Bioequivalence Study of Low-Dose Anagrelide 0.5 mg Capsules under Fasting Conditions in Healthy Turkish Male Subjects

Bioequivalence Study of Low-Dose Anagrelide 0.5 mg Capsules under Fasting Conditions in Healthy Turkish Male Subjects | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Bioequivalence Study of Low-Dose Anagrelide 0.5 mg Capsules under Fasting Conditions in Healthy Turkish Male Subjects Ahmet Inal, Zafer Sezer, Onur Pinarbasli, Burcu Bulut, Martin Reinsch, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6453732/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The pharmacokinetic profiles and relative bioavailability of anagrelide from the test and reference preparations were compared to establish the test preparation's bioequivalence to the reference under fasting conditions. In a randomized sequence, male Caucasian subjects were administered a single dose of either one capsule of the test or reference preparation separated by a washout phase of seven days. Blood samples were collected at pre-specified time points following drug administration to assess anagrelide pharmacokinetics. These samples were collected in K 2 EDTA tubes, centrifuged, and stored at <-70°C. Anagrelide concentrations were quantified using a validated LC-MS/MS method with ESI(+). The pharmacokinetic parameters, including AUC (0−t) , AUC (0−∞) , C max , t max , λ z , t ½ (λ z ), and AUC %−extrapol , were calculated using Phoenix WinNonlin V8.1.0.3530 software. The absorption extent (AUC (0−t) ) and the absorption rate (C max ) of anagrelide from the test and reference preparations were found to be comparable. The 90% confidence intervals for the ln-transformed ratios of AUC (0−t) and C max of the test/reference preparations met the bioequivalence criteria of 80.00–125.00%. Furthermore, the 90% confidence interval for the ln-transformed data of the secondary pharmacokinetic parameter, AUC (0−∞) , of anagrelide was also included in the bioequivalence acceptance range. Bioequivalence study anagrelide healthy volunteers pharmacokinetic bioavailability Figures Figure 1 Figure 2 Key points Bioequivalence of anagrelide 0.5 mg capsules The safety profile of anagrelide 0.5 mg capsules Introduction Anagrelide (ANA) is an inhibitor of phosphodiesterase 3 (PDE3) that possesses both antithrombotic properties and the ability to reduce platelet counts. It is primarily used to treat thrombocythemia in patients with myeloproliferative disorders. The medication is commonly formulated as anagrelide hydrochloride, specifically designed to decrease platelet levels [1,2]. Anagrelide is primarily metabolized by the CYP 1 A 2 enzyme, which leads to the production of its active metabolite, 6,7-dichloro-3-hydroxy-1,5-dihydroimidazol[2,1-b]quinazolin-2-one, commonly referred to as 3-hydroxyanagrelide. Clinical observations indicate that palpitations are a widely reported side effect. However, these palpitations typically lessen with continued use over time [3]. Anagrelide exhibits dose proportionality within the 0.5 mg to 2.5 mg range. It is extensively absorbed, as demonstrated by the recovery of over 70% of a 1 mg oral radiolabeled dose in the urine of healthy individuals. However, the absolute oral bioavailability of anagrelide is estimated to be less than 50%. Peak plasma concentrations typically reach within 1–2 hours when taken on an empty stomach. The drug does not accumulate in the plasma after multiple doses. Pharmacokinetic studies comparing fed and fasting conditions in healthy volunteers indicate that consuming a 1 mg dose with food results in a 14% decrease in C max but a 20% increase in AUC. For its active metabolite, 3-hydroxyanagrelide, food intake lowers the C max by 29% without significantly affecting the AUC. The plasma half-life of anagrelide is about 1.5 hours, whereas the active metabolite has a half-life of approximately 2.5 hours [4]. Thus, this study will determine the bioequivalence of the lowest dose of anagrelide 0.5 mg tablets, which is difficult to assess in low doses. Methods 2.1 Ethics approval The study received approval from Erciyes University's Ethics Committee for bioavailability-bioequivalence trials on February 6, 2019, with the approval number 2019/21. In addition, the Turkish Ministry of Health (The Turkish Medicines and Medical Devices Agency) granted the final permit through an official letter dated March 18, 2019, with reference number 66175679-514.06.01-E.45591. The clinical phase of this study was carried out in compliance with the ethical principles outlined in the Declaration of Helsinki (Fortaleza, Brazil, October 2013), the Turkish Law on Pharmaceutical and Medical Preparations No. 1262, the ICH Harmonised Tripartite Guideline for Good Clinical Practice (ICH-GCP), and the Turkish Good Clinical Practice Guidelines (dated November 13, 2015) [5,6,7,8]. All participants in this clinical trial were thoroughly informed about the study's purpose, with written text and verbal information by the principal investigator/co-investigator. Subsequently, each participant who voluntarily signed the informed consent form was included in the study. 2.2 Identity of Investigational Products The test product was manufactured per Good Manufacturing Practices (GMP) standards. The reference product, purchased from the United States market, was used as the comparator in the study (Table 1 ). Table 1 Study Drugs Test Product ANAGRELIDE 0.5 mg Capsule Batch no.: 1910819001 İlko İlaç Sanayi ve Ticaret A.Ş Sancaktepe – İstanbul, Türkiye Expiry date: 01/2021 Reference Product AGRYLIN® (Anagrelide HCl) Capsule Batch no: AF9326E Shire US Inc- USA Expiry date: 07/2019 2.3 Study Design This study was a single-center, open-label, randomized, two-period crossover bioequivalence trial with a single dose that included 42 healthy male subjects (Fig. 1 ). Between July 1, 2019, and July 2, 2019, 47 healthy male Caucasian volunteers underwent screening. Following comprehensive physical examinations and clinical laboratory assessments, 42 individuals were enrolled in the study. Specific inclusion and exclusion criteria were established and assessed during the pre-study screening to ensure safety and reduce inter-subject variability in anagrelide pharmacokinetics. The study included 42 healthy male participants and was completed by all participants aged 18–54 with a body mass index (BMI) ranging from 19.6 to 29.9 kg/m². Participants were admitted to the Good Clinical Practice (GCP) center the evening before medication administration (Day 0). They were hospitalized until the completion of the 10-hour blood collection period on Day 1. Upon entry, the subjects were given an evening snack of approximately 600 kcal at approximately 7:30 PM, which they were expected to consume by 9:00 PM. After a fasting period of at least 10 hours, participants were given either the reference or test formulation orally, which included 0.5 mg of anagrelide (as anagrelide HCl) with 240 ml of water. After the drug administration, fluid intake was limited to water, and maximum water consumption on the dosing day was limited to a maximum of 1.5 liters. Additionally, water consumption was limited to one hour in the preceding and post-dosing. However, the participants were dosed with 240 ml of water. A standardized 1,200 kcal meal was served four hours after the dosing. Smoking was forbidden throughout the hospitalization, and alcohol consumption was restricted from two days before the hospitalization to the last blood sampling. Participants were prohibited from consuming foods or beverages containing caffeine, other methylxanthines (such as coffee, tea, cola, and chocolate), or fruit juice from two days before the study medication until the last blood sample was collected. Products containing orange or grapefruit were prohibited from seven days before the first dosing the completion of the trial. Use of prescribed systemic or topical medications was not allowed in the until two weeks of the drug administration. On the other hand, over-the-counter medications (including herbal supplements) were prohibited in the prior week of dosing. Participants were also required to refrain from using systemic or topical medications (including herbal products) until the study was completed. Any use of prescription or nonprescription systemic or topical medications due to any medical condition within the four weeks before the survey had to be documented on a case report form (CRF). Furthermore, subjects who had received any investigational drug within two months before the study were excluded. Immediately after pre-dose blood sampling, the test or reference product (0.5 mg of anagrelide as anagrelide HCl) was administered under the supervision of the investigators. Each participant swallowed the capsule, standing with 240 mL of water. A second healthcare practitioner watched drug administration and checked participants' mouths and hands to verify undesired drug disposal. Participants were then asked to sit upright for at least two hours following the drug administration. Subjects were discharged from the clinical center after the 10-hour blood sampling. There was at least a seven-day washout period between treatment periods. To assess the pharmacokinetics of anagrelide, blood samples were taken before dosing (0 h) and at specified intervals: 15 minutes (0.25 h), 30 minutes (0.50 h), 45 minutes (0.75 h), 1 hour, 1 hour 15 minutes (1.25 h), 1 hour 30 minutes (1.50 h), 1 hour 45 minutes (1.75 h), 2 hours, 2 hours 20 minutes (2.33 h), 2 hours 40 minutes (2.67 h), 3 hours, 3 hours 30 minutes (3.50 h), 4 hours, 5 hours, 6 hours, 8 hours, and 10 hours post-dose. Blood samples (approximately 7 mL each) were drawn from an antecubital or forearm vein using an indwelling catheter or individual venipunctures. Samples were placed in tubes containing K 2 EDTA, an anticoagulant, and in a mini refrigerator set at 4 ± 2°C for up to 20 minutes. After centrifugation (3,000 rpm, 4–7°C, 10 minutes), the obtained plasma was transferred to labeled polypropylene storage tubes (two tubes containing at least 1.5 mL of plasma for each sample) within 20 minutes. These tubes were sealed and stored in a deep freezer below <-70°C until shipment to Analytisches Zentrum Biopharm GmbH in Berlin, Germany. Participants were prohibited from taking any medication for adverse events during the study without the investigators’ permission. In case of the need to administer a concomitant treatment for any adverse event, investigators must use proper medication. Then, any potential impact of such treatment on the pharmacokinetics of the study drug was carefully evaluated. The only medicines permitted during the trial were single doses of paracetamol, which could be used to manage adverse events such as headaches. 2.4 Analytical Part: The analytical part of the present study was performed following the EMA Guideline on bioanalytical method validation and FDA Guidance for Industry: Bioanalytical Method Validation [9,10]. All available samples from the 42 participants were analyzed. Plasma samples were analyzed for anagrelide concentrations using a validated LC-MS/MS method with ESI(+). The analytical procedures complied with Good Laboratory Practice (GLP) regulations. The lower limit of quantification for anagrelide was set at 40.0 pg/mL when measuring unknown plasma sample concentrations. The mean relative deviations of quality control (QC) samples, representing inter-assay accuracy (bias%), were calculated as 3.8% for high-quality control (HQC), 1.8% for medium-quality control (MQC), and − 0.7% for low-quality control (LQC) samples. Corresponding inter-assay precision values (CV%) were determined to be 4.5% (HQC), 5.6% (MQC), and 9.9% (LQC). 2.5 Statistics Statistical analysis of pharmacokinetic study data was conducted following the guidelines specified (8). The analysis included all participants who had no significant protocol deviations and had available measurements for all primary target variables (per protocol subject set). The primary target variables were AUC (0−t) and C max , while AUC (0−∞) , t max , λ z , and t ½ (λ z ) were classified as secondary variables. AUC %−extrapol was assessed as an auxiliary pharmacokinetic parameter. Statistical evaluations were performed using Phoenix WinNonlin V8.1.0.3530. To improve adherence to standard distribution assumptions, AUC (0−t) , AUC (0−∞) , and C max data were logarithmically transformed before undergoing parametric analysis (ANOVA). Based on these transformations, 90% confidence intervals were derived using two one-sided t-tests. Bioequivalence was confirmed if the 90% confidence intervals for AUC (0−t) and C max fell within the predefined range of 80.00% – 125.00%. Additionally, statistical assessments of t max values were performed. Non-parametric analysis using two one-sided Wilcoxon tests was also conducted, with the resulting 90% confidence intervals documented as supportive data; however, these results were not used to determine bioequivalence. Pharmacokinetic parameters, including AUC (0−t) , AUC (0−∞) , C max , t max , λ z , t ½ (λ z ), and AUC %−extrapol for anagrelide, were calculated using Phoenix WinNonlin software (version 8.1.0.3530). According to the proposed methodology by Diletti et al. [11] and considering an estimated ANOVA coefficient of variation (intra-subject variability) ranging from 20–25% or higher for the key parameters AUC (0−t) and C max , a test/reference parameter ratio of 0.95–1.05, and statistical power of 80%, a minimum of 36 participants (per protocol population for pharmacokinetic evaluation) was deemed necessary to meet the study's objectives. Forty-two subjects received the study drug, and no dropouts were replaced unless the number of evaluable subjects fell below the required 36. Results 3.1 Pharmacokinetic results Analytical data from the pharmacokinetic study underwent a blind review, confirming that the dataset included all 42 participants who completed the survey without protocol deviations or violations of inclusion criteria. Additionally, all primary target variables were available for analysis. Initially, 47 male Caucasian participants were screened through comprehensive physical examinations and clinical laboratory assessments. Of these volunteers, 42 healthy male individuals aged 18–54 with a BMI between 19.6–29.9 kg/m² were included in the study. Each participant strictly followed the study protocol and completed the trial. Plasma samples from all 42 participants were collected to measure anagrelide concentrations. The mean (± s.d.) demographic data for the 42 participants who completed the study were as follows: 34.12 (± 8.95) years in age, 78.67 (± 9.95) kg in weight, 174.90 (± 5.41) cm in height, and a BMI of 25.71 (± 2.94) kg/m² (Table 2 ). Table 2 Demographic Data of Subjects Parameter Age (a) Weight (kg) Height (cm) BMI (kg/m 2 ) Mean Value 34.12 78.67 174.90 25.71 Standard Deviation 8.95 9.95 5.41 2.94 CV (%) 26.23 12.65 3.10 11.44 Minimum 18 58 164 19.60 Maximum 54 100 186 29.90 Number 42 42 42 42 Pharmacokinetic analysis showed striking similarity between test and reference drugs. There were no significant differences in key pharmacokinetic parameters of anagrelide, including AUC (0−t) , AUC (0−∞) , and C max (Table 3 ). Furthermore, additional parameters such as λ z , t ½ (λ z ), and AUC %−extrapol were evaluated and summarized in a table with corresponding descriptive statistics. Table 3 Pharmacokinetic parameters (arithmetic means ± s.d.) of anagrelide; n = 42 subjects Drugs AUC (0−t) [pg × h/mL] AUC (0−∞) [pg × h/mL] C max [pg/mL] t max [h] λ z [1/h] t ½ (λz) [h] %AUC %−extrapol [%] T: ANAGRELIDE 0.5 mg Capsule batch number: 1910819001 4,533.3 ± 2,379.3 4,666.5 ± 2,394.3 1,997.1 ± 1,159.2 0.94 ± 0.53 0.5222 ± 0.1338 1.47 ± 0.75 3.29 ± 2.33 R: AGRYLIN® (Anagrelide HCl) Capsule batch number: AF9326E 4,515.0 ± 2,392.3 4,638.0 ± 2,447.6 2.061.3± 1,054.0 0.94± 0.59 0.5528± 0.1205 32 ± 30 2.94 ± 1.32 The mean plasma concentrations of anagrelide are illustrated in Figs. 2 a and 2 b, presented on both linear (± SEM) and log-linear scales. Table 4 presents the 90% confidence intervals along with the corresponding point estimators, calculated as the ratios of the test to reference geometric means. Table 4 Statistical Results of Anagrelide (test vs reference); n = 42 Pharmacokinetic parameters: (ANOVA; two one-sided t-tests) 90% confidence intervals point estimators (geometric means; ratios test/reference) Intra-subject variability AUC (0−t) 94.09% − 104.75% 99.28% 14.68% C max 85.62% − 104.03% 94.37% 26.98% AUC (0−∞) 94.50% − 105.10% 99.66% 14.55% 3.2 Safety Results All adverse events (AE) reported by participants, whether spontaneously, during routine questioning before the start of each study period, or during scheduled assessments at specified time points (0-hour, 1 hour, 6 hours, and 10 hours) in both periods, were documented in the AE information sheet within each participant’s CRF. Throughout the study, 13 adverse events were recorded in 11 of the 42 enrolled participants. Among these, eight treatment-emergent adverse events were classified as moderate-intensity headaches. Three headaches occurred in Period I, two following administration of the reference product and one after the test product. Five headaches were reported in Period II, three after receiving the reference product and two after the test product. Of these, five headaches were managed with paracetamol (500 mg). Additionally, five adverse events were classified as mild in intensity, including two headaches, one case of nausea, one instance of vomiting, and one case of hypotension. One headache was observed in Period II after administration of the reference product, while hypotension occurred in Period I following the test product. The remaining three mild events (one headache, nausea, and vomiting) were reported in Period I after administration of the reference product. All treatment-emergent adverse events were assessed as “possible” attributable to study drug, except hypotension, which was determined as unlikely drug related, and all cases resolved. There were no severe adverse events. A summary of observed adverse events is provided in Table 5 . Table 5 A summary of the observed treatment-emergent adverse events Drug Relationship Adverse Event (no. AE) (WHO term) Treatment T Treatment R possible vomiting (1) - 1 possible nausea (1) - 1 possible headache (10) 3 7 unlikely hypotension (1) 1 - Discussion Anagrelide's rate (C max ) and extent (AUC (0−t) ) of absorption from the reference formulation and test formulation are comparable, according to the study's findings. The bioequivalence range of 80.00–125.00% encompasses the 90% confidence intervals for the T/R ratios of the natural In-transformed AUC (0−t) and C max values. Likewise, the secondary pharmacokinetic measure AUC (0−∞) 's 90% confidence interval satisfies the accepted bioequivalence standards [8]. Both formulations demonstrated acceptable safety profiles, with no clinically significant differences in tolerability. Bioequivalence between test and reference was confirmed in 42 healthy male participants following a single oral dose of 0.5 mg anagrelide (as anagrelide HCl) under fasting conditions. The rate and extent of a drug's absorption and the extent to which it becomes available at the site of action are referred to as bioavailability. According to guidelines, two medicinal products are considered bioequivalent if their bioavailability is sufficiently similar after administering an equivalent molar dose, ensuring comparable efficacy and safety [8]. This condition is met when the 90% confidence intervals for AUC (0−t) and C max ratios fall within the pre-defined range of 80.00–125.00%. Common adverse effects associated with anagrelide use include rapid heartbeat, chest pain, breathing difficulties, numbness, tingling, burning sensations, headache, dizziness, weakness, gastrointestinal disturbances (such as stomach pain, gas, indigestion, nausea, vomiting, diarrhea, and loss of appetite), swelling in the hands or feet, fever, cough, general malaise, back pain, rash, and itching [12]. While the likelihood of these adverse effects is lower with single-dose administration, using the lowest effective dose in healthy volunteers remains essential to minimize potential risks [13]. Previous study on healthy participants have utilized higher doses (2 mg) than the 0.5 mg dose used in this study [14]. In contrast, our study employed a lower dose (0.5 mg), reducing the risk of adverse effects in healthy volunteers. The importance of this situation has also been shown in our previous study [15]. While the drug in the extended-release formulation was used in the previous research, the immediate-release formulation was applied in our study [15]. The study was designed under fasting conditions. The earlier study observed that the pharmacokinetic parameters C max , AUC, and t 1/2 were affected when the drug was taken with food [15]. Conclusion This study demonstrated the bioequivalence of the test and reference drugs containing low doses of anagrelide. Likewise, the safety of the test drug is equivalent to the reference drug. The low-dose design in healthy volunteers will guide future studies. Abbreviations AUC Area under the concentration time curve of plasma C max Maximum plasma concentration t max time to achieve C max t 1/2 half life CI Confidence intervals; CRF Case report form EMA European Medicines Agency FDA U.S. Food and Drug Administration HPLC High-performance Liquid Chromatography LLOQ Lower limit of quantification QC Quality control HQC High-quality control MQC Medium-quality control LMQC Low-medium-quality control LQC Low-quality control LC-MS/MS Liquid Chromatography Mass Spectrometer TITCK Turkiye Ilac ve Tıbbi Cihaz Kurumu Declarations Acknowledgements We would like to thank the Proofreading & Editing Office of the Dean for Research at Erciyes University for copyediting and proofreading service for this manuscript. Author contributions All authors contributed to the concept and design of the study. OP and BB took decisions in the name of the sponsor. SK prepared study documentation and carried out the necessary application and submissions for the study. AI carried out the clinical part as the principal investigator, ZS and MM as co-investigator. MR, performed Bioanalysis in plasma. WM performed the Pharmacokinetic analysis, statistics and reporting parts. AI and SK contributed to writing the manuscript. All authors approved the final version of the manuscript. Funding This study was supported by ILKO Pharmaceuticals, Turkiye. Data availability The data are not publicly available due to compromise the privacy of study participants and due to the fact that trade secret. The data that are publicly available are shared in the article. Ethics approval and consent to participate The study was approved by an independent ethics committee-Ethics Committee for Bioavailability-Bioequivalence Trials of Erciyes University (date of approval: 06.02.2019; vote no: 2019/21) and the Turkish Medicines and Medical Devices Agency (TİTCK) of Turkish Ministry of Health, (date of approval: 18.03.2019, note no.: 66175679-514.06.01-E.45591). The purpose of the investigation was fully disclosed to each subject in a written document and oral presentation by the principal investigator or one of the co-investigators. Then, each participant voluntarily signed the informed consent form. Consent for publication Not applicable Competing Interests AI is the principal investigator at Hakan Cetinsaya GCP Center, and ZS and MM are co-investigators at Hakan Çetinsaya GCP Center. OP and BB are the sponsor representatives from ILKO Pharmaceuticals. MR is staff in Analytisches Zentrum Biopharm GmbH. WM is a staff in Pharmakin Consulting Services UG. SAK is staff in IDEAL Biyolojik Urunler ve Ilac Danismanlik Egitim Ltd. Sti. References PDE3A inhibitor anagrelide activates death signaling pathway genes and synergizes with cell death-inducing cytokines to selectively inhibit cancer cell growth. An R, Liu J, He J, Wang F, Zhang Q, Yu Q. Am J Cancer Res. 2019 Sep 1;9(9):1905-1921. eCollection 2019. https://www.ncbi.nlm.nih.gov/books/NBK548467/pdf/Bookshelf_NBK548467.pdf,2025 reached at 12.03.2025 Cardiovascular safety of anagrelide in healthy subjects: effects of caffeine and food intake on pharmacokinetics and adverse reactions. Martínez-Sellés M, Datino T, Figueiras-Graillet L, Gama JG, Jones C, Franklin R, Fernández-Avilés F. Clin Drug Investig. 2013 Jan;33(1):45-54. doi: 10.1007/s40261-012-0032-2. Wagstaff AJ, Keating GM. Anagrelide: a review of its use in the management of essential thrombocythaemia Drugs 2006; 66(1): 111 – 131 WMA Declaration of Helsinki – Ethical Principles for Medical Research Involving Human Participants https://www.wma.net/wp-content/uploads/2024/10/DoH-Oct2013.pdf reached at 11.03.2025 Pharmaceutical and medical preparations law; No : 1262. Publication Date in the Official Gazette: 26th May, 1928 Issue No. 898. https://titck.gov.tr/storage/legislation/meFOxeFD.pdf) GCP regulations. Turkish Pharmaceuticals and Medical Devices Agency, 13 Nov 2015 https://titck.gov.tr/storage/Archive/2020/legislation/KADKLVZ01IKU13.11.2015Rev08_13ac0133-274b-44dc-98cd-33998758cc72.pdf Guideline on the Investigation of Bioequivalence Committee for Medicinal Products for Human Use (CHMP) CPMP/EWP/QWP/1401/98 Rev. 1/Corr**, London, 20 January 2010 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-investigation-bioequivalence-rev1_en.pdf reached at 25.07.2024 Guideline on bioanalytical method validation EMEA/CHMP/EWP/192217/2009- 21 July 2011 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method validation_en.pdf reached at 25.07.2024 Guidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf reached at 28.07.2024 Diletti E, Hauschke D, Steinijans VW Sample size determination for bioequivalence assessment by means of confidence intervals Int J Clin Pharmacol Ther Toxicol 30 (Suppl. 1), 51 - 58 (1992) RxList Internet Drug Index – Anagrelide https://www.rxlist.com/anagrelide/generic-drug.htm, at reached 11.03.2025 Bioequivalence study of low dose drospirenone/ethinyl estradiol 3 mg/0.03 mg film tablets under fasting conditions in Turkish healthy female subjects. Inal A, Sezer Z, Uluözlü B, Oflas M, Reinsch M, Martin W, Mazicioglu MM, Koru SA. Pharmacol Res Perspect.2024 Aug;12(4):e1253 Pharmacokinetics, bioequivalence, tolerability, and effects on platelet counts of two formulations of anagrelide in healthy volunteers and patients with thrombocythemia associated with chronic myeloproliferation. Petrides PE, Gisslinger H, Steurer M, Linkesch W, Krumpl G, Schüller A, Widmann R. Clin Ther. 2009 Feb;31(2):386-98. doi: 10.1016/j.clinthera.2009.02.008. Pharmacokinetics of a Novel Anagrelide Extended-Release Formulation in Healthy Subjects: Food Intake and Comparison With a Reference Product. Petrides PE, Schoergenhofer C, Widmann R, Jilma B, Klade CS Clin Pharmacol Drug Dev. 2018 Feb;7(2):123-131. doi: 10.1002/cpdd.340. Epub 2017 Mar 16. Additional Declarations Competing interest reported. AI is the principal investigator at Hakan Cetinsaya GCP Center, and ZS and MM are co-investigators at Hakan Çetinsaya GCP Center. OP and BB are the sponsor representatives from ILKO Pharmaceuticals. MR is staff in Analytisches Zentrum Biopharm GmbH. WM is a staff in Pharmakin Consulting Services UG. SAK is staff in IDEAL Biyolojik Urunler ve Ilac Danismanlik Egitim Ltd. Sti. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6453732","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":443199258,"identity":"e6e25b52-3de7-48bd-9fa0-213b229d8f53","order_by":0,"name":"Ahmet Inal","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3klEQVRIiWNgGAWjYDACZiB+UAFlAAFjA1FaEs6gaGEmwqbENgSbsBb+dt6DHxLn2eTzt/MYPvzBYCO74QD/sQ/4tEgc5kuWSNyWZjnjMI+xMQ9DmvGGA8zMM/Bac5jHAKjlsIEBM4+ZNJCbCNKCV4c80PAfiXP+g7SY//zB8J+wFoPDPGYSiQ0HwLYw8DAcIKzFEKjFIuFYsoHEYbZiaR6DZOOZh5mN8WqRO3/G+MaHGjsD/v7DGz/+qLCT7Tve+BivFiTAYQB0JwMDUTEJBewPiFc7CkbBKBgFIwoAAPMQQUcOuHYqAAAAAElFTkSuQmCC","orcid":"","institution":"Erciyes University, Erciyes University, Hakan Cetinsaya Good Clinical Practice and Research Center","correspondingAuthor":true,"prefix":"","firstName":"Ahmet","middleName":"","lastName":"Inal","suffix":""},{"id":443199259,"identity":"2ea31038-bc1f-482f-a10a-22b284c533e2","order_by":1,"name":"Zafer Sezer","email":"","orcid":"","institution":"Erciyes University, Erciyes University, Hakan Cetinsaya Good Clinical Practice and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Zafer","middleName":"","lastName":"Sezer","suffix":""},{"id":443199260,"identity":"29e81e3f-2f11-434e-a854-a5ba2002bcca","order_by":2,"name":"Onur Pinarbasli","email":"","orcid":"","institution":"ILKO Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Onur","middleName":"","lastName":"Pinarbasli","suffix":""},{"id":443199261,"identity":"989d9219-fb40-402e-8072-b462c81887c8","order_by":3,"name":"Burcu Bulut","email":"","orcid":"","institution":"ILKO Pharmaceuticals","correspondingAuthor":false,"prefix":"","firstName":"Burcu","middleName":"","lastName":"Bulut","suffix":""},{"id":443199262,"identity":"6874d05e-8069-4c71-98e6-d69fa4af7033","order_by":4,"name":"Martin Reinsch","email":"","orcid":"","institution":"Analytisches Zentrum Biopharm GmbH","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Reinsch","suffix":""},{"id":443199263,"identity":"c9108b49-a1fb-47f0-8684-ab178299d843","order_by":5,"name":"Wolfgang Martin","email":"","orcid":"","institution":"Pharmakin Consulting Services UG","correspondingAuthor":false,"prefix":"","firstName":"Wolfgang","middleName":"","lastName":"Martin","suffix":""},{"id":443199264,"identity":"e8dcae0f-d946-427a-9a55-e2648bbd6721","order_by":6,"name":"Mumtaz M. Mazicioglu","email":"","orcid":"","institution":"Erciyes University, University Hakan Cetinsaya Good Clinical Practice and Research Center","correspondingAuthor":false,"prefix":"","firstName":"Mumtaz","middleName":"M.","lastName":"Mazicioglu","suffix":""},{"id":443199265,"identity":"6d75d8ce-7ff7-4ae2-8ff6-6d0ec6b44271","order_by":7,"name":"Selma Alime Koru","email":"","orcid":"","institution":"IDEAL Biyolojik Urunler ve Ilac Danismanlik Egitimi Ltd. Sti","correspondingAuthor":false,"prefix":"","firstName":"Selma","middleName":"Alime","lastName":"Koru","suffix":""}],"badges":[],"createdAt":"2025-04-15 10:23:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6453732/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6453732/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":80698080,"identity":"e5ee6561-b1e8-4062-a6c3-ce21c207df4a","added_by":"auto","created_at":"2025-04-16 07:12:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":119288,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic study design\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6453732/v1/f7c1c9381869b4c5493bafb4.png"},{"id":80698083,"identity":"c500cd4b-118e-46b0-ae31-2cc2039f4bbe","added_by":"auto","created_at":"2025-04-16 07:12:26","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443077,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Mean concentrations (± sem) of anagrelide in plasma after administration of different (test and reference) anagrelide-containing preparations; linear plot; n = 42\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eb.\u003c/strong\u003e Mean concentrations of anagrelide in plasma after administration of different (test and reference) anagrelide-containing preparations; loglinear plot; n = 42\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6453732/v1/f74fd80133ac2c3c551373e5.png"},{"id":86112239,"identity":"1a5d8235-a4da-4f94-9a9f-f557a632eb0c","added_by":"auto","created_at":"2025-07-06 21:16:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1369931,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6453732/v1/8caddd6d-6436-4b9a-a1c0-66d910d8b06a.pdf"}],"financialInterests":"Competing interest reported. AI is the principal investigator at Hakan Cetinsaya GCP Center, and ZS and MM are co-investigators at Hakan Çetinsaya GCP Center. OP and BB are the sponsor representatives from ILKO Pharmaceuticals. MR is staff in Analytisches Zentrum Biopharm GmbH. WM is a staff in Pharmakin Consulting Services UG. SAK is staff in IDEAL Biyolojik Urunler ve Ilac Danismanlik Egitim Ltd. Sti.","formattedTitle":"Bioequivalence Study of Low-Dose Anagrelide 0.5 mg Capsules under Fasting Conditions in Healthy Turkish Male Subjects","fulltext":[{"header":"Key points","content":"\u003cp\u003eBioequivalence of anagrelide 0.5 mg capsules\u003cbr\u003eThe safety profile of anagrelide 0.5 mg capsules\u003c/p\u003e"},{"header":"Introduction","content":"\u003cp\u003eAnagrelide (ANA) is an inhibitor of phosphodiesterase 3 (PDE3) that possesses both antithrombotic properties and the ability to reduce platelet counts. It is primarily used to treat thrombocythemia in patients with myeloproliferative disorders. The medication is commonly formulated as anagrelide hydrochloride, specifically designed to decrease platelet levels [1,2].\u003c/p\u003e \u003cp\u003eAnagrelide is primarily metabolized by the CYP\u003csub\u003e1\u003c/sub\u003eA\u003csub\u003e2\u003c/sub\u003e enzyme, which leads to the production of its active metabolite, 6,7-dichloro-3-hydroxy-1,5-dihydroimidazol[2,1-b]quinazolin-2-one, commonly referred to as 3-hydroxyanagrelide. Clinical observations indicate that palpitations are a widely reported side effect. However, these palpitations typically lessen with continued use over time [3].\u003c/p\u003e \u003cp\u003eAnagrelide exhibits dose proportionality within the 0.5 mg to 2.5 mg range. It is extensively absorbed, as demonstrated by the recovery of over 70% of a 1 mg oral radiolabeled dose in the urine of healthy individuals. However, the absolute oral bioavailability of anagrelide is estimated to be less than 50%. Peak plasma concentrations typically reach within 1\u0026ndash;2 hours when taken on an empty stomach. The drug does not accumulate in the plasma after multiple doses. Pharmacokinetic studies comparing fed and fasting conditions in healthy volunteers indicate that consuming a 1 mg dose with food results in a 14% decrease in C\u003csub\u003emax\u003c/sub\u003e but a 20% increase in AUC. For its active metabolite, 3-hydroxyanagrelide, food intake lowers the C\u003csub\u003emax\u003c/sub\u003e by 29% without significantly affecting the AUC. The plasma half-life of anagrelide is about 1.5 hours, whereas the active metabolite has a half-life of approximately 2.5 hours [4].\u003c/p\u003e \u003cp\u003eThus, this study will determine the bioequivalence of the lowest dose of anagrelide 0.5 mg tablets, which is difficult to assess in low doses.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Ethics approval\u003c/h2\u003e \u003cp\u003e The study received approval from Erciyes University's Ethics Committee for bioavailability-bioequivalence trials on February 6, 2019, with the approval number 2019/21. In addition, the Turkish Ministry of Health (The Turkish Medicines and Medical Devices Agency) granted the final permit through an official letter dated March 18, 2019, with reference number 66175679-514.06.01-E.45591.\u003c/p\u003e \u003cp\u003e The clinical phase of this study was carried out in compliance with the ethical principles outlined in the Declaration of Helsinki (Fortaleza, Brazil, October 2013), the Turkish Law on Pharmaceutical and Medical Preparations No. 1262, the ICH Harmonised Tripartite Guideline for Good Clinical Practice (ICH-GCP), and the Turkish Good Clinical Practice Guidelines (dated November 13, 2015) [5,6,7,8].\u003c/p\u003e \u003cp\u003eAll participants in this clinical trial were thoroughly informed about the study's purpose, with written text and verbal information by the principal investigator/co-investigator. Subsequently, each participant who voluntarily signed the informed consent form was included in the study.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2.2 Identity of Investigational Products\u003c/h3\u003e\n\u003cp\u003eThe test product was manufactured per Good Manufacturing Practices (GMP) standards. The reference product, purchased from the United States market, was used as the comparator in the study (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStudy Drugs\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTest Product\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eANAGRELIDE 0.5 mg Capsule\u003c/p\u003e \u003cp\u003eBatch no.: 1910819001\u003c/p\u003e \u003cp\u003eİlko İlaç Sanayi ve Ticaret A.Ş Sancaktepe – İstanbul, Türkiye\u003c/p\u003e \u003cp\u003eExpiry date: 01/2021\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eReference Product\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAGRYLIN® (Anagrelide HCl) Capsule\u003c/p\u003e \u003cp\u003eBatch no: AF9326E\u003c/p\u003e \u003cp\u003eShire US Inc- USA\u003c/p\u003e \u003cp\u003eExpiry date: 07/2019\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e \u003cp\u003e\u003c/p\u003e\n\u003ch3\u003e2.3 Study Design\u003c/h3\u003e\n\u003cp\u003eThis study was a single-center, open-label, randomized, two-period crossover bioequivalence trial with a single dose that included 42 healthy male subjects (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Between July 1, 2019, and July 2, 2019, 47 healthy male Caucasian volunteers underwent screening. Following comprehensive physical examinations and clinical laboratory assessments, 42 individuals were enrolled in the study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSpecific inclusion and exclusion criteria were established and assessed during the pre-study screening to ensure safety and reduce inter-subject variability in anagrelide pharmacokinetics. The study included 42 healthy male participants and was completed by all participants aged 18–54 with a body mass index (BMI) ranging from 19.6 to 29.9 kg/m².\u003c/p\u003e \u003cp\u003eParticipants were admitted to the Good Clinical Practice (GCP) center the evening before medication administration (Day 0). They were hospitalized until the completion of the 10-hour blood collection period on Day 1. Upon entry, the subjects were given an evening snack of approximately 600 kcal at approximately 7:30 PM, which they were expected to consume by 9:00 PM. After a fasting period of at least 10 hours, participants were given either the reference or test formulation orally, which included 0.5 mg of anagrelide (as anagrelide HCl) with 240 ml of water.\u003c/p\u003e \u003cp\u003eAfter the drug administration, fluid intake was limited to water, and maximum water consumption on the dosing day was limited to a maximum of 1.5 liters. Additionally, water consumption was limited to one hour in the preceding and post-dosing. However, the participants were dosed with 240 ml of water. A standardized 1,200 kcal meal was served four hours after the dosing.\u003c/p\u003e \u003cp\u003eSmoking was forbidden throughout the hospitalization, and alcohol consumption was restricted from two days before the hospitalization to the last blood sampling. Participants were prohibited from consuming foods or beverages containing caffeine, other methylxanthines (such as coffee, tea, cola, and chocolate), or fruit juice from two days before the study medication until the last blood sample was collected. Products containing orange or grapefruit were prohibited from seven days before the first dosing the completion of the trial.\u003c/p\u003e \u003cp\u003eUse of prescribed systemic or topical medications was not allowed in the until two weeks of the drug administration. On the other hand, over-the-counter medications (including herbal supplements) were prohibited in the prior week of dosing. Participants were also required to refrain from using systemic or topical medications (including herbal products) until the study was completed. Any use of prescription or nonprescription systemic or topical medications due to any medical condition within the four weeks before the survey had to be documented on a case report form (CRF). Furthermore, subjects who had received any investigational drug within two months before the study were excluded.\u003c/p\u003e \u003cp\u003eImmediately after pre-dose blood sampling, the test or reference product (0.5 mg of anagrelide as anagrelide HCl) was administered under the supervision of the investigators. Each participant swallowed the capsule, standing with 240 mL of water. A second healthcare practitioner watched drug administration and checked participants' mouths and hands to verify undesired drug disposal. Participants were then asked to sit upright for at least two hours following the drug administration.\u003c/p\u003e \u003cp\u003eSubjects were discharged from the clinical center after the 10-hour blood sampling. There was at least a seven-day washout period between treatment periods.\u003c/p\u003e \u003cp\u003eTo assess the pharmacokinetics of anagrelide, blood samples were taken before dosing (0 h) and at specified intervals: 15 minutes (0.25 h), 30 minutes (0.50 h), 45 minutes (0.75 h), 1 hour, 1 hour 15 minutes (1.25 h), 1 hour 30 minutes (1.50 h), 1 hour 45 minutes (1.75 h), 2 hours, 2 hours 20 minutes (2.33 h), 2 hours 40 minutes (2.67 h), 3 hours, 3 hours 30 minutes (3.50 h), 4 hours, 5 hours, 6 hours, 8 hours, and 10 hours post-dose.\u003c/p\u003e \u003cp\u003eBlood samples (approximately 7 mL each) were drawn from an antecubital or forearm vein using an indwelling catheter or individual venipunctures. Samples were placed in tubes containing K\u003csub\u003e2\u003c/sub\u003eEDTA, an anticoagulant, and in a mini refrigerator set at 4 ± 2°C for up to 20 minutes. After centrifugation (3,000 rpm, 4–7°C, 10 minutes), the obtained plasma was transferred to labeled polypropylene storage tubes (two tubes containing at least 1.5 mL of plasma for each sample) within 20 minutes. These tubes were sealed and stored in a deep freezer below \u0026lt;-70°C until shipment to Analytisches Zentrum Biopharm GmbH in Berlin, Germany.\u003c/p\u003e \u003cp\u003eParticipants were prohibited from taking any medication for adverse events during the study without the investigators’ permission. In case of the need to administer a concomitant treatment for any adverse event, investigators must use proper medication. Then, any potential impact of such treatment on the pharmacokinetics of the study drug was carefully evaluated. The only medicines permitted during the trial were single doses of paracetamol, which could be used to manage adverse events such as headaches.\u003c/p\u003e\n\u003ch3\u003e2.4 Analytical Part:\u003c/h3\u003e\n\u003cp\u003e The analytical part of the present study was performed following the EMA Guideline on bioanalytical method validation and FDA Guidance for Industry: Bioanalytical Method Validation [9,10].\u003c/p\u003e \u003cp\u003eAll available samples from the 42 participants were analyzed. Plasma samples were analyzed for anagrelide concentrations using a validated LC-MS/MS method with ESI(+). The analytical procedures complied with Good Laboratory Practice (GLP) regulations. The lower limit of quantification for anagrelide was set at 40.0 pg/mL when measuring unknown plasma sample concentrations. The mean relative deviations of quality control (QC) samples, representing inter-assay accuracy (bias%), were calculated as 3.8% for high-quality control (HQC), 1.8% for medium-quality control (MQC), and − 0.7% for low-quality control (LQC) samples. Corresponding inter-assay precision values (CV%) were determined to be 4.5% (HQC), 5.6% (MQC), and 9.9% (LQC).\u003c/p\u003e\n\u003ch3\u003e2.5 Statistics\u003c/h3\u003e\n\u003cp\u003e Statistical analysis of pharmacokinetic study data was conducted following the guidelines specified (8). The analysis included all participants who had no significant protocol deviations and had available measurements for all primary target variables (per protocol subject set).\u003c/p\u003e \u003cp\u003eThe primary target variables were AUC\u003csub\u003e(0−t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e, while AUC\u003csub\u003e(0−∞)\u003c/sub\u003e, t\u003csub\u003emax\u003c/sub\u003e, λ\u003csub\u003ez\u003c/sub\u003e, and t\u003csub\u003e½\u003c/sub\u003e(λ\u003csub\u003ez\u003c/sub\u003e) were classified as secondary variables. AUC\u003csub\u003e%−extrapol\u003c/sub\u003e was assessed as an auxiliary pharmacokinetic parameter. Statistical evaluations were performed using Phoenix WinNonlin V8.1.0.3530. To improve adherence to standard distribution assumptions, AUC\u003csub\u003e(0−t)\u003c/sub\u003e, AUC\u003csub\u003e(0−∞)\u003c/sub\u003e, and C\u003csub\u003emax\u003c/sub\u003e data were logarithmically transformed before undergoing parametric analysis (ANOVA).\u003c/p\u003e \u003cp\u003eBased on these transformations, 90% confidence intervals were derived using two one-sided t-tests. Bioequivalence was confirmed if the 90% confidence intervals for AUC\u003csub\u003e(0−t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e fell within the predefined range of 80.00% – 125.00%. Additionally, statistical assessments of t\u003csub\u003emax\u003c/sub\u003e values were performed. Non-parametric analysis using two one-sided Wilcoxon tests was also conducted, with the resulting 90% confidence intervals documented as supportive data; however, these results were not used to determine bioequivalence. Pharmacokinetic parameters, including AUC\u003csub\u003e(0−t)\u003c/sub\u003e, AUC\u003csub\u003e(0−∞)\u003c/sub\u003e, C\u003csub\u003emax\u003c/sub\u003e, t\u003csub\u003emax\u003c/sub\u003e, λ\u003csub\u003ez\u003c/sub\u003e, t\u003csub\u003e½\u003c/sub\u003e(λ\u003csub\u003ez\u003c/sub\u003e), and AUC\u003csub\u003e%−extrapol\u003c/sub\u003e for anagrelide, were calculated using Phoenix WinNonlin software (version 8.1.0.3530).\u003c/p\u003e \u003cp\u003eAccording to the proposed methodology by Diletti et al. [11] and considering an estimated ANOVA coefficient of variation (intra-subject variability) ranging from 20–25% or higher for the key parameters AUC\u003csub\u003e(0−t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e, a test/reference parameter ratio of 0.95–1.05, and statistical power of 80%, a minimum of 36 participants (per protocol population for pharmacokinetic evaluation) was deemed necessary to meet the study's objectives. Forty-two subjects received the study drug, and no dropouts were replaced unless the number of evaluable subjects fell below the required 36.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003e3.1 Pharmacokinetic results\u003c/h2\u003e\u003cp\u003eAnalytical data from the pharmacokinetic study underwent a blind review, confirming that the dataset included all 42 participants who completed the survey without protocol deviations or violations of inclusion criteria. Additionally, all primary target variables were available for analysis. Initially, 47 male Caucasian participants were screened through comprehensive physical examinations and clinical laboratory assessments. Of these volunteers, 42 healthy male individuals aged 18–54 with a BMI between 19.6–29.9 kg/m² were included in the study. Each participant strictly followed the study protocol and completed the trial. Plasma samples from all 42 participants were collected to measure anagrelide concentrations.\u003c/p\u003e\u003cp\u003eThe mean (± s.d.) demographic data for the 42 participants who completed the study were as follows: 34.12 (± 8.95) years in age, 78.67 (± 9.95) kg in weight, 174.90 (± 5.41) cm in height, and a BMI of 25.71 (± 2.94) kg/m² (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDemographic Data of Subjects\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameter\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAge (a)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeight (kg)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eHeight (cm)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eBMI (kg/m\u003csup\u003e2\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMean Value\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e34.12\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e78.67\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e174.90\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e25.71\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStandard Deviation\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e9.95\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e5.41\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCV (%)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e26.23\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.65\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.10\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.44\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimum\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e58\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e164\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e19.60\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaximum\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e54\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e186\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e29.90\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e42\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003ePharmacokinetic analysis showed striking similarity between test and reference drugs. There were no significant differences in key pharmacokinetic parameters of anagrelide, including AUC\u003csub\u003e(0−t)\u003c/sub\u003e, AUC\u003csub\u003e(0−∞)\u003c/sub\u003e, and C\u003csub\u003emax\u003c/sub\u003e (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Furthermore, additional parameters such as λ\u003csub\u003ez\u003c/sub\u003e, t\u003csub\u003e½\u003c/sub\u003e(λ\u003csub\u003ez\u003c/sub\u003e), and AUC\u003csub\u003e%−extrapol\u003c/sub\u003e were evaluated and summarized in a table with corresponding descriptive statistics.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"±\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePharmacokinetic parameters (arithmetic means ± s.d.) of anagrelide; n = 42 subjects\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrugs\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAUC\u003csub\u003e(0−t)\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[pg × h/mL]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAUC\u003csub\u003e(0−∞)\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[pg × h/mL]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[pg/mL]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003et\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[h]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eλ\u003csub\u003ez\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[1/h]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003et\u003csub\u003e½\u003c/sub\u003e (λz)\u003c/p\u003e \u003cp\u003e[h]\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e%AUC\u003csub\u003e%−extrapol\u003c/sub\u003e\u003c/p\u003e \u003cp\u003e[%]\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT: ANAGRELIDE 0.5 mg Capsule\u003c/p\u003e \u003cp\u003ebatch number:\u003c/p\u003e \u003cp\u003e1910819001\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e4,533.3 ± 2,379.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e4,666.5 ± 2,394.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e1,997.1 ± 1,159.2\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e0.94 ± 0.53\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e0.5222 ± 0.1338\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1.47\u003c/p\u003e \u003cp\u003e± 0.75\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e3.29\u003c/p\u003e \u003cp\u003e± 2.33\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eR: AGRYLIN® (Anagrelide HCl) Capsule\u003c/p\u003e \u003cp\u003ebatch number: AF9326E\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c2\"\u003e \u003cp\u003e4,515.0 ± 2,392.3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c3\"\u003e \u003cp\u003e4,638.0 ± 2,447.6\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c4\"\u003e \u003cp\u003e2.061.3± 1,054.0\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c5\"\u003e \u003cp\u003e0.94±\u003c/p\u003e \u003cp\u003e0.59\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"±\" colname=\"c6\"\u003e \u003cp\u003e0.5528±\u003c/p\u003e \u003cp\u003e0.1205\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e32\u003c/p\u003e \u003cp\u003e± 30\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e2.94\u003c/p\u003e \u003cp\u003e± 1.32\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003cp\u003eThe mean plasma concentrations of anagrelide are illustrated in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb, presented on both linear (± SEM) and log-linear scales.\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e presents the 90% confidence intervals along with the corresponding point estimators, calculated as the ratios of the test to reference geometric means.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical Results of Anagrelide (test vs reference); n = 42\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePharmacokinetic parameters:\u003c/p\u003e \u003cp\u003e(ANOVA; two one-sided t-tests)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e90% confidence intervals\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003epoint estimators\u003c/p\u003e \u003cp\u003e(geometric means; ratios test/reference)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIntra-subject variability\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e(0−t)\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"−\" colname=\"c2\"\u003e \u003cp\u003e94.09% − 104.75%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.28%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.68%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"−\" colname=\"c2\"\u003e \u003cp\u003e85.62% − 104.03%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e94.37%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e26.98%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e(0−∞)\u003c/sub\u003e\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\"−\" colname=\"c2\"\u003e \u003cp\u003e94.50% − 105.10%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e99.66%\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.55%\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e\u003ch3\u003e3.2 Safety Results\u003c/h3\u003e\u003cp\u003eAll adverse events (AE) reported by participants, whether spontaneously, during routine questioning before the start of each study period, or during scheduled assessments at specified time points (0-hour, 1 hour, 6 hours, and 10 hours) in both periods, were documented in the AE information sheet within each participant’s CRF. Throughout the study, 13 adverse events were recorded in 11 of the 42 enrolled participants.\u003c/p\u003e\u003cp\u003eAmong these, eight treatment-emergent adverse events were classified as moderate-intensity headaches. Three headaches occurred in Period I, two following administration of the reference product and one after the test product. Five headaches were reported in Period II, three after receiving the reference product and two after the test product. Of these, five headaches were managed with paracetamol (500 mg).\u003c/p\u003e\u003cp\u003eAdditionally, five adverse events were classified as mild in intensity, including two headaches, one case of nausea, one instance of vomiting, and one case of hypotension. One headache was observed in Period II after administration of the reference product, while hypotension occurred in Period I following the test product. The remaining three mild events (one headache, nausea, and vomiting) were reported in Period I after administration of the reference product.\u003c/p\u003e\u003cp\u003eAll treatment-emergent adverse events were assessed as “possible” attributable to study drug, except hypotension, which was determined as unlikely drug related, and all cases resolved. There were no severe adverse events. A summary of observed adverse events is provided in Table\u0026nbsp;\u003cspan refid=\"Tab5\" class=\"InternalRef\"\u003e5\u003c/span\u003e.\u003c/p\u003e\u003cdiv class=\"gridtable\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003ctable float=\"Yes\" id=\"Tab5\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 5\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eA summary of the observed treatment-emergent adverse events\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003c/colgroup\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDrug Relationship\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdverse Event (no. AE) (WHO term)\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTreatment T\u003c/p\u003e \u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTreatment R\u003c/p\u003e \u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epossible\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003evomiting (1)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epossible\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003enausea (1)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003epossible\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eheadache (10)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eunlikely\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ehypotension (1)\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/table\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAnagrelide's rate (C\u003csub\u003emax\u003c/sub\u003e) and extent (AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e) of absorption from the reference formulation and test formulation are comparable, according to the study's findings. The bioequivalence range of 80.00\u0026ndash;125.00% encompasses the 90% confidence intervals for the T/R ratios of the natural In-transformed AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e values. Likewise, the secondary pharmacokinetic measure AUC\u003csub\u003e(0\u0026minus;\u0026infin;)\u003c/sub\u003e's 90% confidence interval satisfies the accepted bioequivalence standards [8]. Both formulations demonstrated acceptable safety profiles, with no clinically significant differences in tolerability.\u003c/p\u003e \u003cp\u003eBioequivalence between test and reference was confirmed in 42 healthy male participants following a single oral dose of 0.5 mg anagrelide (as anagrelide HCl) under fasting conditions.\u003c/p\u003e \u003cp\u003eThe rate and extent of a drug's absorption and the extent to which it becomes available at the site of action are referred to as bioavailability. According to guidelines, two medicinal products are considered bioequivalent if their bioavailability is sufficiently similar after administering an equivalent molar dose, ensuring comparable efficacy and safety [8]. This condition is met when the 90% confidence intervals for AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e ratios fall within the pre-defined range of 80.00\u0026ndash;125.00%.\u003c/p\u003e \u003cp\u003eCommon adverse effects associated with anagrelide use include rapid heartbeat, chest pain, breathing difficulties, numbness, tingling, burning sensations, headache, dizziness, weakness, gastrointestinal disturbances (such as stomach pain, gas, indigestion, nausea, vomiting, diarrhea, and loss of appetite), swelling in the hands or feet, fever, cough, general malaise, back pain, rash, and itching [12]. While the likelihood of these adverse effects is lower with single-dose administration, using the lowest effective dose in healthy volunteers remains essential to minimize potential risks [13]. Previous study on healthy participants have utilized higher doses (2 mg) than the 0.5 mg dose used in this study [14]. In contrast, our study employed a lower dose (0.5 mg), reducing the risk of adverse effects in healthy volunteers. The importance of this situation has also been shown in our previous study [15]. While the drug in the extended-release formulation was used in the previous research, the immediate-release formulation was applied in our study [15]. The study was designed under fasting conditions. The earlier study observed that the pharmacokinetic parameters C\u003csub\u003emax\u003c/sub\u003e, AUC, and t\u003csub\u003e1/2\u003c/sub\u003e were affected when the drug was taken with food [15].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrated the bioequivalence of the test and reference drugs containing low doses of anagrelide. Likewise, the safety of the test drug is equivalent to the reference drug. The low-dose design in healthy volunteers will guide future studies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAUC \u0026nbsp; \u0026nbsp; Area under the concentration time curve of plasma\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e\u0026nbsp; \u0026nbsp;Maximum plasma concentration\u003c/p\u003e\n\u003cp\u003et\u003csub\u003emax\u003c/sub\u003e\u0026nbsp; \u0026nbsp; time to achieve C\u003csub\u003emax\u003c/sub\u003e\u003c/p\u003e\n\u003cp\u003et\u003csub\u003e1/2 \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/sub\u003ehalf life\u003c/p\u003e\n\u003cp\u003eCI \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Confidence intervals;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCRF \u0026nbsp; \u0026nbsp; Case report form\u003c/p\u003e\n\u003cp\u003eEMA \u0026nbsp; \u0026nbsp;European Medicines Agency\u003c/p\u003e\n\u003cp\u003eFDA \u0026nbsp; \u0026nbsp; U.S. Food and Drug Administration\u003c/p\u003e\n\u003cp\u003eHPLC \u0026nbsp; High-performance Liquid Chromatography\u003c/p\u003e\n\u003cp\u003eLLOQ \u0026nbsp;Lower limit of quantification\u003c/p\u003e\n\u003cp\u003eQC \u0026nbsp; \u0026nbsp; \u0026nbsp; Quality control \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eHQC \u0026nbsp; \u0026nbsp;High-quality control\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMQC \u0026nbsp; \u0026nbsp;Medium-quality control\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLMQC \u0026nbsp;Low-medium-quality control\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eLQC \u0026nbsp;Low-quality control\u003c/p\u003e\n\u003cp\u003eLC-MS/MS \u0026nbsp; Liquid Chromatography Mass Spectrometer\u003c/p\u003e\n\u003cp\u003eTITCK \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Turkiye Ilac ve Tıbbi Cihaz Kurumu\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Proofreading \u0026amp; Editing Office of the Dean for Research at Erciyes University for copyediting and proofreading service for this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors contributed to the concept and design of the study. OP and BB took decisions in the name of the sponsor. SK prepared study documentation and carried out the necessary application and submissions for the study. AI carried out the clinical part as the principal investigator, ZS and MM as co-investigator. MR, performed Bioanalysis in plasma. WM performed the Pharmacokinetic analysis, statistics and reporting parts. AI and SK contributed to writing the manuscript. All authors approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by ILKO Pharmaceuticals, Turkiye. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are not publicly available due to compromise the privacy of study participants and due to the fact that trade secret. The data that are publicly available are shared in the article. \u0026nbsp;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was approved by an independent ethics committee-Ethics Committee for Bioavailability-Bioequivalence Trials of Erciyes University (date of approval: 06.02.2019; vote no: 2019/21) and the Turkish Medicines and Medical Devices Agency (TİTCK) of Turkish Ministry of Health, (date of approval: 18.03.2019, note no.: 66175679-514.06.01-E.45591).\u003c/p\u003e\n\u003cp\u003eThe purpose of the investigation was fully disclosed to each subject in a written document and oral presentation by the principal investigator or one of the co-investigators. Then, each participant voluntarily signed the informed consent form.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAI is the principal investigator at Hakan Cetinsaya GCP Center, and ZS and MM are co-investigators at Hakan \u0026Ccedil;etinsaya GCP Center. OP and BB are the sponsor representatives from ILKO Pharmaceuticals. MR is staff in Analytisches Zentrum Biopharm GmbH. WM is a staff in Pharmakin Consulting Services UG. SAK is staff in IDEAL Biyolojik Urunler ve Ilac Danismanlik Egitim Ltd. Sti.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003ePDE3A inhibitor anagrelide activates death signaling pathway genes and synergizes with cell death-inducing cytokines to selectively inhibit cancer cell growth. An R, Liu J, He J, Wang F, Zhang Q, Yu Q. Am J Cancer Res. 2019 Sep 1;9(9):1905-1921. eCollection 2019.\u003c/li\u003e\n\u003cli\u003ehttps://www.ncbi.nlm.nih.gov/books/NBK548467/pdf/Bookshelf_NBK548467.pdf,2025 reached at 12.03.2025\u003c/li\u003e\n\u003cli\u003eCardiovascular safety of anagrelide in healthy subjects: effects of caffeine and food intake on pharmacokinetics and adverse reactions. Mart\u0026iacute;nez-Sell\u0026eacute;s M, Datino T, Figueiras-Graillet L, Gama JG, Jones C, Franklin R, Fern\u0026aacute;ndez-Avil\u0026eacute;s F. Clin Drug Investig. 2013 Jan;33(1):45-54. doi: 10.1007/s40261-012-0032-2.\u003c/li\u003e\n\u003cli\u003eWagstaff AJ, Keating GM. Anagrelide: a review of its use in the management of essential thrombocythaemia Drugs 2006; 66(1): 111 \u0026ndash; 131\u003c/li\u003e\n\u003cli\u003eWMA Declaration of Helsinki \u0026ndash; Ethical Principles for Medical Research Involving Human Participants https://www.wma.net/wp-content/uploads/2024/10/DoH-Oct2013.pdf reached at 11.03.2025\u003c/li\u003e\n\u003cli\u003ePharmaceutical and medical preparations law; No : 1262. Publication Date in the Official Gazette: 26th May, 1928 Issue No. 898. https://titck.gov.tr/storage/legislation/meFOxeFD.pdf)\u003c/li\u003e\n\u003cli\u003eGCP regulations. Turkish Pharmaceuticals and Medical Devices Agency, 13 Nov 2015 https://titck.gov.tr/storage/Archive/2020/legislation/KADKLVZ01IKU13.11.2015Rev08_13ac0133-274b-44dc-98cd-33998758cc72.pdf\u003c/li\u003e\n\u003cli\u003eGuideline on the Investigation of Bioequivalence Committee for Medicinal Products for Human Use (CHMP) CPMP/EWP/QWP/1401/98 Rev. 1/Corr**, London, 20 January 2010 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-investigation-bioequivalence-rev1_en.pdf reached at 25.07.2024\u003c/li\u003e\n\u003cli\u003eGuideline on bioanalytical method validation EMEA/CHMP/EWP/192217/2009- 21 July 2011 https://www.ema.europa.eu/en/documents/scientific-guideline/guideline-bioanalytical-method validation_en.pdf reached at 25.07.2024\u003c/li\u003e\n\u003cli\u003eGuidance for Industry Bioanalytical Method Validation U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research (CDER), Center for Veterinary Medicine (CVM), May 2018. https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf reached at 28.07.2024\u003c/li\u003e\n\u003cli\u003eDiletti E, Hauschke D, Steinijans VW Sample size determination for bioequivalence assessment by means of confidence intervals Int J Clin Pharmacol Ther Toxicol 30 (Suppl. 1), 51 - 58 (1992)\u003c/li\u003e\n\u003cli\u003eRxList Internet Drug Index \u0026ndash; Anagrelide https://www.rxlist.com/anagrelide/generic-drug.htm, at reached 11.03.2025\u003c/li\u003e\n\u003cli\u003eBioequivalence study of low dose drospirenone/ethinyl estradiol 3 mg/0.03 mg film tablets under fasting conditions in Turkish healthy female subjects. Inal A, Sezer Z, Ulu\u0026ouml;zl\u0026uuml; B, Oflas M, Reinsch M, Martin W, Mazicioglu MM, Koru SA. Pharmacol Res Perspect.2024 Aug;12(4):e1253\u003c/li\u003e\n\u003cli\u003ePharmacokinetics, bioequivalence, tolerability, and effects on platelet counts of two formulations of anagrelide in healthy volunteers and patients with thrombocythemia associated with chronic myeloproliferation. Petrides PE, Gisslinger H, Steurer M, Linkesch W, Krumpl G, Sch\u0026uuml;ller A, Widmann R. Clin Ther. 2009 Feb;31(2):386-98. doi: 10.1016/j.clinthera.2009.02.008.\u003c/li\u003e\n\u003cli\u003ePharmacokinetics of a Novel Anagrelide Extended-Release Formulation in Healthy Subjects: Food Intake and Comparison With a Reference Product. Petrides PE, Schoergenhofer C, Widmann R, Jilma B, Klade CS Clin Pharmacol Drug Dev. 2018 Feb;7(2):123-131. doi: 10.1002/cpdd.340. Epub 2017 Mar 16.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Bioequivalence study, anagrelide, healthy volunteers, pharmacokinetic, bioavailability","lastPublishedDoi":"10.21203/rs.3.rs-6453732/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6453732/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe pharmacokinetic profiles and relative bioavailability of anagrelide from the test and reference preparations were compared to establish the test preparation's bioequivalence to the reference under fasting conditions. In a randomized sequence, male Caucasian subjects were administered a single dose of either one capsule of the test or reference preparation separated by a washout phase of seven days. Blood samples were collected at pre-specified time points following drug administration to assess anagrelide pharmacokinetics. These samples were collected in K\u003csub\u003e2\u003c/sub\u003eEDTA tubes, centrifuged, and stored at \u0026lt;-70\u0026deg;C. Anagrelide concentrations were quantified using a validated LC-MS/MS method with ESI(+). The pharmacokinetic parameters, including AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e, AUC\u003csub\u003e(0\u0026minus;\u0026infin;)\u003c/sub\u003e, C\u003csub\u003emax\u003c/sub\u003e, t\u003csub\u003emax\u003c/sub\u003e, λ\u003csub\u003ez\u003c/sub\u003e, t\u003csub\u003e\u0026frac12;\u003c/sub\u003e(λ\u003csub\u003ez\u003c/sub\u003e), and AUC\u003csub\u003e%\u0026minus;extrapol\u003c/sub\u003e, were calculated using Phoenix WinNonlin V8.1.0.3530 software. The absorption extent (AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e) and the absorption rate (C\u003csub\u003emax\u003c/sub\u003e) of anagrelide from the test and reference preparations were found to be comparable. The 90% confidence intervals for the ln-transformed ratios of AUC\u003csub\u003e(0\u0026minus;t)\u003c/sub\u003e and C\u003csub\u003emax\u003c/sub\u003e of the test/reference preparations met the bioequivalence criteria of 80.00\u0026ndash;125.00%. Furthermore, the 90% confidence interval for the ln-transformed data of the secondary pharmacokinetic parameter, AUC\u003csub\u003e(0\u0026minus;\u0026infin;)\u003c/sub\u003e, of anagrelide was also included in the bioequivalence acceptance range.\u003c/p\u003e","manuscriptTitle":"Bioequivalence Study of Low-Dose Anagrelide 0.5 mg Capsules under Fasting Conditions in Healthy Turkish Male Subjects","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-16 07:12:21","doi":"10.21203/rs.3.rs-6453732/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"b16a8363-e798-489e-ba06-2add59a02b1c","owner":[],"postedDate":"April 16th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-07-06T21:08:18+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-16 07:12:21","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6453732","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6453732","identity":"rs-6453732","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}