Journal of Infectious Diseases Cellular To Extra-Cellular Concentrations Of Doxycycline, Enoxacin And Roxithromycin In Human Polymorphonuclear Neutrophils. *Corresponding Author: Anam Niazi . Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. Email: aynakhan@yahoo.coms. Received: 11-Apr-2026, Manuscript No. JOID - 5601 ; Editor Assigned: 14-Apr-2026 ; Reviewed: 01-May-2026, QC No. JOID - 5601 ; Published: 11-May-2026. DOI: 10.52338/joid.2026.5601. Citation: Anam Niazi . Cellular To Extra-Cellular Concentrations Of Doxycycline, Enoxacin And Roxithromycin In Human Polymorphonuclear Neutrophils. Journal of Infectious Diseases. 2026 May; 17(1). doi: 10.52338/joid.2026.5601. Copyright © 2026 Anam Niazi . This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ISSN 2831-8064 Research Article Anam Niazi 1 1Institute of Basic Medical Sciences, Khyber Medical University, Peshawar, Pakistan. www.directivepublications.org Abstract Introduction: Since bacteria which can penetrate and reproduce within phagocytes are more likely to cause prolonged and serious infections, this emphasize the use of those antibiotics which concentrate inside cells and kill those bacteria. C/E ratio is a parameter to determine the drug concentration inside and outside the cell. In present study we determined the C/E Ratios of doxycycline, enoxacin and roxithromycin in human polymorphonuclear neutrophils. Method: Polymorphonuclear neutrophils from the whole blood was separated by the protocol given by Polymorphprep reagent obtained from Progen Company with few modifications. Viability of cells was tested by trypan blue. Later, these neutrophils were allowed to incubate with drugs at concentration prepared by given C max and T max in literature. After that they were micro-centrifuge, freezed at -80 ͦC and hence been separated into cellular and extracellular concentrations which were quantitatively assayed by RP-High Performance Liquid Chromatography technique. Results: The result shows that doxycycline, enoxacin and roxithromycin have an outstanding ability to concentrate inside the human polymorphonuclear neutrophils. Hence these drugs are proven to be more effective against intracellular pathogens. However, they are dependent upon the conditions and factors provided to them. These results can prove to be an important milestone for further research and are of great importance. It provides us a fair idea towards treatment approach and allows the comparison with other drugs of same group. Conclusion: This study demonstrates that enoxacin and doxycycline achieve substantially higher intracellular accumulation in human polymorphonuclear neutrophils than roxithromycin, supporting their greater potential efficacy against intracellular pathogens. Keywords: Polymorphonuclear neutrophils; Intracellular accumulation; Doxycycline; Enoxacin; Roxithromycin. INTRODUCTION Antibiotics, type of antimicrobials is a large and important group of medicine which act by either killing or arresting growth of susceptible microorganisms inside and outside the cells. Some of the antibiotics also have antiprotozoal activity (1). Antibiotics work mainly through four different ways: they can stop cell wall synthesis, block protein synthesis, interfere with folate synthesis, or disrupt DNA gyrase activity. Each of these actions messes with how bacteria maintain their structure, perform essential functions, produce nucleotides, or replicate their DNA (2). Polymorphonuclear neutrophils are type of white blood cells and the first line of defense along with macrophages in any infection, so those microorganisms which multiply and survive in them are more prone to relapse and elongation of disease period. Only some of the antibiotics are capable of internal localization of cells. Those antibiotics which penetrate and accumulate more intracellularly are believed to kill more efficiently the intracellular pathogens (3). Enoxacin, Doxycycline and Roxithromycin are antibiotics which belong to class flouroquinolones, tetracycline and macrolides respectively. These antibiotics are founds to be more effective against intracellular and atypical pathogens (4-6). Doxycycline is a type of tetracycline that stops bacteria from growing by preventing protein synthesis. It does this by binding to the 30S ribosomal subunit, which blocks the attachment of aminoacyl tRNA. Resistance to doxycycline happens when Gram negative bacteria become less permeable, utilize efflux pumps, produce protective ribosomal proteins, modify the drug with enzymes, or develop mutations in the 16S rRNA that reduce how well the drug binds (7-9). Enoxacin gets into bacteria through porins and inhibits DNA replication by attaching to DNA gyrase and topoisomerase IV. This leads to breaks in DNA strands and problems with separating

Directive Publications Anam Niazi newly formed chromosomes. Bacteria can become resistant through mutations in these enzymes or by reducing the levels of the drug inside the cell due to efflux mechanisms (10-12). Roxithromycin works by binding reversibly to the 50S ribosomal subunit. This prevents the ribosome from moving along the mRNA and affects transpeptidation, ultimately suppressing protein synthesis and the growth of bacteria. Resistance can occur through active efflux pumps, the hydrolysis of the drug by enzymes, or through chromosomal mutations that change the 50S ribosomal target (13-17) This study is finding and comparing ratios of cellular to extracellular concentrations of these three drugs in human PMNs and plasma using HPLC analysis. Cellular to extra cellular Ratio (C/E) is a determining parameter used to compare antibiotics and to find the localization of drug inside the cells and outside in the plasma, Comparison of it give us a fair idea about the drugs cellular uptake and possible advantageous effects against intracellular pathogens. MATERIAL AND METHODS Study setting This study was performed in Pharmacology, Hematology and cell culture laboratories, Institute of Basic Medical Sciences, Khyber Medical University Peshawar. It was approved in the 47th meeting of KMU-AS&RB held on 28 February 2017 under the Reference No. DIR/KMU-AS&RB/EC/000604. Moreover, ethical approval was obtained from the ethical committee of Khyber Medical University under Reference No. DIR/KMU- AS&RB/EC/000604, Held on 06-04-2017. Blood collection 10ml venous blood was collected for each drug assay from different healthy volunteers, fulfilling the requirements of inclusion/exclusion criteria. Consent form in both Urdu and English languages were signed from volunteers after telling them full details and made sure about complete understanding of it (18). HPLC analysis Chemicals, Reagents, and Standards HPLC-grade methanol, acetonitrile, water, acetic acid, potassium dihydrogen phosphate, and diluted hydrochloric acid were used in the experiments. Doxycycline, enoxacin, and roxithromycin were sourced from local pharmaceutical companies in Pakistan, and diazepam used as an internal standard for the doxycycline analysis. Preparation of Calibration Standards Stock solutions (1 mg/mL) of doxycycline, enoxacin, and roxithromycin were prepared using appropriate solvents and serially diluted to obtain calibration ranges of 0.0625–1 mg/ mL. was diluted in HPLC-grade water, while enoxacin was prepared using a mix of water, methanol, and acetic acid in a ratio of 2:5:3 (v/v/v) and then further diluted with a mixture of methanol and water at 8:2 (v/v). Roxithromycin dilutions were made in methanol (18). UV Spectrophotometric Analysis The maximum absorbance wavelength (λmax) using a UV spectrophotometer was determined and turned out to be 229 nm for doxycycline, 290 nm for enoxacin, and 215 nm for roxithromycin. These wavelengths were used for the chromatographic detection later on (18). Plasma Sample Preparation To collect human blood samples, EDTA tubes were used and centrifuged them at 3000 rpm for 10 minutes to separate the plasma. 250 µL of this plasma was taken and spiked it with another 250 µL of the calibration standards. For the doxycycline analysis, 100 µL of the diazepam internal standard was added. Protein precipitation was carried out with acetonitrile (250 µL), mixed it with a vortex, and then centrifuged it at 11,000 rpm for 10 minutes. Finally, the supernatants were filtered through 0.22 µm nylon membrane filters and stored them at −20 °C until ready for the analysis (18). HPLC Conditions Reverse-phase high-performance liquid chromatography (RP- HPLC) using Shimadzu HPLC systems fitted with a C18 column (5 µm, 25 × 0.46 cm) was used in this study. The injection volume was set at 20 µL, and kept the flow rate at 1 mL/min. For doxycycline, the mobile phase was a mixture of water and methanol (50:50, v/v; pH 2). In the case of enoxacin, methanol and water (80:20, v/v), while for roxithromycin, a buffer with 0.03 M KH 2 PO 4 and methanol (40:60, v/v; pH 4.5) was used. The retention time of the compounds at their respective λmax values, was noted at 7.15 minutes for doxycycline, 1.6 minutes for enoxacin, and 18 minutes for roxithromycin. Calibration curves were constructed using the peak area responses that obtained (18). Preparation of Drug Suspensions for Assay Drug suspensions were prepared based on known pharmacokinetic parameters, including maximum plasma concentration (C_max), time to reach maximum concentration (T_max), and elimination half-life. The stock solutions of doxycycline, enoxacin, and roxithromycin at 100 µg/mL in Hanks’ Balanced Salt Solution (HBSS) were prepared, then diluted them to reach final assay concentrations that matched their respective C_max values. Specifically, (19) and enoxacin (20) final concentrations were set at 2 µg/mL (C_max ≈ 2 ± 1 mg/L; T_max = 2 h) and 2.741 µg/mL (C_max = 2.741 ± 0.960 mg/L; T_max ≈ 1 h), respectively. For roxithromycin, a stock solution based on a reported C_max of 6.34 mg/L; T_max ≈ 1 h was prepared, and diluted it to get a final assay concentration of 6.34 µg/mL, using 5 mL for the analysis (21). Page - 2Open Access, Volume 15 , 2026

Anam Niazi Directive Publications Isolation, Purification, and Drug Treatment of Human Polymorphonuclear Neutrophils Isolation of Polymorphonuclear Neutrophils Polymorphonuclear neutrophils (PMNs) were isolated from peripheral venous blood in two healthy adult volunteers. All reagents and solutions were equilibrated to room temperature prior to use. Blood was collected aseptically using sterile syringes into EDTA-containing blood collection tubes that contained 5 mL of the blood. Whole blood was carefully layered over Polymorphprep (a 13.8 % w/v sodium diatrizoate and 8.0 % w/v polysaccharide solution) using a Pasteur pipette. This procedure used 5 mL of whole blood and was done using a 15 mL Falcon tubes, the two solutions had limited mixing at the interface before centrifugation. Centrifuge was set at 550 g for 30-35 minutes at 19 °C without using the brake to stop the centrifuge. Centrifugation resulted in the sedimentation and separation of whole blood into six distinct layers, including the following: plasma, peripheral blood mononuclear cells (PBMCs), the isolation medium, and polymorphonuclear neutrophils, the isolation medium, and erythrocytes at the bottom of the tube. The plasma and PBMC layers were aspirated using a Pasteur pipette. The neutrophil layer was collected using a serological pipette and included 1 to 2 mL of the isolation medium by orienting the tube upwards and gently rotating it. The erythrocyte layer of the tube was discarded (22, 23). Washing and Purification of Neutrophils The neutrophil fractions, from both Falcon tubes, were pooled, using a new 15 mL Falcon tube containing 9 mL of Calcium- and Magnesium-Free, 1x Phosphate buffer Saline (PBS). The total volume was adjusted to 14mL by adding PBS, and centrifuged at 450gav for 10 min at 19°C. After the centrifugation process, red pellet containing neutrophils and residual erythrocytes were formed at the bottom of tube. The supernatant was aspirated very carefully without disrupting the sediment. To lyse the erythrocytes, the sediment was re-suspended in 3 mL of sterile distilled water and agitated for 30 seconds (longer than 30 seconds was avoided to prevent lysis of neutrophils). Immediately after, 700 µL of 5x PBS was added and the mix was agitated gently. The resulting suspension was centrifuged at 250gav for 5 minutes at 19°C, and the supernatant was aspirated to leave a sediment approximately 2mL in diameter. This lysis procedure was repeated three additional times until the white sediment was obtained and identified as purified neutrophils. The remaining neutrophils were washed with 9 mL of Calcium and Magnesium Free PBS and centrifuged at 250g av for 5 minutes. The supernatants were then aspirated to leave approximately 2 mL of neutrophil retentate, which was then re-suspended in 5mL of Hank's Balanced Salt Solution (HBSS) (22, 23). Assessment of Cell Viability by Trypan Blue Exclusion The Trypan Blue exclusion test was used to assess neutrophil viability prior to final resuspension in HBSS (i.e. to determine if the cells could be used for functional assays). 10 µl of the neutrophil cell suspension, with equal volume (10 µl) of 0.4% Trypan Blue solution, was added to a cryovial and mixed gently by pipetting. The cell-dye mix was then incubated for 3 min at room temperature. The hemocytometer was prepared by placing a coverslip on it, and the cell-dye mix was carefully added through the notch, being careful not to form air bubbles. The hemocytometer and microscope were cleaned with 70% ethanol and 30% distilled water before use. The cells were viewed on a light microscope at 10X magnification. The viable neutrophils were white and the non-viable cells were blue. Each corner square (total of four) of the hemocytometer (each corner square containing 16 smaller squares) was counted as a total of 64 squares; hence, each corner square contained a viable neutrophil count as well as a total neutrophil count. Cell counts were made using a tallied method (22, 23). Cell viability was calculated using the following formula: Incubation of Neutrophils with Drugs Purified and viable neutrophils suspended in 5 mL of HBSS were mixed with an equal volume (5 mL) of previously prepared drug solutions in HBSS in centrifuge tubes. The suspensions were incubated in a shaking incubator (WiseCube WIG-105, Germany) at 37 °C with constant agitation. Incubation durations were based on the maximum time (Tmax) for each drug: doxycycline (2 hours), enoxacin (1 hour), and roxithromycin (1.04 hours) (22, 23). Separation of Neutrophils and Extracellular Fluid Following incubation, the tubes were centrifuged at 250 × g for 10 minutes at 4 °C. The supernatant was carefully removed, leaving approximately 1 mL, and the cell pellet was resuspended by gentle tapping. In a microcentrifuge tube, 40 µL of 88% formic acid was placed at the bottom, followed by careful layering of 300 µL of silicone oil. Subsequently, 400 µL of the resuspended drug-treated neutrophil suspension was layered over the silicone oil. The tubes were centrifuged in a refrigerated microcentrifuge (MSE Harrier 18/18, UK) at 12,000 × g for 5 minutes at 4 °C. During centrifugation, neutrophils passed through the silicone oil and were lysed by formic acid, while extracellular fluid remained above the oil layer. The microcentrifuge tubes were then frozen at −80 °C. The following day, frozen tubes were carefully cut at the interface between the two layers using a sharp sterile razor. The upper (extracellular) and lower (cellular) layers were transferred into separate microcentrifuge tubes (22, 23). Page - 3Open Access, Volume 15 , 2026

Anam Niazi Directive Publications RESULTS The present study was conducted to evaluate the intracellular accumulation of three antibiotics—doxycycline, enoxacin, and roxithromycin—by determining their cellular/extracellular (C/E) concentration ratios at maximum concentration (C_max) and time to maximum concentration (T_max). Quantitative analysis was performed using a validated highperformance liquid chromatography (HPLC) method. For each antibiotic, a set of standard dilutions was prepared to construct calibration curves. The assay yielded consistent retention times and satisfactory precision and accuracy within all three drugs’ concentration ranges. Diazepam (the internal standard) produced consistent retention times of approximately 5.6 minutes. Average accuracy of diazepam was between 74.04% and 95.13% and the coefficients of variation were within acceptable limits confirming the reliability of the analytical method (Table 1). Table 1. Assay of Internal Standard, Diazepam. Sr # Actual Concentration on (mg/ml) Expected Concentration (mg/L) RT (min) SD %Accuracy Mean ± %Coefficient of variance %Yield C1 C2 C3 1 10 8.985 8.8768.924 5.6 0.546 89.28 8.928±0.61 89.28 2 10 7.381 7.4527.384 5.6 0.04015 74.04 7.406±0.54 74.04 3 10 9.414 9.6149.512 5.6 0.1000 95.13 9.513±1.05 95.13 4 10 9.137 9.2019.313 5.6 0.08908 92.17 9.217±0.97 92.17 5 10 8.539 8.5418.562 5.6 0.01274 85.47 8.547±0.15 85.47 For doxycycline, standard concentrations ranging from 6.25 to 100 mg/L produced mean measured concentrations between 5.92 ± 0.53 mg/L and 95.02 ± 2.25 mg/L. Accuracy ranged from 88.4% to 99.76%, with coefficients of variation below 9%, indicating good linearity and repeatability of the assay (Table 2). Table 2. Assay of Standard Doxycycline Dilutions. Sr#Actual Concentration (mg/L) Expected Concentration (mg/L) RT (min)Mean ± SD % Accuracy % Coefficient of variance %Yield C1 C2 C3 1 100 97.507 94.412 93.13111.6 95.02±2.250 95.02 2.37 95.02 2 50 44.228 49.905 48.40311.6 47.51±2.942 95.02 6.19 95.02 3 25 25.001 26.459 23.36711.6 24.94±1.547 99.76 6.20 99.76 4 12.5 11.011 11.518 10.62711.6 11.052±0.446988.4 4.04 88.4 5 6.25 6.451 5.929 5.39711.6 5.92±0.5270 95.58 8.89 95.58 Similarly, enoxacin calibration standards (6.25–100 mg/L) yielded mean measured concentrations between 6.2 ± 0.10 mg/L and 96.67 ± 1.53 mg/L, with accuracy values ranging from 91.2% to 96.67% ( Table 3). Table 3. Assay of Standard Enoxacin dilutions. Sr #Actual Concentration (mg/L) AUC Expected Concentration (mg/L) RT (min)Mean±SD Accuracy % %Coefficient of variance %Yield C1 C2 C3 1 100 1247940398 97 95 1.6 96.67±1.52896.67 1.58 96.67 2 50 6237015 49.2 47.8 46.2 1.6 47.73±1.50195.46 3.14 95.46 3 25 4158010 23.5 24.2 23.2 1.6 23.63±0.513294.52 2.17 94.52 4 12.5 2120902 11.2 10.9 12.1 1.6 11.40±0.624591.2 5.48 91.2 5 6.25 1178278 6.2 6.3 6.1 1.6 6.2±0.1000 95.38 1.61 95.38 Roxithromycin standards over the same concentration range showed mean concentrations from 6.20 ± 0.10 mg/L to 96.77 ± 1.55 mg/L, with accuracy exceeding 91% in all cases (Table 4). Page - 4Open Access, Volume 15 , 2026

Anam Niazi Directive Publications Table 4. Assay of Standard Roxithromycin dilutions. Sr#Actual Concentration (mg/L) AUC Expected Concentration (mg/L) RT (min)Mean±SD Accuracy % %Coefficient of variance %Yield C1 C2 C3 1 100 349567298.20 97.0095.1218 96.77±1.55296.77 1.60 96.77 2 50 158944249.2 47.8 46.218 47.73±1.50195.46 1.34 95.46 3 25 836285 23.5 24.2 23.218 23.63±0.513294.52 2.17 94.52 4 12.5 398231 11.2 10.9 12.118 11.40±0.624591.2 5.48 91.2 5 6.25 76738 6.2 6.3 6.1 18 6.20±0.10 99.2 1.61 99.2 The intracellular and extracellular concentrations were determined by using peak area and height data from the standard calibration curve from each of the drugs respectively. Doxycycline demonstrated very high levels of intracellular accumulation. The intracellular concentration was between 18.10 mg /L and 18.96 mg /L and the extracellular concentration was between 2.50mg /L and 2.81mg /L giving C/E ratios that were between 6.72 and 7.54 thus indicating significant cellular penetration (Table 1) with an average C/E of 7.040 ± 0.4386 ( Table 5). Table 5. Cellular/ Extracellular concentration Ratio of Doxycyline. Sr#AUC INT Height INTIntracellular Concentration mg/L AUC EXT Height EXTExtracellular Concentration mg/L C/E Ratio 1 9057495 131440 18.840 1202044 9137 2.8056 6.72 2 8954308 102587 18.102 1181701 8764 2.639 6.86 3 9265834 255232 18.960 1969653 9723 2.501 7.54 Enoxacin also showed substantial intracellular accumulation however there was less of a difference between the two concentrations compared to doxycycline where intracellular levels were between 9.16 mg /L and 9.97 mg /L versus extracellular concentrations which were between 0.98 mg /L and 1.11 mg /L, therefore C/E ratios ranged from 8.97 to 9.40 with a mean C/E ratio of 9.146 ± 0.2245 (Table 6). Consequently enoxacin has the highest level of intracellular accumulation compared to the three antibiotics. Table 6. Cellular/ Extracellular concentration Ratio of Enoxacin. Sr#AUC INT Height INT Intracellular Concentration mg/L AUC EXT Height EXT Extracellular Concentration mg/L C/E Ratio 1 1239823 85528 9.9679 198558 14140 1.1115 8.97 2 149856 86543 9.2672 176544 16342 1.0212 9.07 3 139876 83465 9.1645 129874 13986 0.975 9.399 In contrast, roxithromycin showed significantly lower levels of intracellular accumulation compared to both doxycycline and enoxacin with intracellular concentration ranging between 1.47 mg /L and 1.97 mg /L and extracellular concentrations ranging between 0.82 mg /L and 0.86 mg /L; producing a range of C/E ratios (1.71 to 2.36) and an average C/E ratio of 2.006 ± 0.3303 (Table 7). Table 7. Cellular/ Extracellular concentration Ratio of Roxithromycin. Sr#AUC INTHeight INTIntracellular Concentration mg/L AUC EXTHeight EXTExtracellular Concentration mg/L C/E Ratio 1 42075 1702 1.4706 20275 860 0.8601 1.709 2 54064 2301 1.9651 19145 845 0.8321 2.3616 3 45134 1692 1.5982 23965 898 0.8209 1.9468 The results from a comparative study of C/E ratios indicate pronounced differences in the intracellular levels of the various antibiotics. Enoxacin had the greatest amount of cellular accumulation compared to doxycycline and roxithromycin, which had the lowest C/E ratios. These observations demonstrate that there are drug-specific differences in cellular uptake and retention, and thus may impact their intracellular antimicrobial activity. Page - 5Open Access, Volume 15 , 2026

Anam Niazi Directive Publications DISCUSSION Intracellular pathogens are great source of prolonged lethal and recurrent infections because of their capability of coping environmental challenges and surviving even after phagocytic ingestion. Optimal therapy approach to deal with this situation is, the use of particularly those antibiotics which are capable of intracellular uptake and accumulation inside the granulocytic phagocytes specially neutrophils to inactivate and kill those microorganisms. However, this relationship is not always directly proportional. C/E ratio is an imperative parameter to determine the drug concentration inside and outside the relevant cell. Different method approaches were used for quantitative analysis of drug to determine cell concentration, which were Radio assay, HPLC and flourometric techniques. HPLC has replaced radioassay and flourmetric techniques to avoid the risks linked with radiolabelled compounds and restriction of fluorescence associated structure detection, respectively. Three main drugs Doxycycline, Enoxacin and Roxithromycin were chose from different classes of antibiotics to give us a major idea for the potential of that class and specifically that drug to treat and cure cellular pathogens. Cmax and Tmax are chief parameters of pharmacokinetics that ultimately determine and affect bioavailability, were reviewed from literature for each drug. HPLC method for doxycycline was developed and validated and the peaks of drug and internal standard diazepam were well resolved. Retention time of doxycycline was found to be on 11.6mins and diazepam on 5.6mins. Calibration curve was plotted Area under curve against known concentration of doxycycline(100mg-6.25mg/L) and regression equation was r=0.96. This suggests the reproducibility of results. Samples were prepared from the above-mentioned procedure for cellular and extracellular concentrations and were run on HPLC. C/E ratio of doxycycline was calculated from the calculated values of Cellular and extracellular concentration, and it was found to be C/E Ratio of DOX= 7.040±0.4386 (Mean ± Standard deviation). This result fair comply with results of research done on Minocycline transport into Human neutrophils and suggests 7 times more intracellular penetration of DOX than extracellular fluid concentration which made it more preferable for killing of intracellular microorganisms (24). Fluoroquinolones, have outstanding ability to penetrate more in phagocytic cells and eradicate microorganism more effectively that resist phagocytic killing (25). Many researches have been done up till now for finding C/E ratio of different drugs of fluoroquinolones and showed worthy results (26-28). For enoxacin, method was developed and calibration curve was plotted manually in software Graph Pad Prism. Standard curve was plotted by area under curve against known concentration of enoxacin (100mg-6.25mg/L) and regression equation was r=0.99 which suggested it reproducible. C/E ratio of enoxacin is found to be 9.146±0.2245 (Mean ± Standard deviation). This value depicts that enoxacin concentrate 9 times more inside the neutrophils than extracellular. It’s a significant value which allows making a comparison with other fluoroquinolones and making more favorable to use against intracellular bacteria like Str. pneumoniae and Staphylococcus aureus (29). Roxithromycin belong to group macrolids, and this class also found to possess high ability to accumulate inside the phagocytes than non-phagocytes (30). Calibration curve was plotted manually in Graph Pad Prism software against concentration(100mg-6.25mg/L) and regression equation was found to be r=0.99. C/E ratio of roxithromycin is found to be C/E Ratio= 2.006±0.3303 (Mean ± Standard deviation). It illustrates the accumulation of drug twice inside the neutrophils than to extracellular. This can be high upto 7 to 8 times depending upon the conditions provide to them. CONCLUSION The present study concludes that doxycycline, enoxacin and roxithromycin have different rates of accumulation inside polymorphonuclear neutrophils, indicated by the ratios of drug concentration inside cells compared to outside. Of the three tested antibiotics, the drug with the most accumulation inside neutrophils was enoxacin (C/E = approx 9), followed by doxycycline (C/E = approx 7), while roxithromycin (C/E = approx 2) showed much less accumulation within neutrophils. Therefore, the physicochemical properties of specific drugs and their pharmacokinetics are responsible for differences in their ability to be taken up and retained inside neutrophils. Doxycycline and enoxacin exhibited substantial concentrations within neutrophils, suggesting a greater efficacy against intracellular or phagocyte-associated pathogens, which can cause prolonged and recurrent infections that are difficult to treat. Although roxithromycin exhibited less accumulation than doxycycline and enoxacin under the conditions used here, the localization of roxithromycin in immune cells provides evidence of its therapeutic relevance; intracellular accumulation may vary according to the condition of the specific immune cell and experimental conditions. Therefore, these findings will aid in selecting appropriate therapy when considering infections caused by intracellular organisms and will support additional research looking at antimicrobial efficacy and using larger sample sizes and testing other antibiotics for functional activity to better define the clinical relevance of drug accumulation within immune cells. Page - 6Open Access, Volume 15 , 2026

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