PHARMACEUTICAL AND PHARMACOLOGICAL ASPECTS OF CREATING A NEW COMBINATION DRUG WITH ANTIVIRAL ACTIVITY

Abstract

References

1. substances. The most optimal mobile phase was ethanol-methylene chloride- used as a non-polar component of the mobile concentrated ammonia solution (6:3:1), which phase, the fractional part of which in the eluent ensured the selective separation of the three ac- significantly affected the retention coefficient tive substances and impurity A of acyclovir. of ACV, and to a lesser extent GA and AA. Adsorption zones, visible under UV light, are Thus, chromatography was carried out in the formed as round, clearly defined violet spots following solvent systems of neutral and basic on a greenish background. The detection limit nature: phosphate buffer-acetonitrile (7:3), for GA is 1 μg, for AA – 0.5 μg, and for ACV (8:2), (6:4); chloroform-methanol-water – 0.1 μg, impurity A – 0.1 μg. The Rf values (25:20:5), (30:17:3); isopropanol methylene were 0.33 for GA at the level of the SS GA, chloride-water (6:3:1); isopropanol-methylene 0.66 for AA, 0.54 for ACV, and 0.75 for impu- rity A. Figure 2. Chromatogram of the test solution and comparison solutions I.R. – Test solution (model tablet mix- ture) Validation was carried out in accordance a – SS GA with recommendations, taking into account the b – SS АA specifics of TLC methods [13,14], determining c – SS АЦК the following analytical characteristics: speci- d – SS impurity A ficity, precision (intermediate) and robustness. e – ACV solution for standardization of The values of the validation characteristics of impurities the developed TLC method are presented be- f – solution for determining the suitalow (Table 2-6, Figure 3). bility of the chromatographic system Table 2 –Resolution and selectivity characteristics in the determination of active ingredients in CD Ingredient Rf Spot volume, cm Sensitivity Specificity Impurity A 0.75 0.06 - - ACV 0.66 0.35 PrA-ACV: 4 PrA-ACV: 1.1 AA 0.54 0.93 PrA-AA: 3.6 PrA-AA: 1.2 GA 0.33 0.14 PrA -GA: 8 PrA-GA: 1.6 Table 3 –Retention coefficient values taking into account the standard deviation Analyte sr (%) Rf avg (n=5) Open rate (recovery) (%) Impurity A 0.02 0.75 99.9 ACV 0.02 0.66 101 AA 0.05 0.54 99.8 GA 0.01 0.33 100 Figure 3.Probability distribution graph of Rf values for GA, AK and ACV Table 4Precision of Rf values corresponding to GA on the model mixture of CD Laboratory of the Department of Pharmaceutical Disci- Lab plines Date October 5, 2023 October 5, 2023 October 6, 2023 Intralaboratory Type of precision On one record On different records precision dimension 1, Rf 0.323 0.333 0.345 dimension 2, Rf 0.344 0.356 0.356 dimension 3, Rf 0.333 0.375 0.311 Average value Rf X
2. 32 0.35 0.34 mid Standard deviation of
3. 01 0.02 0.02 repeatability Relative Standard Er-
4. 15 5.93 6.95 ror, % (CV, RSD) Rf max-Rf min 0.02 0.04 0.05 Table 5 -Precision of Rf values corresponding to the AA on the model mixture of CD Laboratory Laboratory of the Department of Pharmaceutical Disciplines Date October 5, 2023 October 5, 2023 October 6, 2023 Type of precision On one record On different records Intralaboratory precision dimension 1, Rf 0.66 0.68 0.66 dimension 2, Rf 0.65 0.66 0.67 dimension 3, Rf 0.66 0.67 0.66 Average value of 0.55 0.58 0.66 Rf, Xmid Standard deviation 0.01 0.03 0.05 of repeatability Relative Standard Error, % (CV, 2.09 4.31 9.09 RSD) Rf max-Rf min 0.01 0.02 0.01 Table 6 -Precision of Rf values corresponding to the ACV on the model mixture of CD Laboratory Laboratory of the Department of Pharmaceutical Disciplines Date October 5, 2023 October 5, 2023 October 6, 2023 Type of precision On one record On different records Intralaboratory precision dimension 1, Rf 0.56 0.58 0.55 dimension 2, Rf 0.56 0.56 0.5 dimension 3, Rf 0.54 0.61 0.6 Average value of 0.55 0.58 0.55 Rf, Xmid Standard deviation 0.01 0.03 0.05 of repeatability Relative Standard Error, % (CV, 2.09 4.31 9.09 RSD) Rf max-Rf min 0.02 0.05 0.10 The robustness of a method is a measure of its ability to remain unchanged by small but It was established that chromatograms obtained on Sorbfil and FilterBio plates, with non-random changes in the method paramedifferent chamber saturation times - 20, 30, 40, ters, demonstrating the suitability and reliabil60 min, under different drying conditions of ity of the method under normal use. When de- termining the robustness of a method for iden- the developed plate - at room temperature and in a drying cabinet, give comparable results tifying active ingredients and impurities in a and can be used in analysis. drug product, the influence of chromatography The obtained validation characteristics conditions (sorbents of similar plates from difdemonstrate the suitability and reliability of ferent manufacturers, the type and saturation the developed method, which was tested on la- of the chromatographic chamber, the separaboratory samples of effervescent tablets. tion band length, and different drying condiC) UV spectrophotometry for quantita- tions) on the final result was assessed. tive determination of active ingredients in CD. The study of the optical characteristics of Differences in the solubility of the com- the samples in the ultraviolet range of the specposition's components were used to separate trum was carried out using an SF-2000 spec- trophotometer (Russia), equipped with soft- the active ingredient—GA—from the inactive compounds. The latter, unlike GA, are highly ware from OKB Spektr (Russia). soluble in water, allowing for their effective The components of the studied combined separation from the active substance. composition are characterized by the following Quantitative analysis also required pre- spectral properties in the ultraviolet region: - liminary separation of the active ingredients, for GA, the maximum absorption in a 3% soas the electronic absorption spectra of GA and lution of trichloroacetic acid is (258 ± 2) nm AA partially overlap, precluding their simulta- (State Pharmacopoeia of the Russian Federaneous determination. It should be noted that tion XIV, FS-42-2636); AA is prone to oxidation in neutral and alka- — AA in a 0.1 M solution of hydrochloric acid line environments. Therefore, precipitation has an absorption maximum at 247 nm (SP with a 0.1 M hydrochloric acid solution was RK, Vol. 2, 2008); used to isolate the active components from the — ACV in a 0.1 M hydrochloric acid solution CD, followed by dissolution of AA and ACV. is characterized by an absorption maximum at The resulting filtrate was used for quantitative 254 nm with a shoulder at 272 nm (State Phardetermination of ACV and AA, while the pre- macopoeia of the Republic of Kazakhstan, cipitate, freed from inactive components by Vol. 2, 2008, “Acyclovir Tablets”). dissolution in water, was used to analyze the GA content. The spectra of the active ingredi- ents are presented in Figures 4-6. Figure 4. UV spectra of the solution of the SS GA (1) and the test solution of the medicinal product in the form of effervescent tablets (2) Figure 5. UV absorption spectra of the test solution (2) and SS AK (1) in 0.1 M hydrochloric acid solution Figure 6. UV absorption spectra of the test solution (2) and ACV SS (1) in 0.1 M hydrochloric acid solution The content of active ingredients was determined by the formula: Where is - the optical density of the test solution; – optical density (OD); - weight of the drug sample, in g; – sample weight (SW), in g; – average weight of 1 tablet, in g. Tables 7-10 below show the results of testing parameters such as identity, linearity, accuracy and precision. Table 7– Characteristics of the linearity of the method Ingredient D E Yesr Openability GA 35 0.4050 134.8 35 0.4312 135.2 34 0.4645 135.2 0.4901 0.5412 135.3 135.4 133.5 0.9997 41 0.5702 135.6 46 0.5981 135.7 0.6276 0.6542 135.8 136.4 AA 13 0.2845 212.7 0.3312 0.3775 212.6 211.4 20 0.4325 211.9 215.2 0.9996 21 0.4826 212.0 25 0.5402 211.8 25 0.5841 210.9 29 0.6312 212.6 28 0.6875 212.6 ACV 400 0.5455 323.5 402 0.5503 324.1 410 0.6234 323.6 415 0.6901 324.0 324.0 0.9998 420 0.7434 324.5 432 0.7753 323.5 435 0.8123 324.9 445 0.8509 325.1 450 0.9207 323.6 Table 8characteristics of the correctness of the method on tablets Quantity Tablet, composition,G Found,G Open rate, % of sub- stance GA AA ACV GA AA ACV GA AA ACV found in the table, % 70 0.0280 0.070 280 0.0281 0.0693 0.2767 99.6 99.5 99.3 80 0.0320 0.080 320 0.0323 0.0792 0.3182 99.8 99.4 99.5 90 0.0360 0.090 360 0.0367 0.089 0.3583 99.9 99.0 99.2 100 0.0410 0.100 400 0.0419 0.099 0.3989 99.3 99.3 99.0 110 0.0440 0.110 440 0.0442 0.1089 0.4378 99.7 99.5 99.6 0.0470 0.120 0.0540 0.130 0.0479 0.1188 0.4776 99.6 0.0547 0.1287 0.5174 99.8 99.2 99.7 99.4 99.3 Table 9– characteristic of the ACVuracy of the method on a model mixture The amount detected, m Y mid R ∆ Y mid Rf Y mid∆ Y mid rср% mg According to the GA 40.27 40.84 41.12 41.54 9 35.92 1.04 0.82 2.36 35.92±0.82 1.96 41.92 42.62 42.79 43.02 43.19 According to AA 100.34 99.11 99.45 9 99.70 1.73 1.36 2.36 99.701.36 2.74 100.23 99.60 99.84 100.31 99.54 100.92 According to the ACV 399.34 400.11 399.45 400.23 9 399.53 1.52 1.24 2.23 399.531.24 2.53 399.60 399.84 401.31 399.54 399.92 Table 10– study of the accuracy of the method for quantitative determination of active substances in several batches of a medicinal product Series No. GA AA ACV 010363 40.27 99.34 399.34 020363 40.84 100.11 400.11 030363 41.12 99.45 399.45 040363 41.54 100.23 400.23 050363 41.92 99.61 399.60 42.62 99.84 399.84 060363 Average value, mg 41,385 99,763 399.54 Differences, mg 2.35 2.5 2.4 5,341 Relative difference, % 5,678 5,127 Average square Off, mg 0.83 0.964 0.765 Rel. Average square Off, % 2,006 1,977 1,234 Table 11– Study of the accuracy of analysis in the quantitative determination of active ingredients in several batches of CD Series No. GA AA ACV 010363 40.27 99.34 399.34 020363 40.84 100.11 400.11 030363 41.12 99.45 399.45 040363 050363 41.54 41.92 100.23 99.61 400.23 399.60 060363 42.62 99.84 399.84 Average value, mg Differences, mg 41,385 2.35 99,763 2.5 399.54 2.4 Relative difference, % 5,678 5,127 5,341 Average square Off, mg 0.83 0.964 0.765 Rel. Average square Off, % 2,006 1,977 1,234 The identity of the active ingredients of The linear dependence of the method the effervescent tablets GA, AA, and ACV was characterizes the possibility of obtaining an an- confirmed by matching the absorption maxima alytical signal in the form of optical density and minima of the analyzed samples with the proportional to the content of the analyte in the corresponding RSO preparations under optisample being studied. mally selected spectrophotometric conditions. The linearity of the test method was Statistical processing of the experimental restudied using model mixtures in the range of sults was performed in accordance with the 70-130% of the declared GA, AA, and ACV State Pharmacopoeia of the Republic of Kacontent in effervescent tablets. Statistical anal- zakhstan, Volume 1. ysis of the model mixture results revealed a linin Vero E6 cell culture. The combinations ear relationship between the active ingredient studied included GA, AA, αIFN and ACV. The concentration and optical density. main criterion for the antiviral activity of indi- The percentage levels of regeneration vidual substances and their combinations was were observed within the range of 99.6-99.9% the ability of the drugs to prevent the death of for GA, 99.0-99.7% for AA, 99.2-101.8% for infected cells by inhibiting the development of ACV. virus-induced CPE. As shown in Table 9, the relative error The experiment was conducted using a of the average results for active substances is monolayer culture of Vero E6 cells grown in within the range of 1.90-2.74%, which indi96-well polymer plates. Infection was per- cates good reproducibility of the developed formed at a multiplicity of infection of 0.1 method. PFU/cell. Incubation was carried out for 48 Thus, it was established that the devel- oped method for determining GA, AA and hours at 37°C. In control samples, the CPE de- velopment rate was 95–100%, corresponding ACV in pharmaceutical preparations has the to complete destruction of the cell monolayer. correct accuracy and reproducibility in relation The efficacy of the studied compounds to the declared content of the active substance was expressed as ID₅₀ and ID₉₅—the concen- in the dispersion, with a linear correlation of trations of the substances that inhibit the devel- ±30% in the analytical range of the tablet, opment of virus-induced CPE by 50% and which makes them useful for a reliable assess95%, respectively. When studying the com- ment of the quality of drugs. bined effect of the drugs, the concentrations of the components that provide these levels of in- Study of the specific activity of CD in in hibition when used together were determined. vitro experiments The antiherpetic activity of the combina- tions was assessed by the fractional inhibitory The antiviral activity of four compounds, concentration (FIC) index, calculated using the both individually and in triple combinations, formula: was studied against two strains of herpes sim- plex virus type 1 (HSV-1)—sensitive and reFIC = ID50 of compound A in combina- sistant to acyclovir. Activity was assessed ustion/ID50 of compound A + ID50 of compound ing microtiter assays to determine the inhibiB in combination/ID50 of compound B tion of virus-induced cytopathic effect (CPE) Table 12Antiherpetic activity of triple combinations of a number of compounds in the model of HSV-1 with different drug sensitivities in Vero E6 cell culture № Compoun CD50 mcg/ml ID50 μg/ml ID95 µg/ml FIC Effect 1 ACV+A >15.6+31.25 7.8+15.6+3.91/7.8 15.6+31.2+7.81/ 0.681/ Synergistic + αIFN +7.8 +31.25+7.82 15.6+62.5+7.82 0.682 / Synergetic 2 ACV+A >125+1000+ 7.8+15.6+1251/15. 15.6+15.6+2501 0.871/ Synergistic/ A+GA 125 6+31.25+125 /31.2+15.6+250 1.02 additive 3 ACV+G >31.2+250+1 7.8+62.5+1.951/5.6 15.6+125+3.91/ 0.871/ Synergistic + αIFN 5.6 +62.5+3.92 31.25+125+3.92 0.872 / Synergetic An analysis of the data presented in TaACV + GA + AA is of greatest interest. The ble 12 reveals that the triple combination of combined use of these compounds reduces the concentration of the most toxic component, synergistic and additive effects, confirming its ACV, demonstrating the combination's potenfeasibility for use in the development of a com- tial to increase the therapeutic index. This bination drug in effervescent tablet form. combination has been shown to exhibit both AS A LUBRICANT REDUCED THE LABOR AND DISCUSSION. THE CONDUCTED STUDIES ENERGY INTENSITY OF THE PRODUCTION PRO- SUBSTANTIATED THE CHOICE OF AN EFFERCESS AND INCREASED THE WETTABILITY AND VESCENT FORMULATION FOR A COMBINATION SOLUBILITY OF THE RESULTING TABLETS , DRUG CONTAINING GA, AA, AND ACV. THE WHICH IS ESPECIALLY IMPORTANT WHEN CRE- USE OF DIRECT COMPRESSION ALLOWED FOR ATING EFFERVESCENT DRUGS . THE NATURAL THE PRODUCTION OF A STABLE AND TECHNOSWEETNESS AND AROMA OF GA ELIMINATED LOGICALLY REPRODUCIBLE DOSAGE FORM THE NEED FOR ADDITIONAL FLAVORINGS AND WITH OPTIMAL QUALITY INDICATORS . THE SWEETENERS , MAKING THE DRUG SAFER AND USE OF PRUV® SODIUM STEARYL FUMARATE MORE PHARMACOLOGICALLY PURE . The absence of exothermic and endosaturation, drying conditions), demonstrating thermic peaks in the DSC curves of the studied its reliability in analytical practice. mixtures confirms the absence of chemical inThe UV spectrophotometric method teractions between the components of the drug, demonstrated high accuracy, precision, and demonstrating the pharmaceutical compatibillinearity in the range of 70–130% of the nomi- ity of GA, AA, and ACV in the selected comnal active ingredient content. Correlation coef- position. The obtained results are consistent ficient values (r = 0.9996–0.9998) indicate the with published data on the stability of these method's high analytical sensitivity. The anal- compounds under moderate temperature conysis results for tablets from different batches ditions. The "accelerated aging" method allowed demonstrate stability of GA, AA, and ACV content within acceptable tolerances, confirm- us to establish a shelf life of the drug of 3 years ing the reproducibility of the technological when stored at a temperature not exceeding 25process. 26°C. Throughout the entire testing period, the samples maintained their physicochemical An in vitro study of antiviral activity demonstrated that the combination of GA, AA, properties, as demonstrated by the stability of and ACV exhibits a significant synergistic or their average weight, homogeneity, active inadditive effect against herpes simplex virus gredient content, and the absence of impurities. Thus, the selected excipient composition and type 1, including acyclovir-resistant strains. This demonstrates the potential of the devel- direct compression technology ensure the oped composition as an effective preventative physical and chemical stability of the dosage and therapeutic agent for herpesvirus infec- form. tions. GA likely enhances the penetration and Validation of the TLC method confirmed effectiveness of ACV due to its membrane-sta- its selectivity, specificity, and precision. Sepabilizing and anti-inflammatory properties, ration of active components and impurities was while AA provides antioxidant protection and achieved in the optimal solvent system of ethincreases cellular resistance to viral aggres- anol–methylene chloride–ammonia solution sion. (6:3:1), yielding clear adsorption zones and reOverall, the study results confirm the ra- producible Rf values. Low relative standard tionale for the chosen composition and tech- deviations (2–9%) confirm the high repeatabilnology for producing effervescent tablets. The ity of the method. Robustness of the method developed drug is characterized by high stabil- was demonstrated with changes in non-essenity, reproducibility, safety, and potential for tial analytical conditions (plate type, chamber further preclinical and clinical trials. The pre- sented data can serve as a scientific and practiglycyrrhizic acid, ascorbic acid and acyclovir cal basis for the development of a domestic has been developed. This form is distinguished combination antiviral drug with increased bioby confirmed pharmaceutical compatibility of availability and an improved profile of action. the components, validated quality control Conclusion. methods and a pronounced synergistic antivi- Based on the conducted research, a staral effect, which justifies the prospects for its ble and technologically reproducible effervesfurther implementation in pharmaceutical cent form of a combination drug containing practice. Bibliography: 1. Nosik, N. N. Viral infections and disinfection / N. N. Nosik, D. N. 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