facebook
twitter
vk
instagram
linkedin
google+
tumblr
akademia
youtube
skype
mendeley
Wiki
Global international scientific
analytical project
GISAP
GISAP logotip
Перевод страницы
 

A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

 A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS
Александр Присяжнюк, студент

Николай Блажеевский , профессор, доктор химических наук, профессор

Харьковский национальный фармацевтический университет, Украина

Участник первенства: Национальное первенство по научной аналитике - "Украина";

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop, and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying, on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).

Fig. 1. Structure of diperoxyazelaic acid
 

The basis for ointments of diperoxyazelaic acid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble bases on Pharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands, are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidin activation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinic acid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly (ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly (ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006), 20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consisting is determined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1.

The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

  • 1. Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.
  • 2. Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 
  • 3. Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.
  • 4. Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.
  • 5. Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php
  • 6. Potekaev N.N., Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.
  • 7. T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.
  • 8. Kolyadenko V.G., Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.
  • 9. Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.
  • 10. Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.
  • 11. Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.
  • 12. Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.
  • 13. Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.
  • 14. Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.
  • 15. A Guidance Ministry of Health in Ukraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

A COMPARATIVE

 

A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Picture1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

 

A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Fig.1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Fig.1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

 

A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Fig.1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Fig.1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

A COMPARATIVE STUDY OF THE ANTIBACTERIAL ACTIVITY OF OINTMENT BASED ON DIPEROXYAZELAIC AND UGRESOL 10% AND ACNESTOP 20% PREPARATIONS

Oleksandr Prysiazhniuk, the 4th year student of Biotechnology Speciality;

National University of Pharmacy, Ukraine;

Blazheyevskiy Mykola, Dr. of Chemistry, Full Prof.

National University of Pharmacy, Ukraine;

National Research Analytics Championship,

Open European-Asian Research Analytics Championship,

 

The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

Keywords: acne, benzoyl peroxide, azelaic acid, diperoxyazelaic acid, antibacterial preparations.

Introduction. It is known that inflammation of the skin are accompanied by inflammation bacterial contamination and therefore require the use of antimicrobial prolonged action. This effect can be achieved by using external antimicrobial agents in the form of ointments, creams and gels. [1] In present among the large number of proposed drugs for the treatment of acne stipulates the dosage form surface action based on azelaic acid and benzoyl peroxide. Azelaic acid (HOOC(CH2)7COOH) - the active pharmaceutical ingredient (API) of a famous cream called Skinoren® 20% (Schering) is the antibiotic drug, which is produced by the yeast Pityrosporum acne, it inhibits the growth of propionic bacterias and the formation of fatty acids contribute to the occurrence of acne. In turn the API of a drug called Ugresol 10% (Pharmascience Inc., Canada), benzoyl peroxide (C6H5C(O)OO(O)C6H5) effectively regulates the processes of keratinization in the sebaceous follicles, improves oxygenation of tissues, reduces the level of free fatty acids in the lipid tissues has antimicrobial effect, especially with regard to Propionibacterium acne and Staphylococcus epidermidis [2-10].

Research of interest clarifies the applicability as an active pharmaceutical ingredient in preparations of surface action regarding acne diperoxyazelaic acid, which, unlike benzoyl peroxide, not capable of radical decomposition, and therefore will not have the ability to enhance carcinogenesis by UV radiation.

The aim of the study was a comparative study of antimicrobial activity of the ointment on the basis of our proposed new substance - diperoxyazelaic acid (hereinafter both DPAA) (Fig. 1) and two European branded drugs - AkneStop 20% (Corporation "Arterium", Kyiv) and Ugresol Lotion 10% (Brij 30, disodium EDTA, carbomer 940) (Pharmascience Inc., Canada).


Fig.1 – Structure of diperoxyazelaicacid

The basis for ointmentsofdiperoxyazelaicacid was elected polyethylene oxide (PEO). This is the most common excipient of all known water-soluble basesonPharmacopoeias most part of the world. The advantages of PEO: a satisfactory water solubility, the ability to dissolve the hydrophilic and hydrophobic drugs, well mixed with wax and glycerides to form stableof pseudo-emulsion well applied to the skin, does not prevent gas exchange and does not affect the activity of glands are weak bactericidal effect due to the presence of primary hydroxyl groups osmotically active. All PEO baseareneutral, non-toxic, physiologically neutral, long-term use does not damage the skin, easy to release drugs is not the environment for the development of microflora. Through active mixed with other substances, these bases can be used for all soluble and most water-insoluble drugs.

Peroxyacids mechanism of action is as follows (the following). Compounds with active oxygen used in the composition of disinfectants and antimicrobial agents, representing hydrogen peroxide, perborate, persulfate, percarbonate et al., As well as peracids, hydrogen peroxide oxidation obtaining carboxylic acids. They can be seen as a replacement product or just the two hydrogen atoms in a molecule of hydrogen peroxide to acyl groups.

Compounds with active oxygen belong to the group of disinfectants oxidants. Under the influence Lane oxide group is oxidinactivation of lipids and proteins essential elements of microorganisms, bacterial cytoplasmic membrane and membranes of the spore forms of bacteria. Lipid peroxidation, most pronounced on unsaturated fatty acids, this leads to a decrease hydrophobicity and increasing membrane permeability. And changing membrane proteins through the formation of protein-lipid complexes, oxidation and denaturation of proteins containing -SH group and the possible formation of cross-links in amino (NH2). Inhibition of redox enzymes leads to respiratory failure microbial cells. Study of oxygen-containing drugs on the cells of bacteria and spores of bacillus showed that all tested microorganisms lose protein and 90%; DNA - to (30-50)% and the RNA- to 30%, and spore forms also lose 25% dipicolinicacid. Equally significant washout of intracellular components demonstrates the rapid violating the integrity of bacterial cells and spores [11-12].

  Material and methods. Diperoxyazelaic acid was synthesized by a known method of Swern [13] according to the scheme (in the present sulfuric acid):

НО(О)С(СН2)7С(О)ОН+Н2О2 = НОО(О)С(СН2)7С(О)ООН + H2O.

Diperoxyazelaic acid (nonanebis (peroxoic acid), T mp. + 90-90,5ºC (with decomp.), the content of active oxygen species (AOC) 14,2%; рКа1=8,08, рКа2=9,19.

To confirm the stability of DPAA, one sample was kepted in a self-sealing bag at room temperature (30–35 °C). The active oxygen content of the sample was determined by iodometric titration after every 10-15 days. It was found that it retains its active oxygen content over a period of 50 days (14,2-14,1%). There was no change observed in the physical appearance, too. This confirms the stability of DPAA at room temperature.

An ointment composition: 0.8-1.2 wt.% DPAA; Poly(ethylene oxide), MW400(PEO 400) (Specif. 2483 007 71150986 2006), 78,0-79,4 wt.%, Poly(ethylene oxide), MW1500(PEO-1500) (Specif. 2483-008-71150986-2006),20,19-19,38 wt.%; disodium EDTA (0,1-0,02 wt.%). The content of DPAA ointment consistingisdetermined experimentally reduce its content in the composition of the drug leads to a marked reduction of antimicrobial activity. The composition of water-soluble hydrophilic ointment base with the specified content PEO 400, PEO-1500, disodium EDTA was also chosen experimentally. Due to optimal components of this framework is the most appropriate, since promotes optimal release of active substances, chemically indifferent with respect to the drug and is convenient in terms of technology in the production of ointments.

The study of antimicrobial activity of the objects carried by agar diffusion, based on the ability of active substances to diffuse in the culture medium, previously inoculated microorganisms. According to WHO recommendations for evaluation of antibacterial and antifungal activity of drugs as a test culture was used by museum strains (gram-negative bacillus Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Gram-positive cocci Staphylococcus aureus ATCC 25923 and spore-forming bacillus Bacillus subtilis ATCC 6633, yeast fungi Candida albicans ATCC 653/885) and clinical isolates (Candida tropicalis, Candida krusei, Candida glabrata). In the experiment used daily suspension of microorganisms in saline. Microbial load was 1 ∙ 107 colony forming units in 1 ml of nutrient medium [14,15].

Antimicrobial activity of 1% ointment DPAA studied in 1 day after preparation, 2 weeks and 1 month deposit. The results determine the antimicrobial activity of 1% ointment DPAA and preparations comparison presented in Table 1 showsthat 1% ointment DPAA detect antimicrobial action with respect to C. albicans and C. tropicalis level comparator AkneStop 20%. While antibiotic ointment investigated the effect DPAA against Gram-negative (E. coli, Ps. Aeruginosa) and gram-positive bacteria (St. aureus, B. subtilis) far exceeds the effect of other investigational drugs. As seen from the data in Table 1, ointment based DPAA revealed high antimicrobial activity to the St. aureus, E. coli, Ps. aeruginosa and C. albicans, less pronounced - as B. subtilis, C. tropscalis, C. krusei and C. glabrata.

Table 1. The results determine the antimicrobial activity

Testculture

Zones of growth inhibition test cultures mm (n = 3)

Ointment based DPAA 1%

After 1 month of storage

Staphylococcus aureus

45,4±1,3

Escherichia  coli

45,0±0,4

Pseudomonas  aeruginosa

38,8±2,6

Bacillus subtilis

22,7±2,8

Candidatropscalis

23,7±1,4

Candida albicans

31,3±3,7

Candida krusei

21,5±1,5

Candida glabrata

14,8±1,7

Aknestop 20%

 

Staphylococcus aureus

35,1±0,4

Escherichia  coli

21,3±0,7

Pseudomonas  aeruginosa

29,0±1,2

Bacillus subtilis

15,0±0,7

Candidatropscalis

22,3±0,3

Candida albicans

27,1±1,1

Candidakrusei

22,4±0,6

Candida glabrata

9,0±0,5

Ugresol 10%

 

Staphylococcus aureus

15,1±1,2

Escherichia  coli

12,8±1,3

Pseudomonas  aeruginosa

-

Bacillus subtilis

16,1±1,2

Candida tropscalis

17,7±0,5

Candida albicans

24,5±0,5

Candida krusei

-

Candida glabrata

-

Note. "-" - A zone of stunted growth of microorganisms available.

Consequently, studies have shown that the new DPAA ointments based on hydrophilic matrix sufficiently high inherent antimicrobial activity, which dominated European brands, and therefore suggests its promising for further in-depth research.

 

Conclusions. The diperoxyazelaic acid was synthesised. Ointment on the polyethylenoxide basis containing 1% diperoxyazelaic acid was obtained. The antimicrobial activity of the ointment and branded drugs Acne Stop and Ugresol have been explored in comparative perspective. The shelf life of creams was clarified and was made a conclusion about the prospects for further deep studying on the subject of it application as extemporal dosage form.

 

References:

1.      Drohovoz S.M, Shchokina K.G. Pharmacologyinpalms. Handbook for pharmacists, doctors and students of medical and pharmaceutical universities. Kharkiv. 2009. - 110 p.

2.      Fluhr J.W., Degitz K. // J. Dtsth. Dermatol. Ges. – 2010. – V. 8, Suppl. 1. – P. 24-30. 

3.      Worret W.I., Fluhr J.W. // J. Dtsth. Dermatol. Ges. – 2006. – V. 4, № 4. – P. 293-300.

4.      Harper J.C. // J. Dtsth. Dermatol. Ges. – 2010. – V.9,  № 5. – P. 482-487.

5.      Usenko G.D., Zupanets I.A., Bezdetko N.V. // pharmacist. - 2002. - №19. Access mode: http://www.provisor.com.ua/archive/2002/N19/art_17.php

6.      Potekaev N.N.,Andreeva L.V.// A Journal of Dermatology and Venereology - 2001. - № 6. - P. 32-35.

7.      T. Protsenko, A. Kaplan // Ukrainian Journal of Dermatology, Venereology, Сosmetology - 2007. - № 2 (25). - P. 12-14.

8.      Kolyadenko V.G.,Usenko G.D.// Weekly Pharmacy. - 1997. - № 27. - P. 42-43.

9.      Stepanenko V.І. // Klіnіchna іmunologіya, alergіya, іnsektologіya. - 2010. - №1. - P. 86-96.

10.  Usenko G.D. // Practitioners Magazine - 1997.- № 2.- p.p.11-14.

11.  Paly H.K., Antisepticsin the prevention and treatment of infections // Authors; Ed. GK Paly. - Kyiv .: Health. - 1997. -201 p.

12.  Krasilnikov A.P.Handbook on antiseptic. - App. : High society. HQ., 1995. - 367 p.

13.  Winfred E.,  Witnauer L. P.,  Swern D.//J. Am. Chem. Soc.- 1957.- Vol. 79, №8.- P. 1929–1931.

14.  Guidelines "Studying the specific activity of antimicrobial drugs." - Kyiv., 2004. - 38 p.

15.  A Guidance Ministry of Health inUkraine "Determination of the sensitivity of microorganisms to antibiotics." - Kyiv., 2007, N 167. MB 9.9.5 - 143-2007.

 

1. PrysiazhniukOleksandr(Присяжнюк Олександр Васильович)

2. 4thyearstudent, «Biotechnology» specialіty(студент 4 курсу, спеціальність «Біотехнологія»)

NationalUniversityofPharmacy(Национальный фармацевтический университет)

3.National University of Pharmacy (Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,   м. Харків,  вул. Валентинівська 4

Mobtel. /тел.  +380500113461.Email: prisyaga07@gmail.com

4. Контактна інформація для одержання збірника і сертифікату:

Блажеевский Николай

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 

1.BlazheyevskіyMykola, (Блажеєвський Микола)

2.Doctor of Chemistry, professor, professor at the department of physical and colloid chemistry

(Доктор хімічних наук, професор, професор кафедри фізичної та колоїдної хімії)

3.NationalUniversityofPharmacy, Національний фармацевтичний університет (м. Харків) National University of Pharmacy,

Ukraine, Kharkiv, st. Valentynivs’ka 4

індекс: 61168,  м. Харків,  вул. Валентинівська, 4

4. Контактна інформація для одержання збірника:

Блажеевский Николай Евстафьевич

проспект Юбилейный, 70 кв. 79

Харьков-118, Украина, 61118

Tel. (0572) 97-98-38

Mob.tel: (098)974-99-28

 Email:  blazejowski@ukr.net

 

0
Ваша оценка: Нет Средняя: 9 (2 голоса)
Комментарии: 4

Сметанина Екатерина Ивановна

Уважаемые коллеги! Чудесная перспективная работа, которая представляет собой фармацевтическую разработку нового перспективного ЛС. Удачи Вам в дальнейших научных поисках

Присяжнюк Александр Васильевич

Уважаемая Екатерина Ивановна, благодарим Вас за интерес, проявленный к нашей работе и высокую оценку! С уважением Присяжнюк Александр.

Ковальская Елена Васильевна

У статті наведені результати вивчення протимікробної активості мазі на основі синтезованої нової оригнальної сустанції дипероксиазелаїнової кислоти у порівнянні з європейськими брендовими препаратами на основі азелаїнової кислоти та бензоїлу пероксиду. Покана суттєво вища активність запропонованої мазі і зроблений висновок про перспективність її на предмет подальшого поглибленого вивчення для застосування як лікарського засобу, виготовленого в умовах аптеки. Відмінна робота! Вітаю студента Олександра і бажаю подальших успіхів у науковій роботі під керівництвом професора Миколи Блажеєвського.

Присяжнюк Александр Васильевич

Шановна Елена Василівна! Дякую за проявлений інтерес до публікіації. Бажаю і Вам, у подальшому, прогресивної роботи та натхнення! З повагою, Присяжнюк Олескандр.
Комментарии: 4

Сметанина Екатерина Ивановна

Уважаемые коллеги! Чудесная перспективная работа, которая представляет собой фармацевтическую разработку нового перспективного ЛС. Удачи Вам в дальнейших научных поисках

Присяжнюк Александр Васильевич

Уважаемая Екатерина Ивановна, благодарим Вас за интерес, проявленный к нашей работе и высокую оценку! С уважением Присяжнюк Александр.

Ковальская Елена Васильевна

У статті наведені результати вивчення протимікробної активості мазі на основі синтезованої нової оригнальної сустанції дипероксиазелаїнової кислоти у порівнянні з європейськими брендовими препаратами на основі азелаїнової кислоти та бензоїлу пероксиду. Покана суттєво вища активність запропонованої мазі і зроблений висновок про перспективність її на предмет подальшого поглибленого вивчення для застосування як лікарського засобу, виготовленого в умовах аптеки. Відмінна робота! Вітаю студента Олександра і бажаю подальших успіхів у науковій роботі під керівництвом професора Миколи Блажеєвського.

Присяжнюк Александр Васильевич

Шановна Елена Василівна! Дякую за проявлений інтерес до публікіації. Бажаю і Вам, у подальшому, прогресивної роботи та натхнення! З повагою, Присяжнюк Олескандр.
Партнеры
 
 
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
image
Would you like to know all the news about GISAP project and be up to date of all news from GISAP? Register for free news right now and you will be receiving them on your e-mail right away as soon as they are published on GISAP portal.