COMPUTERIZED CALCULATION OF BIOLOGICAL POTENCY OF ANTI- BIOTICS: INTERACTIVE COMPUTER PROGRAM '' POTENCY v.2'' (TEST:''5+3'' DOSES): CALCULATION OF

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1 COMPUTERIZED CALCULATION OF BIOLOGICAL POTENCY OF ANTI- BIOTICS: INTERACTIVE COMPUTER PROGRAM '' POTENCY v.2'' (TEST:''5+3'' DOSES): CALCULATION OF BIOLOGICAL POTENCY OF NEO- MYCIN AND BACITRACIN ANTIBIOTICS IN ENBECIN OINTMENT Milan B. Arambašić and Radmila Mandić Pharmaceutical factory '' GALENIKA a.d''., Quality Control Center, Dep. Biological Control, YU Beograd-Zemun, Batajnički put b.b., Serbia and Montenegro Contact person: Milan B. Arambašić Pharmaceutical factory '' GALENIKA a.d''. Quality Control Center Department of Biological Control YU Beograd-Zemun Batajnički put b.b. Serbia and Montenegro tel fax

2 1 REZIME IZRAČUNAVANJE BIOLOŠKE AKTIVNOSTI ANTIBIOTIKA POMOĆU RAČU- NARA: PRIKAZ INTERAKTIVNOG RAČUNARSKOG PROGRAMA ''AKTIV- NOST v. 2 '' (TEST ''5+3'' DOZE): IZRAČUNAVANJE BIOLOŠKE AKTIVNOSTI ANTIBIOTIKA NEOMICINA I BACITRACINA U ENBECIN MASTI U radu je prikazan interaktivni računarski program ''AKTIVNOST v. 2'', napisan u programskom jeziku BASIC, na osnovu algoritma za kvantifikovanje rezultata bioloških eksperimenata primenom testa ''5+3'' doze. Izvršavanje programa je prikazano na test-primeru izračunavanja biološke aktivnosti antibiotika neomicina i bacitracina u Enbecin masti (''GALENIKA a.d.'', Beograd). SOMMARIO CALCOLARE DELL' ATTIVITA' BIOLOGICA DEGLI ANTIBIOTOCI MEDIATE IL COMPUTER: LA PRESENTAZIONE DEL PROGRAMMA INTERATTIVO DI COMPUTER ''ATTIVITA v.2 '' (TEST ''5+3'' DOSI): CALCOLARE DELL' ATTIVITA' BIOLOGICA DEGLI ANTIBIOTICI NEOMICINA E BACITRACINA NELL' UNGUENTO ENBECIN Nell' elaborato è stato presentato il programma interattivo di computer ''ATTIVI- TA v. 2'' scrito in lingua di programma BASIC in base all' algoritmo per la quantificazione dei resultati degli esami biologici con l'applicazione del test ''5+3'' dosi. L'esecuzione del programma è stata presentata all'esempio del calcolare delle attività biologiche di antibiotici neomicina e bacitracina nell'inguento Enbecin (''GA- LENIKA a.d.'', Beograd). SUMMARY This paper presents an interactive computer program ''POTENCY v.2'' written in BASIC program language, based on the algorhythm for quantification of biological test results using ''5+3'' dose test. The program execution is presented by an example of the calculation of biological potency of antibiotics neomycin and bacitracin in the medicinal product ENBECIN ointment (''GALENIKA a.d.'', Beograd). Keywords: BASIC program; Biological assay; Microbiological assay; Diffusion method; Zone of inhibition; Assay ''5+3'' doses; Biological potency; Ointment; Antibiotic; Neomycin; Bacitracin

3 2 INTRODUCTION Quality control of starting materials and finished medicinal products can be: chemical, physico-chemical and biological. Biological control includes the assessment of biological potency of antibiotics using the methods of diffusion on agar and turbidimetry. Experimental results comprise a great number of numerical data that should be processed using suitable statistical methods both to evaluate the biological potency of a specified antibiotic and to assess experiment reliability (error limit of the experiment). Both the biological potency of a tested antibiotic and the limit of error of experiment can be calculated manually (based on the existing algorhythms), but a computerized procedure makes the calculation significantly faster and simpler. This paper presents an interactive computer program ''POTENCY v.2'' written in BASIC program language, based on the algorhythm for quantification of biological test results using ''5+3'' dose test. The program execution is presented by an example of the calculation of biological potency of antibiotics neomycin and bacitracin in the medicinal product ENBECIN ointment (''GALENIKA a.d.'', Beograd) using microbiological assay (diffusion method). THEORETICAL DESCRIPTION OF THE PROGRAM POTENCY v.2 The program is designed in such a way to request basic identification to be entered first: name of the tested product (substance), its declared potency and batch number; name of the standard solution, its potency and batch number (code) and, finally test date. The microbiological assay (diffusion method) is based on the comparison of sensitive microorganism inhibition zones obtained with identical standard and test concentrations (1,2). Since the test is carried out with a great number of Petri plates, due to potentially different agar thickness, inhibition zones obtained with identical antibiotic concentrations (standard or sample) may differ in size. To avoid this problem, inhibition zones are subjected to correction by entering first the inhibition zone values obtained with the following concentrations: S1S3, S2S3, S4S3, S5S3, T2S3, T3S3 and T4S3. Based on this information, the program is able to calculate the mean value for inhibition zone diameters obtained with the reference standard concentration S3. Then, based on this, it corrects the values for inhibition zone diameters obtained with standard concentrations S1, S2, S4 and S5 and test concentrations T2, T3 and T4 (3). The program is interactive since, to execute it, it is necessary to enter the following information: 1) values for the standard concentrations S1, S2, S3, S4 and S5; 2) assumed (declared) potency of the tested product. As neither Yugoslav Pharmacopoeia (1) nor European Pharmacopoeia (2) define which statistical method sholud be used for experimental data evaluation ( calculate the potency using appropriate statistical methods ), program ''POTENCY v.2'' was designed applying algorhitm for quantification of biological assays data, wich fulfill

4 3 the requirements of the US Pharmacopoeia: use a log transformation, straight-line method with least squares fitting procedure, and a test for linearity (4). After the values of standard concentrations S1- S5 have been entered, based on the pairs of experimental values for X(i) (the values for standard concentrations S1 (X1), S2 (X2), S3 (X3), S4 (X4) and S5 (X5) and the value for Y(i) (the corrected values for inhibition zone diameters made considering the standard concentrations S1 (Y1), S2 (Y2), S4 (Y4), S5 (Y5) and the reference one S3 (Y3) using the logarithm function with a free member Y = a + b * log X (Eq.1) as the mathematical model which perform linearization of experimental data using log transformation of indipendend variable X only (semi-log transformation), the program determines the values for the following parameters:a (Y-axis intercept) and b (line slope) of linear regression, the relevant value for square error (E), coefficient of correlation (R) and the value for Student test (T) for the calculated coefficient of correlation. Further, based on the parametric values (a and b) obtained by experimental data processing obtained for the set YOBS values (corrected zone inhibition values based on test concentrations T2, T3 and T4) the program calculates XCALC (the relevant test concentration values T2, T3 and T4) and compares them (calculates percentual deviation) to the set values for test concentrations T2, T3 and T4. Then the mean deviation value (%) for calculated test concentrations T2, T3 and T4 is calculated starting from the set T2, T3 and T4 values and, based on the declared potency of the test preparation, calculates the biological potency of the tested antibiotic in appropriate units. RESULTS Results of the application of the ''POTENCY v. 2 '' program (execution of assayexamples measure of program correctness). Neomycin (5) The standard solution was prepared in the following concentrations (in I.U./ml): 6.4 (S1), 8 (S2), 10 (S3), 12.5 (S4) and 15 (S5), while the test sample (assumed potency: 3300 I.U./g) was prepared in concentrations (in I.U./ml): 8 (T2), 10 (T3) and 12.5 (T4). Each concentration was tested on 3 agar plates each with 6 holes. Three holes in each Petri plate were filled with the reference standard concentration S3 and the remaining three holes were filled either with one of the standard or sample concentrations. The test was done twice from a single homogenous sample. Staphylococcus aureus ATCC 6538-P was used as the test organism. Monitored parameter: size of diameter of inhibition zones (in mm).

5 4 FIRST TEST: GROUP S1S NUMBER OF SAMPLES S1S3 = 9 SUM OF SAMPLES S1S3 = MEAN VALUES S1S3 = DISPERSION S1S3 = GROUP S2S NUMBER OF SAMPLES S2S3 = 9 SUM OF SAMPLES S2S3 = MEAN VALUES S2S3 = DISPERSION S2S3 = GROUP S4S NUMBER OF SAMPLES S4S3 = 9 SUM OF SAMPLES S4S3 = MEAN VALUES S4S3 = DISPERSION S4S3 = GROUP S5S NUMBER OF SAMPLES S5S3 = 9 SUM OF SAMPLES S5S3 = MEAN VALUES S5S3 = DISPERSION S5S3 = GROUP T2S NUMBER OF SAMPLES T2S3 = 9

6 5 SUM OF SAMPLES T2S3 = MEAN VALUES T2S3 = DISPERSION T2S3 = GROUP T3S NUMBER OF SAMPLES T3S3 = 9 SUM OF SAMPLES T3S3 = MEAN VALUES T3S3 = DISPERSION T3S3 = GROUP T4S NUMBER OF SAMPLES T4S3 = 9 SUM OF SAMPLES T4S3 = MEAN VALUES T4S3 = DISPERSION T4S3 = NUMBER OF SAMPLES STANDARD GROUP S3 = 63 SUM OF STANDARD GROUP S3 = 1280 MEAN VALUES OF STANDARD GROUP S3 = STANDARD GROUP S NUMBER OF SAMPLES S1 = 9 SUM OF SAMPLES S1 = MEAN VALUES S1 = DISPERSION S1 = CORRECTED MEAN VALUE OF STANDARD GROUP S1 = STANDARD GROUP S NUMBER OF SAMPLES S2 = 9

7 6 SUM OF SAMPLES S2 = MEAN VALUES S2 = 19.9 DISPERSION S2 = CORRECTED MEAN VALUE OF STANDARD GROUP S2 = STANDARD GROUP S NUMBER OF SAMPLES S4 = 9 SUM OF SAMPLES S4 = MEAN VALUES S4 = DISPERSION S4 = CORRECTED MEAN VALUE OF STANDARD GROUP S4 = STANDARD GROUP S NUMBER OF SAMPLES S5 = 9 SUM OF SAMPLES S5 = MEAN VALUES S5 = DISPERSION S4 = CORRECTED MEAN VALUE OF STANDARD GROUP S5 = TEST GROUP T NUMBER OF SAMPLES T2 = 9 SUM OF SAMPLES T2 = MEAN VALUES T2 = DISPERSION T2 = CORRECTED MEAN VALUE OF TEST GROUP T2 = TEST GROUP T NUMBER OF SAMPLES T3 = 9 SUM OF SAMPLES T3 =

8 7 MEAN VALUES T3 = DISPERSION T3 = CORRECTED MEAN VALUE OF TEST GROUP T3 = TEST GROUP T NUMBER OF SAMPLES T4 = 9 SUM OF SAMPLES T4 = MEAN VALUES T4 = DISPERSION T4 = CORRECTED MEAN VALUE OF TEST GROUP T4 = ENTER VALUE OF STANDARD CONCENTRATION S1 =? 6.4 ENTER VALUE OF STANDARD CONCENTRATION S2 =? 8 ENTER VALUE OF STANDARD CONCENTRATION S3 =? 10 ENTER VALUE OF STANDARD CONCENTRATION S4 =? 12.5 ENTER VALUE OF STANDARD CONCENTRATION S5 =? 15.6 PAIRS (X VALUES OF STANDARD CONCENTRATIONS S1-S5, Y COR- RECTED MEAN VALUES OF INHIBITION ZONES OF STANDARD GROUPS S1-S5) FOR REGRESSION ANALYSIS X,Y? 6.4, X,Y? 8.0, X,Y? 10, X,Y? 12.5, X,Y? 15.6, LOGARITHM FUNCTION WITH A FREE MEMBER Y = A + B * LOG X NUMBER OF DATA POINTS N = 5 SUM OF X = SUM OF X*X = SUM OF Y = SUM OF Y*Y = SUM OF X*Y =

9 8 SLOPE (b) = INTERCEPT ON Y AXIS (a) = Y = a + b * X = * X S.D. (X/Y) = ± SQUARE ERROR = COEF. OF CORR.= T = D.F.= 3 WOULD YOU LIKE A LIST OF REAL VALUES DIFFERENCES? TYPE YES OR PRESS RETURN? YES X YOB YCALC DIFFER E SQUARE ERROR = ASSUMED (DECLARED) POTENCY OF THE SAMPLES: 3300 T2,T3,T4 OBS S2,S3,S4 OBS S2,S3,S4 CALC % POTENCY OF SAMPLES: %, I.E On the same manner was done the 2 nd test with following results: regression equation parameters: (a) , (b) , square error (E) , coefficient of correlation (R) , Student (T) test for 3 degrees of freedom.the biological potency of neomycin in ENBECIN ointment is I.U./g, i.e % The mean biological potency of neomycin in ENBECIN ointment is I.U./g., i.e %. Bacitracin (5) The standard solution was prepared in the following concentrations (in I.U./ml): 0.64 (S1), 0.8 (S2), 1.0 (S3), 1.25 (S4) and 1.5 (S5), while the test sample (assumed potency: 500 I.U./g) was prepared in concentrations (in I.U./ml): 0.8 (T2), 1.0 (T3) and

10 (T4). Microccocus flavius ATCC was used as the test organism.each concentration was tested on 3 agar plates each with 6 holes. Three holes in each Petri plate were filled with the reference standard concentration S3 and the remaining three holes were filled either with one of the standard or sample concentrations. The test was done twice from a single homogenous sample. Monitored parameter: size of diameter of inhibition zones (in mm). FIRST TEST: GROUP S1S NUMBER OF SAMPLES S1S3 = 9 SUM OF SAMPLES S1S3 = MEAN VALUES S1S3 = DISPERSION S1S3 = GROUP S2S NUMBER OF SAMPLES S2S3 = 9 SUM OF SAMPLES S2S3 = MEAN VALUES S2S3 = DISPERSION S2S3 = GROUP S4S NUMBER OF SAMPLES S4S3 = 9 SUM OF SAMPLES S4S MEAN VALUES S4S3 = DISPERSION S4S3 = GROUP S5S NUMBER OF SAMPLES S5S3 = 9 SUM OF SAMPLES S5S3 = MEAN VALUES S5S3 =

11 10 DISPERSION S5S3 = GROUP T2S NUMBER OF SAMPLES T2S3 = 9 SUM OF SAMPLES T2S3 = MEAN VALUES T2S3 = DISPERSION T2S3 = GROUP T3S NUMBER OF SAMPLES T3S3 = 9 SUM OF SAMPLES T4S3 = MEAN VALUES T3S3 = DISPERSION T3S3 = GROUP T4S NUMBER OF SAMPLES T4S3 = 9 SUM OF SAMPLES T4S3 = MEAN VALUES T4S3 = DISPERSION T4S3 = NUMBER OF SAMPLES STANDARD GROUP S3 = 63 SUM OF STANDARD GROUP S3 = MEAN VALUES OF STANDARD GROUP S3 = STANDARD GROUP S NUMBER OF SAMPLES S1 = 9 SUM OF SAMPLES S1 = MEAN VALUES S1 = DISPERSION S1 = 0.389

12 11 CORRECTED MEAN VALUE OF STANDARD GROUP S1 = STANDARD GROUP S NUMBER OF SAMPLES S2 = 9 SUM OF SAMPLRES S2 = MEAN VALUES S2 = DISPERSION S2 = CORRECTED MEAN VALUE OF STANDARD GROUP S2 = STANDARD GROUP S NUMBER OF SAMPLES S4 = 9 SUM OF SAMPLES S4 = MEAN VALUES S4 = DISPERSION S4 = CORRECTED MEAN VALUE OF STANDARD GROUP S4 = STANDARD GROUP S NUMBER OF SAMPLES S5 = 9 SUM OF SAMPLES S5 = MEAN VALUES S5 = DISPERSION S4 = CORRECTED MEAN VALUE OF STANDARD GROUP S5 = TEST GROUP T NUMBER OF SAMPLES T2 = 9 SUM OF SAMPLES T2 = MEAN VALUES T2 = DISPERSION T2 = CORRECTED MEAN VALUE OF TEST GROUP T2 =

13 12 TEST GROUP T NUMBER OF SAMPLES T3 = 9 SUM OF SAMPLES T3 = MEAN VALUES T3 = DISPERSION T3 = CORRECTED MEAN VALUE OF TEST GROUP T3 = TEST GROUP T NUMBER OF SAMPLES T4 = 9 SUM OF SAMPLES T4 = MEAN VALUES T4 = DISPERSION T4 = CORRECTED MEAN VALUE OF TEST GROUP T4 = ENTER VALUE OF STANDARD CONCENTRATION S1 =? 0.64 ENTER VALUE OF STANDARD CONCENTRATION S2 =? 0.8 ENTER VALUE OF STANDARD CONCENTRATION S3 =? 1.0 ENTER VALUE OF STANDARD CONCENTRATION S4 =? 1.25 ENTER VALUE OF STANDARD CONCENTRATION S5 =? 1.56 PAIRS (X VALUES OF STANDARD CONCENTRATIONS S1-S5, Y COR- RECTED MEAN VALUES OF INHIBITION ZONES OF STANDARD GROUPS S1-S5) FOR REGRESSION ANALYSIS X,Y? 0.64, X,Y? 0.8, X,Y? 1.0, X,Y? 1.25, X,Y? 1.56, LOGARITHM FUNCTION WITH A FREE MEMBER Y = A + B * LOG X NUMBER OF DATA POINTS N = 5

14 13 SUM OF X = E-04 SUM OF X*X = SUM OF Y = SUM OF Y*Y = SUM OF X*Y = SLOPE (b) = INTERCEPT ON Y AXIS (a) = Y = a + b * X = * X S.D. (X/Y) = ± SQUARE ERROR = E-03 COEF. OF CORR.= T = D.F.= 3 WOULD YOU LIKE A LIST OF REAL VALUES DIFFERENCES? TYPE YES OR PRESS RETURN? YES X YOB YCALC DIFFER E SQUARE ERROR = E-03 ASSUMED (DECLARED) POTENCY OF THE SAMPLES: 500 T2,T3,T4 OBS S2,S3,S4 OBS S2,S3,S4 CALC % POTENCY OF SAMPLES: %, I.E On the same manner was done the 2 nd test with following results: regression equation parameters:(a) , (b) , square error (E) , coefficient of correlation (R) , Student (T) test for 3 degrees of freedom. The biological potency of bacitracin in ENBECIN ointment is I.U./g, i.e %.

15 14 The mean biological potency of bacitracin in ENBECIN ointment is I.U./g., i.e %. ACKNOWLEDGEMENT The authors thanks Mrs. Zlatica Milutinović for translating the paper into English, and Mrs. Tatjana Josifović for translating the summary into Italian. REFERENCES 1) Pharmacopoeia Jugoslavica 2000, editio quinta (Ph. Jug. V), Book 1, Biological determinations, Mikrobiological determinations of antibiotics, Savezni Zavod za zaštitu i unapređenje zdravlja, Beograd, 2000, p.101.(serbian) 2) European Pharmacopoeia (Eur. Ph. 2002), 4 th Ed., V Microbiological assay of antibiotics, Counsil of Europe, Strasbourg, 2002, p ) Pharmacopoeia Jugoslavica, editio quatra (Ph. Jug. IV), Vol. I, part 9-102, Examination of penicillin contamination, Savezni Zavod za zdravstvenu zaštitu, Beograd, 1984, p.159. (Serbocroatian) 4) USP 26, NF 21: Potencies interpolated from a standard curve, In:<111> Design and analysis of biological assays, United States Pharmacopoeial Convention Inc., Rockville, 2002, p ) USP 26, NF 21: < 81> Antibiotics Microbial assays, United States Pharmacopo- eial Convention Inc., Rockville, 2002, p.2016.