Lenalidomide (IUPAC 3-(7-amino-3-oxo-1H-isoindol-2-yl)piperidine-2,6-dione), a thalidomide analog, is an immunomodulatory (IMiD) and antineoplastic agent used in multiple myeloma therapy¹. Multiple myeloma is a malignant B-cell neoplasia characterized by the excess of monotypic plasma cells in the bone marrow. Established for the 1^st time as agents with antiangiogenic properties, thalidomide, and the other IMiDs inhibit the production of interleukin, which is a growth factor for the proliferation of myeloma cells. In addition, they activate apoptotic pathways through cellular death by means of caspases 8².

• ¹
  PubChem Compound Summary for CID 216326, Lenalidomide,
  1. PubChem Compound Summary for CID 216326, Lenalidomide. Available from:https://pubchem.ncbi.nlm.nih.gov/compound/lenalidomide [Last accessed on 2022 Oct 27].
• ²
  Lenalidomide and thalidomide:mechanisms of action--similarities and differences

  Semin Hematol, 2005
  2. Anderson KC. Lenalidomide and thalidomide:mechanisms of action--similarities and differences. Semin Hematol. 2005;42:S3-8.

In 2005, the Food and Drug Administration (FDA) approved the marketing of 5 mg and 10 mg Revlimid^® (Lenalidomide) capsules for the treatment of transfusion-depending anemia patients due to low-risk myelodysplastic syndrome (MDS) associated to 5q deletion abnormality with or without additional cytogenetic abnormalities and multiple myeloma in combination with dexamethasone³,⁴. The European Medicines Agency issued the authorization to market lenalidomide in 2007⁵. In Brazil, ANVISA issued a public opinion for the approval of Revlimid in 2018 for the treatment of patients with relapsed/refractory multiple myeloma who had received at least one prior treatment; and for the treatment of patients with transfusion-depending anemia derived from low to intermediate-1 risk MDS associated to 5q deletion cytogenetic abnormality, with or without additional cytogenetic abnormalities⁶.

• ³
  Lenalidomide as a novel treatment of acute myeloid leukemia

  Expert Opin Investig Drugs, 2013
  3. Chen Y, Borthakur G. Lenalidomide as a novel treatment of acute myeloid leukemia. Expert Opin Investig Drugs. 2013;22:389-97.
• ⁴
  Drug Approval Package

  Revlimid (Lenalidomide) Capsules. Celgene Corporation, 2005
  4. FDA-Food and Drug Administration. Drug Approval Package. Revlimid (Lenalidomide) Capsules. Celgene Corporation;2005. Available from:https://www.accessdata.fda.gov/drugsatfda_docs/nda/2005/021880s000_RevlimidTOC.cfm [Last accessed on 2005 Dec 27].
• ⁵
  Revlimid Lenalidomide, 2007
  5. EMA. Revlimid Lenalidomide;2007. Available from:https://www.ema.europa.eu/en/medicines/human/EPAR/revlimid#authorisation-details-section
• ⁶
  Agência Nacional de Vigilância Sanitária

  REVLIMID, 2017
  6. ANVISA. Agência Nacional de Vigilância Sanitária. REVLIMID. Brazil:ANVISA;2017. Available from:https://consultas.anvisa.gov.br/#/medicamentos/25351757160201560/?substancia=25181%20

In 2017, ANVISA issued resolution RDC 191 (regarding the control of lenalidomide and any drugs containing it, among other provisions), later revoked in 2022 by a resolution with the same name (RDC 735/2022). Due to the risks associated to the teratogenic effects of lenalidomide-based drugs, the resolution stated, among other provisions, the design of a Risk Management Plan, including a pregnancy prevention program, which requires training of the physicians prescribing the drug by the register holder⁷.

• ⁷
  Agência Nacional de Vigilância Sanitária:resolução DA DIRETORIA COLEGIADA - RDC No 735, DE 13 DE JULHO DE 2022

  Dispõe Sobre o Controle da Substância Lenalidomida e de Medicamento Que a Contenha, e dáOutras Providências, 2022
  7. ANVISA. Agência Nacional de Vigilância Sanitária:resolução DA DIRETORIA COLEGIADA - RDC No 735, DE 13 DE JULHO DE 2022. Dispõe Sobre o Controle da Substância Lenalidomida e de Medicamento Que a Contenha, e dáOutras Providências. Brazil:ANVISA;2022.

In 2020, ANVISA issued RDC 393/2020, which included lenalidomide therapeutic indications for the treatment of multiple myeloma, in combination with bortezomib and dexamethasone, for patients with no prior treatment; for the treatment of follicular lymphoma or previously treated patients with marginal zone lymphoma, in combination with rituximab (anti-CD20 antibody), and relapsed/refractory mantle cell lymphoma⁸.

• ⁸
  Agência Nacional de Vigilância Sanitária

  Lenalidomida:autorizadas Novas Indicações Terapêuticas, 2020
  8. ANVISA. Agência Nacional de Vigilância Sanitária. Lenalidomida:autorizadas Novas Indicações Terapêuticas. Brazil:ANVISA;2020. Available from:http://antigo.anvisa.gov.br/resultado-de-busca?p_p_id=101&p_p_lifecycle=0&p_p_state=maximized&p_p_mode=view&p_p_col_id=column-1&p_p_col_count=1&_101_struts_action=%2fasset_publisher%2fview_content&_101_assetentryid=5893220&_101_type=content&_101_groupid=219201&_101_urltitle=lenalidomida-autorizadas-novas-indicacoes-terapeutic-1&inheritredirect=true

As to the security profile, the authors report that lenalidomide is well tolerated and that most usual adverse events include hematologic toxicity with controllable neutropenia and thrombocytopenia⁹,¹⁰. According to the panel released by ANVISA through VigiMed, in the period between January 01, 2018, and September 09, 2022, 652 lenalidomide suspected adverse events were informed, out of which 16.10% corresponded to neutropenia and 4.45% to thrombocytopenia¹¹.

• ⁹
  Lenalidomide in the treatment of multiple myeloma:a review

  J Clin Pharm Ther, 2008
  9. Armoiry X, Aulagner G, Facon T. Lenalidomide in the treatment of multiple myeloma:a review. J Clin Pharm Ther. 2008;33:219-26.
• ¹⁰
  A phase I trial of lenalidomide in patients with recurrent primary central nervous system tumors

  Clin Cancer Res, 2007
  10. Fine HA, Kim L, Albert PS, Duic JP, Ma H, Zhang W, et al. A phase I trial of lenalidomide in patients with recurrent primary central nervous system tumors. Clin Cancer Res. 2007;13:7101-6.
• ¹¹
  Agência Nacional de Vigilância Sanitária:vigiMed - Eventos Adversos, 2022
  11. ANVISA. Agência Nacional de Vigilância Sanitária:vigiMed - Eventos Adversos. Brazil:ANVISA;2022. Available from:https://www.gov.br/anvisa/pt-br/acessoainformacao/dadosabertos/informacoes-analiticas/notificacoes-de-farmacovigilancia

The objective of this trial was to verify whether the rate and extent of absorption of the 25 mg lenalidomide immediate release capsule formulation manufactured by Eurofarma Laboratórios S.A. are equivalent to those of the reference product, REVLIMID^® when administered in one single dose and under fasting conditions in adult healthy male subjects.

Bioequivalence studies are a type of study that follows specific rules dictated by the regulatory agencies in each country. Thus, study protocol drafting requirements follow the SPIRIT Statement¹²-¹⁴, where applicable. Data reporting is also carried out in the form of standardized reports by the agencies, but they contain all the information recommended for reporting according to the CONSORT Statement¹⁴,¹⁵. In Brazil, the following standards must be followed: (1) RE 894/23¹⁶, which defines the standardization for the preparation of bioequivalence study protocols, and (2) RE 895/23¹⁷, which defines the form of study reporting.

• ¹²
  SPIRIT 2013 statement:defining standard protocol items for clinical trials

  Ann Intern Med, 2013
  12. Chan AW, Tetzlaff JM, Altman DG, Laupacis A, Gøtzsche PC, Krleža-JerićK, et al. SPIRIT 2013 statement:defining standard protocol items for clinical trials. Ann Intern Med. 2013;158:200-7.
• ¹⁴
  An update to SPIRIT and CONSORT reporting guidelines to enhance transparency in randomized trials

  Nat Med, 2022
  14. Hopewell S, Boutron I, Chan AW, Collins GS, de Beyer JA, Hróbjartsson A, et al. An update to SPIRIT and CONSORT reporting guidelines to enhance transparency in randomized trials. Nat Med. 2022;28:1740-3.
• ¹⁴
  An update to SPIRIT and CONSORT reporting guidelines to enhance transparency in randomized trials

  Nat Med, 2022
  14. Hopewell S, Boutron I, Chan AW, Collins GS, de Beyer JA, Hróbjartsson A, et al. An update to SPIRIT and CONSORT reporting guidelines to enhance transparency in randomized trials. Nat Med. 2022;28:1740-3.
• ¹⁵
  CONSORT 2010 statement:updated guidelines for reporting parallel group randomised trials

  BMJ, 2010
  15. Schulz KF, Altman DG, Moher D. CONSORT 2010 statement:updated guidelines for reporting parallel group randomised trials. BMJ. 2010;340:c332.
• ¹⁶
  Nacional de Vigilância Sanitária:resolução-RE No 894, DE 29 DE MAIO DE 2003:guia Para Elaboração de Protocolo de Estudo de Biodisponibilidade Relativa/Bioequivalênci, 2003
  16. ANVISA. Nacional de Vigilância Sanitária:resolução-RE No 894, DE 29 DE MAIO DE 2003:guia Para Elaboração de Protocolo de Estudo de Biodisponibilidade Relativa/Bioequivalênci. Brazil:ANVISA;2003.
• ¹⁷
  Agência Nacional de Vigilância Sanitária:resolução-RE No 895, de 29 de Maio De 2003:ANEXO - Guia Para Elaboração de Relatório Técnico de Estudo de Biodisponibilidade Relativa/Bioequivalência, 2003
  17. ANVISA. Agência Nacional de Vigilância Sanitária:resolução-RE No 895, de 29 de Maio De 2003:ANEXO - Guia Para Elaboração de Relatório Técnico de Estudo de Biodisponibilidade Relativa/Bioequivalência. Brazil:ANVISA;2003.

The test drug −25 mg lenalidomide immediate release capsule−, was manufactured by Eurofarma Laboratórios S/A. The reference product used in the study was Revlimid^® (25 mg lenalidomide immediate release capsule), manufactured by Celgene Europe B.V. and registered in Brazil by Bristol-Myers Squibb Farmacêutica LTDA.

Adult male healthy volunteers willing to participate in the study were selected based on the protocol eligibility criteria 90 days before the first study period. A sufficient number of eligible volunteers appeared in the research center facilities, and the 32 volunteers who fulfilled the protocol requirements were given information regarding the study, after having their inquiries clarified and having decided to willingly take part in the study, each subject signed the informed consent form, previously approved by the Avant Santé Research Center Research Committee (COFEPRIS 18 CI 19 019 021) along with the study protocol. Initially, 32 study subjects were selected and randomized, out of which only 28 completed all the study procedures due to exclusion and/or dropout reasons.

Single dose, randomized, open-label, two-treatment, two-sequence, two-period crossover bioequivalence study of 25 mg lenalidomide capsules manufactured by Eurofarma Laboratórios S.A. versus REVLIMID^® (25 mg lenalidomide immediate release capsule) manufactured by Celgene Europe B.V. and registered in Brazil by Bristol-Myers Squibb Farmacêutica LTDA in adult male healthy volunteers under fasting conditions.

Study subjects were kept in fasting conditions for 10 h before the dose administration and at least 4 h afterward, on each period. The study was conducted under fasting conditions as required by ANVISA - Agência Nacional de Vigilância Sanitária¹⁸.

• ¹⁸
  Agência Nacional de Vigilância Sanitária:lista 1 - Forma de Administração (Formas Farmacêuticas de Liberação Imediata). RESOLUÇÃO - RE no 1170, de 19 de Abril de 2006, 2022
  18. ANVISA. Agência Nacional de Vigilância Sanitária:lista 1 - Forma de Administração (Formas Farmacêuticas de Liberação Imediata). RESOLUÇÃO - RE no 1170, de 19 de Abril de 2006. Brazil:ANVISA;2022.

On each period of the study, a single dose (25 mg) of the test or the reference product was administered orally to the volunteers, in a seated position, with 200 mL of water at room temperature, under fasting conditions. The washout period was 7 days.

A total number of 19 blood samples were collected from each volunteer in each period in tubes with K₂EDTA. Blood samples (4 mL each) were collected at time points 0.00 h (before dose) and after administration at time points 0.25, 0.33, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.00, 2.50, 3.00, 4.00, 6.00, 8.00, 10.00, 12.00, 16.00, and 24.00 h.

After collection, blood samples were processed in a refrigerated centrifuge at 3000 rpm for 10 min at 4 ± 2°C. The plasma obtained from the blood samples was transferred to two different cryogenic tubes (aliquot 1 and aliquot 2) previously identified and stored at a temperature below −50°C.

The validation of the bioanalytical method for quantification of lenalidomide in human plasma using Lenalidomide-d5 as internal standard and K₂EDTA as anticoagulant through extraction in solid phase (Strata-X^® 33 μm cartridge) and ultra-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed in compliance with the acceptance criteria for selectivity, calibration curve, precision, accuracy, residual effect, matrix effect, and stability test in solution and the biological matrix (Table 1).

Chromatographic conditions adopted for the validation and quantification of the study subject samples included the use of a Luna omega 1.6 μm PS C18 50 × 2.1 mm chromatographic column, at 35 ± 2°C. Samples were kept at 5 ± 4°C. Mobile phase A used was Formic Acid 0.1% and mobile phase B was LC-MS grade Acetonitrile in an 87:13 v/v proportion. The injection volume was 3.0 μL and retention times were 1.07 ± 0.3 min for the analyte and 1.05 ± 0.3 for the internal standard, the running time being 2.10 min.

The method proved linear between concentrations of 2.044 ng/mL to 1015.506 ng/mL according to equation y = a + bx [1/x], where "y" is the response, "x" is the analyte concentration and "1/x" is the selected weight.

The lower limit of quantification (LLQ) established for the method was 2.044 ng/mL and validated quality control samples were 5.148 ng/mL, 411.867 ng/mL, and 792.052 ng/mL.

Stability analysis was carried out in plasma in concentrations of 5.148 ng/mL and 792.052 ng/mL and they complied with the acceptance criteria when the samples were subjected to 6 h and 18 min at room temperature (approximately 25°C) (short-term stability), for 2 days 8 h and 38 min in auto-injector (5 ± 4°C) after sample extraction completion (post-processing stability) 4 freeze-and-thaw cycles and 106 long-term days.

Reference standards: lenalidomide (TRC/Canada) was used as an analyte and Lenalidomide-d5 (TRC/Canada) was used as an internal standard for the preparation of the primary standard solutions in methanol HPLC grade. Working solutions were prepared using milli-Q water: methanol (20:80; v/v) as eluent. All solutions were stored at a temperature between 2 and 8°C.

Compounds were extracted from human plasma samples and quantified through LC-MS/MS using the Xevo TQ-S/Acquity UPLC I-Class (Waters) spectrometer, equipped with positive electrospray (ESP+) ionization source, and the analyte and internal standard were detected using multiple reaction monitoring with m/z transitions 260.21 > 149.11 and 265.23 > 151.13, respectively.

Software Masslynx version 4.1 was used for calculating sample concentrations in the analytic phase.

Software Phoenix WinNonlin^™ version 8.3 and Statistical Analysis System (SAS^®) version 9.4 were used to perform the statistical analysis.

The study began with 32 volunteers and ended with 28 healthy male volunteers between 18 and 42 years of age, who complied with the inclusion and exclusion criteria set forth in the protocol.

Pharmacokinetic parameters C_max and AUC_0-t were established using software Phoenix WinNonlin^™ version 8.3 and Sistema de Análise Estatística (SAS^®) version 9.4.

Pharmacokinetic parameters are shown in table 2.

The maximum concentration of C_max obtained for the reference product Revlimid^® was 439.761 ng/mL in 1.5 h. For the test product, lenalidomide, a C_max of 477.742 ng/mL occurred at 1.1 h. Figure 1 shows intermediate concentrations of lenalidomide for the 28 study subjects along collection times.

Eight adverse events were reported during the study: 3 headaches, 1 erythema, 1 rash, 1 hypertriglyceridemia, 1 rectal hemorrhage, and 1 normochromic normocytic anemia. No serious adverse events were reported; 6 events were classified as mild and 2 as moderate.

As to the relation with the study drug, 1 adverse event was classified as possibly related, 2 as probably related, and 5 as unlikely to be related to the study medication.

The study was properly planned and conducted, obtaining pharmacokinetic parameters C_max and AUC0-t which confidence interval values (90%) are within the acceptable limit for the ratio between the geometric means of the test and reference products (80−125%), according to the national legislation, ANVISA resolution RE n^o 1170/2006¹⁹.

• ¹⁹
  Agência Nacional de Vigilância Sanitária:resolução - RE 1.170, de 19 De Abril De 2006

  Guia Para Provas De Biodisponibilidade Relativa/Bioequivalência De Medicamentos, 2006
  19. ANVISA. Agência Nacional de Vigilância Sanitária:resolução - RE 1.170, de 19 De Abril De 2006. Guia Para Provas De Biodisponibilidade Relativa/Bioequivalência De Medicamentos. Brazil:ANVISA;2006.

The planned number of 32 healthy male subjects is consistent with studies published by other authors²⁰,²¹. The population selected for the study was male gender only, as recommended by the FDA guidelines²² and consistent with resolution ANVISA RDC 735/2022⁷.

• ²⁰
  Single-dose comparative pharmacokinetics of two formulations of lenalidomide 25 mg in healthy subjects:a randomized crossover study

  Adv Ther, 2018
  20. Lee S, Hwang JG, Park SY, Lim HJ, Lee SW, Seo MH, et al. Single-dose comparative pharmacokinetics of two formulations of lenalidomide 25 mg in healthy subjects:a randomized crossover study. Adv Ther. 2018;35:210-7.
• ²¹
  Bioequivalence study of single-dose lenalidomide capsule vs. Revlimid® capsule in healthy Chinese males

  Cancer Chemother Pharmacol, 2018
  21. Wang J, Qi L, Wang Z, Chen G, Liu C, Liu Y, et al. Bioequivalence study of single-dose lenalidomide capsule vs. Revlimid® capsule in healthy Chinese males. Cancer Chemother Pharmacol. 2018;82:159-64.
• ²²
  Draft Guidance on Lenalidomide, 2013
  22. FDA-Food and Drug Administration. Draft Guidance on Lenalidomide;2013 Available from:https://www.accessdata.fda.gov/drugsatfda_docs/psg/lenalidomide_draft_oral%20cap_rld%2021880_rc11-13.pdf
• ⁷
  Agência Nacional de Vigilância Sanitária:resolução DA DIRETORIA COLEGIADA - RDC No 735, DE 13 DE JULHO DE 2022

  Dispõe Sobre o Controle da Substância Lenalidomida e de Medicamento Que a Contenha, e dáOutras Providências, 2022
  7. ANVISA. Agência Nacional de Vigilância Sanitária:resolução DA DIRETORIA COLEGIADA - RDC No 735, DE 13 DE JULHO DE 2022. Dispõe Sobre o Controle da Substância Lenalidomida e de Medicamento Que a Contenha, e dáOutras Providências. Brazil:ANVISA;2022.

Both formulations were well tolerated during the study and no serious adverse events were reported. Headache was the most frequent adverse event, contrary to the reports of other authors and the ANVISA panel, who stated neutropenia and thrombocytopenia as the most frequent adverse events⁹,¹¹,²³.

• ⁹
  Lenalidomide in the treatment of multiple myeloma:a review

  J Clin Pharm Ther, 2008
  9. Armoiry X, Aulagner G, Facon T. Lenalidomide in the treatment of multiple myeloma:a review. J Clin Pharm Ther. 2008;33:219-26.
• ¹¹
  Agência Nacional de Vigilância Sanitária:vigiMed - Eventos Adversos, 2022
  11. ANVISA. Agência Nacional de Vigilância Sanitária:vigiMed - Eventos Adversos. Brazil:ANVISA;2022. Available from:https://www.gov.br/anvisa/pt-br/acessoainformacao/dadosabertos/informacoes-analiticas/notificacoes-de-farmacovigilancia
• ²³
  Lenalidomide plus dexamethasone versus thalidomide plus dexamethasone in newly diagnosed multiple myeloma:a comparative analysis of 411 patients

  Blood, 2010
  23. Gay F, Hayman SR, Lacy MQ, Buadi F, Gertz MA, Kumar S, et al. Lenalidomide plus dexamethasone versus thalidomide plus dexamethasone in newly diagnosed multiple myeloma:a comparative analysis of 411 patients. Blood. 2010;115:1343-50.

The 7-day washout period seemed adequate, as all base collection samples of the volunteers of the second period had a concentration below the LLQ.

As in other published works, the analytical technique selected for the study of lenalidomide quantification of in human plasma samples was LC-MS/MS²⁴-²⁶.

• ²⁴
  Development and validation of Lenalidomide in human plasma by LC-MS/MS

  Saudi J Biol Sci, 2019
  24. Ranganathan P, Gunasekaran V, Singhvi I, Ansari MJ. Development and validation of Lenalidomide in human plasma by LC-MS/MS. Saudi J Biol Sci. 2019;26:1843-7.
• ²⁶
  Development and validation of LC-MS/MS method for the quantitation of lenalidomide in human plasma using Box-Behnken experimental design

  Analyst, 2013
  26. Hasnain MS, Rao S, Singh MK, Vig N, Gupta A, Ansari A, et al. Development and validation of LC-MS/MS method for the quantitation of lenalidomide in human plasma using Box-Behnken experimental design. Analyst. 2013;138:1581-8.

Lenalidomide was quantified in its unaltered form, as required by the national legislation²⁷.

• ²⁷
  Agência Nacional de Vigilância Sanitária:lista 2 - Analito Para Estabelecimento da Biodisponibilidade Relativa/Bioequivalência

  Resolução - RE no 1170, de 19 de abril de 2006, 2022
  27. ANVISA. Agência Nacional de Vigilância Sanitária:lista 2 - Analito Para Estabelecimento da Biodisponibilidade Relativa/Bioequivalência. Resolução - RE no 1170, de 19 de abril de 2006. Brazil:ANVISA;2022. Available from:https://www.gov.br/anvisa/pt-br/setorregulado/regularizacao/medicamentos/equivalencia-terapeutica/bioequivalencia/listas [Last accessed on 2022 Mar 31].

Both reference and test drugs showed a maximum plasma concentration C_max of 439.761 ng/mL and 477.742 ng/mL, respectively, consistent with those found in the literature²⁰,²¹.

• ²⁰
  Single-dose comparative pharmacokinetics of two formulations of lenalidomide 25 mg in healthy subjects:a randomized crossover study

  Adv Ther, 2018
  20. Lee S, Hwang JG, Park SY, Lim HJ, Lee SW, Seo MH, et al. Single-dose comparative pharmacokinetics of two formulations of lenalidomide 25 mg in healthy subjects:a randomized crossover study. Adv Ther. 2018;35:210-7.
• ²¹
  Bioequivalence study of single-dose lenalidomide capsule vs. Revlimid® capsule in healthy Chinese males

  Cancer Chemother Pharmacol, 2018
  21. Wang J, Qi L, Wang Z, Chen G, Liu C, Liu Y, et al. Bioequivalence study of single-dose lenalidomide capsule vs. Revlimid® capsule in healthy Chinese males. Cancer Chemother Pharmacol. 2018;82:159-64.

The results show test and reference formulations are considered bioequivalent regarding the absorption rate and extent, as the criteria required by the Brazilian regulatory authority have been complied with (CI 90% between 80 and 125%). Consequently, the test formulation, i.e. lenalidomide manufactured by Eurofarma Laboratórios S/A and the reference product Revlimid^®, both in 25 mg capsules, are bioequivalent and therefore, interchangeable.

C. Sverdloff participated in the study design and supervision. V. Marcondes Rezende performed the data curation and wrote the manuscript and reviews. L. de Cassia Val and L. Nerath Bonanato participated in the Project Administration and supervision. M.E. Cedano Limón conducted the clinical investigation. S. Kakarla performed bioanalytical method validation and supervision of sample analysis. M.H. Badii performed the statistical analysis. M. Pendela performed general site supervision. N.C. Lemus Castro wrote the clinical protocol, supervised clinical investigation, and was the principal investigator. All authors contributed with data and revision and approved the final manuscript.