +) §74 sixteen §75 (-) (-) (-) (-) (-) (-) 0 00 (-) 0 (-) (-X +) (+ X +) (+) (+) (+) (+) (+ X +) (+ X +) §75 32 §76 0000 (-) (-) (-) (-) (-) (-) (-) (-) (-) <-) (+ X +) (+) (+) (+) (+) (+ X +) (+) (+) 876 sixteen 878 "AND HE (-) (-) (-) (-) (-) (-) (-) (-) 00 (-X +) (+ X +) (+) (+) (+) (+) (+ X +) 878 sixteen 380 0 (-) (-) (-) 00 000 (-) (-) (-) 00 (-) (+) (+ X +) (+) (+) (+) (+) (+) (+) 880 sixteen §85 0 (-) (-) (0 00 (-) (-) 0 (-) (-) (-) (-) (-) (-X +) (+ X +) (+ X +) (+) (+) (+ X +) 885 sixteen 886 (-) () (-) (-) 00 000 (-) (-) 0 (-X-) (-X +) (+) (+) (+ X +) (+ X +) (+ X +) 886 sixteen 888 (-) (-) (-) (·) 0 0 (-) 00 (-) (-) 0 (-X-) (-) (+) (+ X +) (+ X +) (+) (+) (+ X +) 888 sixteen 889 (-) 00 (-) 0 0 (-) (-) (-) (-) (-) (-) (-X-) (-) (+) (+ X +) (+) (+) (+ X +) (+) (+) §89 sixteen §90 (-) (-) (-) (-) (-) (-) 0 (-) 00 (-) (-) (-) (-) (-X +) (+) (+) (+) (+) (+) (+) (+) (+) 890 sixteen 891 0 00 (-) 0 0 (-) 0000 (-) (-) (-) (+) (+) . (+ X +) (+) (+) (+) (+) (+ X +) 891 sixteen MVSz (ALLIMAX) (+) ' = height (-) = = no growth

Breeding (parts (oh) -Oxford control

δίαρΕΝ about 1000 500 per million) • 250 125 62.5 31.25 15.6 7.3 3.7 1.8 0.9 81N.No 0 mws 825 (oh) (-) (-) (-) (-) 00 (+) (+) (+) (+) (+) (+) (+ X +) (+) (+) - - - ----- 825 250 867 0000 00 (-X-) (+ X + X + X +) (+ X +) (+) (+) - - - ----- 267 128 868 0000 (-) (-) (-) (-) 0 (+) (+) (+) (+ X +) (+ X +) - - - ----- 868 128 869 (-) (-) (-) (-) 00 00 (-) (+) (+) (+) (+ X +) (+ X +) - - ----- 869 128 870 (-X-XX-X) (-) (-) 00 (+ X +) (+) (+) (+) (+) (+ X +) - - ----- 870 128 872 (-) (-) (-) (-) 00 00 (+) (-) (+) (+) (+ X +) (+ X +) - - - .— 572 128 873 (-) (-) (-) (-) 06) (-X-) (+) (+) (+) (+) (+) (+) (+) (+) - ----- 873 128 874 (-) 00 (-) (-) (-) (-X-) (+ M + X + X +) (+ X +) (+ X +) - - ----- 874 128 575 0000 00 6) 0 (+ XK + X +) (+) (+) (+) (+) - - - ----- 875 128 876 (-) (-) (-) (-) (-) (-) (-) <-) (+ X + X + X +) (+ X +) (+) (+) - - - ----- 876 128 877 0000 (-) (-) 6) 6) (+) (-) (+) (+) (+ X +) (+) (+) -----, - - - 877 128 878 0 (-) (-) (-) (-) (-) (-) (-) (+ X + X + X +) (+) (+) (+ X +) - ----- 878 128 880 000 (-) 00 (-X-) (+ X + X + X +) (+ X +) (+ X +) —— - ----- 880 128 885 (-) (-) (-) (-) 00 (-X-) (+ X + X + X +) (+) (+) (+) (+) - - ----- 585 128 886 () (-) (-) (-) (-X-) (-X-) (+ X + X + X +) (+) (+) (+ X +) - - - —- 886 128 588 <-) (-) (-) (-) (-X-) (-) (-) (+) (+) (+) (+) (+) (+) (+) (+) - - —- 888 128 889 (-) (-) (-) (-) 00 (-) (+) (+) (-) (+) (+) (+) (+) (+) (+) - - —- 889 256 890 000 (-) 00 (-) (+) (+ X +) (+) (+) (+) (+) (+ X +) - - ..... 890 256 591 (-) (-) (-) (-) (-) (-) 00 00 (+ X-) (+) (+) (+) (+) - - ..... 891 128

On the basis of the results obtained, it was obvious that the activity of allicin at a concentration of 500 and 1000 ppm corresponded to the activity of 1 to 10 and 1 to 5 dilutions of coarse garlic extract;

88% of the strains showed minimal inhibitory concentrations for allicin 16 µg / ml and 100% of the strains were inhibited by allicin at a concentration of 32 µg / ml;

88% of the strains showed minimal bactericidal concentrations for allicin 128 μg / ml and 100% of the strains were destroyed by allicin at a concentration of 256 μg / ml.

Allicin has proven to be highly effective against both characterized and wild MK8A strains.

In addition to determining the minimum inhibitory concentration (M1C), allicin was tested for a number of gram-positive and gram-negative bacterial species in relation to the selection of nine bacterial isolates at closer dilutions of aqueous solutions of allicin (allimax). The results are presented in table. 6. Used isolates:

Gram-positive:

81yrossaic aiteic: Oxford control strain (OH) and 2 lab. isolate MK8A (102 & 103) (cocci)

81ar11u1osossi5 inter1bett1b1k (cocci)

81aryuososik rugodepek (cocci)

8ettaba teksseksepk (sticks) gram-negative:

8a1tope11a (orypit (sticks)

Rykeibopak aeshdtok (sticks)

Soybeans (sticks)

Oxford isosensitive agar medium was sterilized and cooled to approximately 45-50 ° C before use. Serial dilutions of aqueous allicin (allimax) were obtained at a concentration of 5000 ppm in isosensitive broth.

- 10 013519

Table 6

Gram-positive 81Arbu1osossi5 aigeik (OH, 102 and 103) and 81ArbuSaussi5

- 11 013519

ml from each dilution of allicin was added to each sterile Petri dish (each test was performed in two copies). 18 ml of the cooled medium was added to allicin, mixed and left to cool and solidify. Bacterial samples were prepared as inoculum of 10 ⁶ CFU / ml and 0.02 ml were sown on each dish using a multipoint inocuator. Negative controls were included in each series of experiments.

Studies have identified a number of M1SK among selected samples of gram-positive and gram-negative bacterial species, the most sensitive species were staphylococci, characterized by M1C between 15 and 41 ppm.

The most resistant Gram-negative species turned out to be Rkeikotopak Aetidshock (M1C 378 ppm) and the most resistant Gram-positive species was the bacterium (13a tagseksepk (M1C 170 ppm).

Allicin liquid extracts were highly active against clinical isolates of antibiotic resistance, including those strains that were identified as resistant to mupirocin.

Cream preparations showed suitable levels of activity at a concentration of 500 µg / ml, which confirms the use of allicin cream as a local remedy for mupirocin-resistant and mupirocin-sensitive strains of parasiticus with multiple resistance to antibiotics.

Following a similar procedure, similar tests were also carried out to compare the effectiveness of allicin with the effectiveness of streptomycin against six strains of ΜΌΚ.ΤΒ (multidrug resistance of tuberculosis). The results are presented in table. 7

- 12 013519

Table 7

Strain ICTV Colonial unit Control Streptomycin Allicin 3221 100 200 0 31222 147 one 0 3632 37 29 0 37018 300 9 0 37010 350 6 0 37007 65 2 0

In the presence of antimicrobial agents, any growth of Mycobacterium tuberculosis is very important. From the results, it is clear that while growth was observed on all the canted agars on which the strains were treated with streptomycin, no growth was detected on any of the allicine canned agars (aqueous solution at a concentration of 500 ppm).

In addition, then, studies have been conducted on the insecticidal properties of allicin, in particular its pediculocidal activity against head lice (Reb1ci1i8 yitapik). Control of infection with head lice, according to tradition, was carried out using ordinary insecticides with some excess. However, head lice strains have emerged in some parts of the world that are resistant to one or more of these insecticides.

In testing adult males and female lice were used in approximately equal amounts. The lice were fed at the beginning of the test and left for at least four hours to acclimatize, the time during which they could excrete the excess water absorbed with the portion of blood they sucked. The experimental series included twenty lice and placed them on the squares of the open mesh nylon fabric (tulle) as on the substrate. Each batch was placed on a labeled 30 mm plastic Petri dish. An aliquot of approximately 5-10 ml of allicin at a concentration of 5000 ppm in an aqueous solution was poured onto the base of a clean 30 mm plastic Petri dish. The lice-carrying tissue was immersed in the liquid for 10 s, during which the tissue was rotated at least twice to ensure the removal of air bubbles. After the tissue and insects were removed from the liquid, they were slightly blotted to eliminate excess liquid and returned to the marked Petri dish. The procedure was repeated on other similar squares of fabric in that series.

Tissue squares bearing lice were incubated under normal conditions of containment (30 ° ± 2 °, Saib 50% ± 15% increased humidity) overnight. At the end of the exposure period, the insects and tissue were washed using a soft cosmetic shampoo (Vooy® Ehes. | Iep1 \ uaz11 8yatroo), diluted one part of the shampoo to fourteen parts of water, after which they were washed three times using 250 ml of warm tap water (34 ° C), which was poured through or on tissue squares. Then the tissue squares were blotted dry using medical wiping cloth and incubated under normal conditions in a clean plastic Petri dish of the appropriate size for one hour. Provided a portion of blood. The lice were left for four hours to recover, before being processed again, as described above. The results of the three tests are presented in Table. 8 in comparison with the control series, which were treated with 60% isopropyl alcohol after 24 h and 48 h.

Table 8

Test Dead Alive Painful Mortality Day 1 A1 five eleven 7 52.2% A2 9 7 five 66.7% AZ 7 9 four 55.0% Total 21 27 sixteen 57.8% C1 2 18 0 11.0% Day 2 A1 22 one one 95.8% A2 nineteen 0 2 100% AZ 15 0 five 100% Total 56 one eight 98.4% C1 four 14 0 28.6%

As can be seen, allicin shows acceptable efficacy overnight with a total mortality rate of 57.8% compared with a control mortality rate of 11%. However, after a portion of the absorbed blood and the second treatment, the effectiveness of allicin becomes 98.4% against the control of 28.6%.

2. Synergistic action of allicin in combination with other antimicrobial agents.

To assess the activity of allicin with respect to the control strains and clinical isolates, 8 aryu1osossi8 aigeik and Ezsiepsoy was examined for a wide variety of antibiotics using the methods of diffusion in the cups, relative to antibiotic resistance and sensitivity to antibiotics.

- 13 013519 tikam bacteria.

They used the Oxford isosensitive medium (Ι8Α, СМ471, χοίά, Va81id81oke, IR). The named medium is recommended for antimicrobial susceptibility testing by the British Society of Antimicrobial Chemotherapy (B8AC). Used the methods prescribed by B8AC (B8AC, 2001). For each series of experiments, preliminary tests were carried out to optimize the seed, the concentration of allicin and the distance on the plate between the tested agents.

Bacterial strains: one antibiotic-sensitive control (81ap11. Aitez, YSTS 6571), 2 methicillin-resistant 81ary. Aitez (MK.8A) and E. Soi, K12.

Antibiotics: tested 22 antibiotics; see tab. 9.

Procedure.

For each test (carried out in three copies).

1. Prepare a culture grown overnight.

2. Prepare inoculum to obtain dense, but not continuous growth on standardized 25-ml agar plates. Dry the surface of the cup.

3. Use a 6 mm sterile cork drill to cut the wells in the cup, add 150 µl of allicin.

4. Add an antibiotic disk.

5. Inoculate overnight at 37 ° C.

6. Assess synergism (zone fusion), antagonism (decrease in the zone of inhibition) or no effect. Get a large number of results to select those combinations that should be tested in phase 2 of the test.

Results.

The first series of experiments was carried out to determine if the change in the concentration of allicin had any effect on possible synergism. In tab. 10 shows the interaction of 8 antibiotics with allicin at two concentrations (500– 250 ppm). Zone sizes are in millimeters.

81ary. Notes: The sizes of the zones for the allicin concentration of 250 ppm were between 5 and 9 mm and were smaller than those of the 500 ppm of allicin zones.

E. soi: the sizes of the zones for the allicin concentration of 250 ppm were between 1 and 12 mm and were smaller than the sizes of the zones for the 500 ppm of allicin. In general, the sizes of the zones were smaller than those obtained with 81are. aitez.

A concentration of 500 ppm was chosen for further testing, so that the area of interaction between allicin and the antibiotic was larger. Also, no inconclusive results were found when using a concentration of 500 ppm, but which were obtained using a concentration of 250 ppm (Table 10 - are the results marked?).

The results presented in table. 9 show antibiotic combinations selected for further research. Twelve combinations were selected, all of which had an assessment of the degree of reaction greater than 5. Combinations with ratings above 5 that were not selected contained antibiotics belonging to the group already studied (for example, the group of aminoglycosidases). Gentamicin and tobramycin, both, were chosen because of their usual use in treating patients and their high grade scores.

In tab. 9 shows the results of a study of the comparative synergistic activity of 22 antibiotics with allicin at a concentration of 500 ppm. Evaluation of the degree of reaction was associated with a possible degree of synergism, which was determined using diffusion tests in agar.

Table 9

Antibiotic Oxford ΜΚ3Α103 Mnza102 E con Evaluation Test Noticing. Tetracycline 3 3 3 2 eleven Yes Gentamicin 3 3 3 one ten Yes aminoglycosidase Fuzidinovy acid 3 3 3 0 9 Yes Rifampicin 3 3 3 0 9 Yes Tobramycin 3 2 3 one 9 Yes aminoglycosidase Novobiocin 3 3 3 0 9 Yes Amikacin 3 3 2 0 eight aminoglycosidase Vancomycin 2 3 2 0 7 Yes Ciprofloxacin 3 3 0 0 6 Yes Erythromycin 3 0 3 0 6 Yes Cefuroxime 3 2 0 0 five Yes Moxolactam 3 2 0 0 five Yes Piperacillin 3 one 0 one five Yes Streptomycin one one 2 0 four aminoglycosidase Ampicillin 3 one 0 0 four Carbenicillin 3 one 0 0 four Cefamendol 3 one 0 0 four Chloramphenicol 3 one 0 0 four Amoxicillin 3 0 0 0 3 Methicillin 3 0 0 0 3 Penicillin 6 3 0 0 0 3 Metronidazole 0 0 0 0 0

- 14 013519

Although a control strain sensitive to antibiotics (Oxford) showed the highest number of high grades for the degree of reaction, MK8A strains also showed good potential and possible synergism of tetracycline with E. soi was noted. FIG. Figures 5–9 demonstrate how the various synergistic interactions are distributed across a degree.

These trials clearly demonstrated the potential antimicrobial synergistic effect of allicin and a wide variety of antibiotics against arrhythmia, including MK8A.

Syograpr-c

EGU-E

RCB as] 8PA

Table 10

MEnME

micr-gyurganism a cup WITH AND I E A1 K RA BUT! TO Me ΑΙ TH OhTOTs 8a 250 31 Sv N 26 38? 35 39 8 27 38 5 500 25 45? 26 48 N 33 48 8 thirty 48 5 MKZA102 250 22 40 N 24 37? 32 37 5 0 37 N 500 26 48 5 25 43? 32 45 3 ten 45? 1ЛН8А103 250 25 40 5 ten 42 N 33 40 3 15 40 N 500 25 47 8 ten 48 N 37 47 3 ten 47 N SOP K12 250 2P 23 N but 23 N 14 23 N 17 14 N 500 25 26 N 25 26 N 25 26 N 25 26 N

Rep © -P ©

Ϊ́βΐt

MouoNo '

microorganism a cup But BUT! TO Ro A / C 81 m K T AND I OhTog <1 For 250 29 38 3 32 38 8 20 39 N 37 33 N 500 33 45 3 36 45 3 20 45 N 38 45 8 MPZ A102 250 26 40? 0 37 " 15 36 N 29 36 3 500 31 45 8 0 43 N sixteen 42 N thirty 45 8 Mnza103 250 23 403 0 40 N 18 40 3 thirty thirty? 500 thirty 45 3 0 44 N sixteen 46 3 32 46 5 E con K12 250 20 14 N 20 14 N 17 23 N 23 1B N 500 25 28 N 27 26 N 18 24 N 25 26 N

Sytoshr - chloramphenicol; EGU111 - erythromycin; EIC asI is fuzidinic acid; Me111 - methicillin; Νονοό - novobiocin; Rep O - penicillin O; 8 (ger - streptomycin; Te! - tetracycline.

K = result, A1 = size of allicin zone.

Results - 8 = synergism, N = no synergism,? = inconclusive data, A = possible antagonism.

Claims

1. An antibacterial or pediculocidal preparation containing allicin or an allicin metabolite and a pharmaceutically acceptable excipient and additionally containing at least one other antibacterial or pediculocidal agent.

2. The drug according to claim 1, wherein the other agent is selected from (ί) penicillins, (ίί) aminoglycosidases, (w) tetracyclines, (ίν) macrolides, (ν) cephalosporins and cefamycins and (νί) fusidic acid, rifampicin, novobiocin , vancomycin, ciprofloxacin, chloramphenicol or metronidazole.

3. The drug according to claim 1 or 2, in which the metabolite of allicin is selected from ΌΑΌ8 (diallyldisulfide), ΌΑΤ8 (diallyl trisulfide), ezo, allitridium and vinyl dithiines.

4. The preparation according to any one of claims 1 to 3, in which the pharmaceutically acceptable excipient is a solid composition on which allicin is bound.

5. The preparation according to claim 4, in which the solid composition contains a modified starch, such as maltodextrin, gum arabic, silicon dioxide and an emulsifier, such as magnesium stearate.

6. The preparation according to any one of claims 1 to 3, in which the pharmaceutically acceptable excipient is a cream or soap.

- 15 013519

7. The drug according to claim 6, in which the excipient includes white semi-solid paraffin, emulsifier, glycerin, water, yellow semi-solid paraffin and a stabilizer.

8. The preparation according to claim 7, in which the emulsifier is a stearate, preferably magnesium stearate.

9. The drug of claim 8, in which the excipient includes oxysulfate, cocamide and cocobetaine.

10. The preparation according to any one of claims 4 to 9, in which the ratio of allicin to excipient is such that it provides a concentration of allicin from 1 to 2000 ppm, preferably from 50 to 1000 ppm, more preferably from 250 to 500 ppm