Evaluation of in vitro and in vivo antibacterial and antifungal activity of “camelyn m”

Benedikte Maglakelidze1, Guguli Abashidze1, Inga Dadeshidze2, Vakxtang Mshvildadze3, Andre Pichete3, Vincent Perreten4, Shota Tsanava5, Nata Shubladze6, Koba Gurielidze1* 1*. Scientific-Pharmaceutical Company “Camelyn”, 0127, Kindzmarauli str. 7, Tbilisi, Georgia. 2. Kutateladze Institute of Pharmacochemistry, 0159, sarajishvili str. 36, Tbilisi, Georgia. 3. Laboratoire LASEVE, Universiteґ du Queґbec a` Chicoutimi, 555, Boulevard de l’ Universiteґ, Chicoutimi, Que.,

Canada G7H 2B1. 4. Institute of Veterinary Bacteriology,University of Berne,Postfach Länggass-Strasse 122, CH-3001 Berne,Switzerland. 5. National Center for Disease Control & Medical Statistics, 0177, Asatiani 9, Tbilisi, Georgia. 6. National Center for Tuberculosis and Lung Diseases, 0101, Maruashvili 50, Tbilisi, Georgia. *Corresponding author: Scientific-Pharmaceutical Company “Camelyn”, 0127, Kindzmarauli str. 7, Tbilisi, Georgia. E-

mail: HUkgurielidze@yahoo.comU

The minimum inhibitory concentration (MIC) of “Camelyn M” active compound obtained from special sort of honey in one of region of Georgia, was determined both by agar and broth dilution methods against some of strains of bacteria and fungy. The antibacterial action of “Camelyn M” was further tested in animal models. “Camelyn M” was seen to possess powerful inhibitory action (0,012-0,150 µg/mL ) against most test bacteria in in vitro studies. In vivo studies showed that the drug offered significant protection (p<0.001) to mice challenged with a virulent bacterium.

“Camelyn M” exhibited potent in vitro activities against fluconazole-resistant strains of Candida albicans , Candida

glabrata, Candida tropicalis, Candida parapsilosis and Candida krusei, with MICs at which 90% of isolates were inhibited of 0.012 µg/ml, respectively.

Key words: “Camelyn M”, honey, antimicrobial, Candida, MIC.

Introduction

Antibiotics are one of our most important weapons in fighting bacterial and fungal infections and have greatly benefited the health-related quality of human life since their introduction. However, over the past few decades these health benefits are under threat as many commonly used antibiotics have become less and less effective against certain illnesses not only because many of them produce toxic reactions but also due to emergence of drugresistant bacteria and fungy(2,3,4,5,6,7,8). “Camelyn M” were isolated from special species of honey from one of region of Georgia (1). “Camelyn M” is a mixture of different biologically active compounds: aldehydes, ketones, bioorganyc acides, (6). Different physicochemical parameters of Camelin’s compounds, such as solubility, primary structure, molecular weight, and other properties play a significant role in the biological activities of “Camelyn M” (6 ). Our studies demonstrated that “Camelyn M” has strong antibacterial and antifungal activity in a wide variety of bacterial and fungal species. “Camelyn M” acts by as detergent on the bacterial and fungal cell membranes and selectively inhibiting wearies of living mechanism of bacterial and fungal cells. The present paper describes the detailed in vitro and in vivo antibacterial and antifungal activity of “Camelyn M”.

Materials and Methods

Media

Liquid media used for this study were peptone water [PW; Oxoid brand bacteriological peptone 1%(w/v) plus Analar NaCl 0.5%(w/v)], nutrient broth (NB; Oxoid), Mueller Hinton broth (MHB; Difco). Solid media were peptone agar (PA), bromothymol blue lactose agar media (BLA), nutrient agar (NA) and Mueller Hinton agar (MHA), obtained by solidifying the liquid media with 1.2%(w/v) agar (Oxoid No.3). Antibacterial and Antifungal agents. “Camelyn M” was isolated from honey as described previously ( 1 ) for the in vitro and in vivo studies. For the in vitro study, fluconazole (FLC) and itraconazole (ITC) were extracted from commercial preparations purchased from PSP Pharmaceuticals, Inc. (Tbilisi, Georgia.)

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Organisms:

Bacteria and fungy:A total of strains of bacteria and fungy were obtained from the American Type Culture Collection .

Media

Liquid media used for this study were peptone water [PW; Oxoid brand bacteriological peptone 1%(w/v) plus Analar NaCl 0.5%(w/v)], nutrient broth (NB; Oxoid), Mueller Hinton broth (MHB; Difco). Solid media were peptone agar (PA), bromothymol blue lactose agar media (BLA), nutrient agar (NA) and Mueller Hinton agar (MHA), obtained by solidifying the liquid media with 1.2%(w/v) agar (Oxoid No.3). In case of BLA. In vitro tests. The MICs for the test organisms were determined by the broth microdilution method described in NCCLS document M27-A2 (3,4,5). The MICs of “Camelyn M” were defined as the lowest concentration that resulted in slight growth (approximately 90% inhibition) or the absence of growth at 48 h. In vivo tests Swiss strain of male white mice weighing 18-20g were used for the in vivo studies. The virulence of the test strain S.typhimurium NCTC 74 was exalted by repeated mouse passage and the median lethal dose (MLD or LD50) of the passaged strain corresponding to 0.95x109 CFU/ mouse suspended in 0.5mL NB served as the challenge dose17 for all the groups of animals. Reproducibility of the challenge dose was ensured by standardization of its optical density in a Klett-Summerson colorimeter at 640nm and determination of the CFU count in NA. To determine the toxicity of “Camelyn M”, 40 mice were taken, 20 of which were injected 60g of the drug, and the rest 20 received 30g of “Camelyn M”. They were kept under observation upto 100 hours. Two groups of mice, 20 animals per group (each mouse weighing about 20g) were kept in separate cages. Group I was intraperitoneally administered 30g “Camelyn M” per mouse (0.1mL from 300g/mL solution of “Camelyn M”), and group II was given 60g of the drug per mouse (0.1mL from 600g/mL solution of “Camelyn M”). After 3 hours, each group I and II was challenged with 50 MLD of S.typhimurium NCTC 74. A control group of 60 mice was also injected similarly with the same bacterial strain, and 0.1mL sterile saline instead of “Camelyn M” The protective capacity of the drug was determined by recording the mortality of the mice in different groups upto 100 hours of the treatment, and statistically by x2 test. In another experiment, 4 groups of mice, 5 animals per group, were taken. Groups 1 and 3 were administered 60 g of “Camelyn M”, while groups 2 and 4 were given 0.1mL sterile saline. After 3 hours, all the groups were given a 50 MLD challenge of S.typhimurium NCTC 74. After 2 hours, groups 1 and 2 were sacrificed. Their heart blood was collected aseptically; their livers and spleens were removed aseptically and homogenised in tissue homogenisers. CFU counts of the individual organs were determined separately. The same procedure was applied on groups 3 and 4, 18 hours after the challenge. Statistical analysis of the in vivo data was done by Student’s t-test. The concentration of “Camelyn M” in mouse blood was assayed by measuring the diameter of the inhibition zones by serum-soaked filter paper discs (6 mm diameter, 3 mm thick, Millipore, absorbing 0.0 3mL volume) on a lawn flooded with bacteria from an 18 hours broth culture of S. typhimurium 74 on peptone agar. The drug concentrations in the sera were determined by referring these values to a standard calibration curve prepared with known concentrations of the drugs.

Results:

All the bacterial strains tested were found to be resistant to many antibiotics. However, “Camelyn M” showed powerful antimicrobial action against all the bacteria“Camelyn M” at a range of concentration of 0,156-3.0 µg/ml inhibited all bacterial strains:

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Fig 1. MICs of “Camelyn M” for specific antibiotic resistant bacterias as determined by broth dilution in Müller-Hinton broth . In vertical- strains of: In horizontal- MICs of “Camelyn M” A) - E. faecium SF11770 1) - 0.002 9) - 0.62 B) - B. antracis ChadA8 2) - 0.004 10) - 1.25 C) - S. aureus BM3318 3) - 0.009 11) - 2.50 D) - B. antracis JFS854 4) - 0.019 E) - S.typhimurium NCTC 74 5) - 0.039 F) - S. sciuri CSLA3 6) - 0.078 G) - S. haemolyticus VPS617 7) - 0.156 H) - B. cereus AND934 8) - 0.310

A B

Fig 2. MICs of “Camelyn M” for specific antibiotic resistant bacterias of Bacilus anthracis

A-after 4 houers inoculation and B-after 4 houers inoculation

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Fig 3. MICs of “Camelyn M” for specific multi and monoresistent bacterias of M.tuberculosis

In vitro antifungal activity.

Table 1 shows the spectrum of activities of “Camelyn M” and other reference against various fungal strains. “Camelyn M” exhibited potent activities against C. albicans, C. glabrata, C. tropicalis, C. guilliermondii, and C. neoformans, with MICs ranging from 0.012 µg/ml for all the above listed cases. Table 2 shows the MICs of “Camelyn M” and other reference agents for clinical yeast isolate of C. albicans. Table 2 shows the results for C. albicans separately for FLC-susceptible (FLC-S) strains (FLC MICs, 8 µg/ml) and FLC-susceptible dose-dependent (FLC-S-DD) and FLC-resistant (FLC-R) strains (FLC MICs, 16 µg/ml), according to the guidelines of NCCLS document M27-A2 (14). “Camelyn M” exhibited potent activities against C. albicans (FLC-S), with MICs at which 90% of isolate are inhibited (MIC90s) of

0.012 µg/ml. “Camelin M” also exhibited potent activities against the FLC-S-DD and FLC-R strains of C. albicans (MIC range, 0.012 µg/ml).

TABLE 1. Antifungal spectrum of “Camelyn M”

Organism and strain MIC (µg/ml)

Camelyn M FLC ITC AMB Candida albicans ATCC 24433 0.012 0.25 0.016 0.12 Candida glabrata ATCC 90030 0.012 4.00 0.12 0.12 Candida tropicalis ATCC 750 0.012 2.00 0.06 0.25 Candida parapsilosis ATCC 22019 0.012 2.00 0.03 0.5 Candida krusei TIMM3378 0.012 32.00 0.06 0.25 Candida guilliermondii ATCC 9390 0.012 2.00 0.03 0.06

TABLE 2. In vitro antifungal activity of “Camelyn M” against clinical isolate of Candida albicans.

Organism (clinical isolates) and agent MIC (µg/ml)

50% 90% Candida albicans “Camelyn M” 0.012 0.012 FLC 0.25-8 1.00 ITC 0.016 0.03 AMB 0.12 0.12

”Camelyn M” belongs to the natural, antibacterial and antifungal agents with a complex mechanism of action. The fact that "Camelyn M" comprises biologically very active phenolic compounds is very interesting. This compounds may be met in leaves and rootstocks of many plants and ensure of resistance of these plant with respect to different pathogens. Natural phenolyc compoumds is characterized with strong detergent properties and at occurrence in small

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size organisms cause destruction of the cell membrane. Resulting from the mentioned, it may be presumed that the biological activity of "Camelyn" includes the above mechanisms, which of cause is a subject of deeper investigations. As it is seen from Tables 1 and 2 "Camelyn M" displays strong in vitro activity with respect to all the test strains in the same concentration -MIC (µg/ml)=0.012. This concentration is more low than all the antibiotics tested by us. In conclusion, the results of the present study suggest that “Camelyn M” is a promising compound, in particular, for the treatment of disseminated or bacterial and mucosal infections induced by C. albicans, including FLC-resistant strains.

References:

[1] B.Maglakelidze. “Antitumoral properties of “Camelyn” and mechanism of activity” Monograf, Tbilisi, 2004 (in Georgian) [2] Berdy, J., 1967. Recent developments of Antibiotic Research and Classification of Antibiotics According to Chemical Structure,

Advances in Applied Microbiology, 309.

[3] Bergey’s manual of determinative bacteriology, 2000. Actinomycetales. 9th

edition.

[4] 4. Hugo, W.B., Russel, A. D., 1983. Pharmaceutical Microbiology, 3rd

edition, Blackwell Scientific publications. [5] Barchiesi, F., A. M. Tortorano, L. F. Di Francesco, M. Cogliati, G. Scalise, and M. A. Viviani. 1999. In-vitro activity of five

antifungal agents against uncommon clinical isolates of Candida spp. J. Antimicrob. Chemother. 43:295-299. [6] Kamai, Y., M. Kubota, T. Fukuoka, Y. Kamai, N. Maeda, T. Hosokawa, T. Shibayama, K. Uchida, H. Yamaguchi, and S.

Kuwahara. 2003. Efficacy of CS-758, a novel triazole, against experimental fluconazole-resistant oropharyngeal candidiasis in mice. Antimicrob. Agents Chemother. 47:601-606

[7] Herreros, E., M. J. Almela, S. Lozano, F. Gomez de las Heras, and D. Gargallo-Viola. 2001. Antifungal activities and cytotoxicity studies of six new azasordarins. Antimicrob. Agents Chemother. 45:3132-3139.

[8] Herreros, E., C. M. Martinez, M. J. Almela, M. S. Marriott, F. Gomez de las Heras, and D. Gargallo-Viola. 1998. Sordarins: in vitro activities of new antifungal derivatives against pathogenic yeasts, Pneumocystis carinii, and filamentous fungi. Antimicrob. Agents Chemother. 42:2863-2869.

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