Skip to main content

Advertisement

SpringerLink
Log in

Co-Delivery of Teriflunomide and Methotrexate from Hydroxyapatite Nanoparticles for the Treatment of Rheumatoid Arthritis: In Vitro Characterization, Pharmacodynamic and Biochemical Investigations

  • Research Paper
  • Published:
Pharmaceutical Research Aims and scope Submit manuscript

Abstract

Purpose

The present investigation was aimed at developing Teriflunomide (TEF) and Methotrexate (MTX) loaded hydroxyapatite nanoparticles and increasing tolerability towards combination therapy against rheumatoid arthritis by reducing hepatotoxicity.

Methods

Drug-loaded HAp-NPs were synthesized by wet-chemical precipitation method and optimized by Box-Behnken experimental design. The developed NPs were subjected to in vitro and in vivo characterization. In-vivo pharmacodynamics and biochemical studies were performed on adjuvant- induced arthritis model treated with different formulations; MTX-TEF-SOL, TEF-HAp-NP, MTX-HAp-NP, TEF-MTX-HAp-NP, FOLITRAX-10 and AUBAGIO.

Results

The size of the optimized formulations, TEF-HAp-NP and MTX-HAp-NP, was found to be 224.3 ± 83.80 nm and 268.3 ± 73.86 nm with drug loading 53.11 ± 0.84% and 67.04 ± 1.12% respectively. In vitro release of TEF from TEF-HAp-NP (70.41 ± 1.22%) and MTX from MTX-HAp-NP (82.43 ± 1.31%) up to 24 h revealed sustained release pattern. Results of the arthritic assessment study showed a significant (P < 0.05) reduction in ankle diameter (61.30 ± 7.42) and arthritis score (2.35 ± 0.24) with a marked restoration of ankle joint micro-architecture in TEF-MTX-HAp-NP treated group. During Hepatotoxicity studies, liver histopathology revealed that the formulation MTX-TEF-HAp-NP was least hepatotoxic with less hepatocyte swelling and fibrous connective tissue proliferation while Folitrax-10 was found to be most hepatotoxic. Biochemical studies revealed that Folitrax-10 significantly (P < 0.05) increased the GOT (313.64 ± 16) and GPT level (334.46 ± 13) while insignificant (P > 0.05) change in GOT (263.68 ± 17) and GPT (229.38 ± 10) level was recorded with TEF-MTX-HAp-NP.

Conclusions

We report that the subcutaneous delivery of TEF-MTX-HAp-NP was most effective as it successfully reduced the dosage by half for maximizing therapeutic efficacy and minimizing side effects.

ᅟ

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

AIA:

Adjuvant Induced Arthritis

CFA:

Complete Freund’s adjuvant

GOT:

Glutamic Oxaloacetate Transaminase

GPT:

Glutamic Pyruvic Transaminase

GSH:

Reduced Glutathione

GST:

Glutathione-S-Transferase

HAp-NP:

Hydroxyapatite nanoparticles

MDA:

Malondialdehyde

MTX:

Methotrexate

MTX-HAp-NP:

MTX-loaded hydroxyapatite nanoparticles

PBS:

Phosphate buffer solution

PDI:

Polydispersity index

ROS:

Reactive oxygen species

SEM:

Scanning Electron Microscopy

TCA:

Trichloroacetic acid

TEF:

Teriflunomide

TEF-HAp-NP:

TEF-loaded hydroxyapatite nanoparticles

TEF-MTX-HAp-NP:

Combination of TEF and MTX loaded hydroxyapatite nanoparticles

TEF-MTX-SOL:

Solution of TEF and MTX

TEM:

Transmission Electron Microscopy

References

  1. Pandey S, Rai N, Rawat P, Ahmad FJ, Talegaonkar S. Nanofacilitated synergistic treatment for rheumatoid arthritis: a ‘three pronged’ approach. Med Hypotheses. 2016;92:44–7.

    Article  CAS  PubMed  Google Scholar 

  2. Gibofsky A. Overview of epidemiology, pathophysiology, and diagnosis of rheumatoid arthritis. Am J Manag Care. 2012;18:S295–302.

    PubMed  Google Scholar 

  3. Silman AJ, Pearson JE. Epidemiology and genetics of rheumatoid arthritis. Arthritis Res. 2002;4:265–72.

    Article  Google Scholar 

  4. Pandey S, Mahtab A, Rai N, Rawat P, Ahmad FJ, Talegaonkar S. Emerging role of CD44 receptor as a potential target in disease diagnosis: a patent review. Recent Patents Inflamm Allergy Drug Discov. 2017;11(2):77–91.

    Article  CAS  Google Scholar 

  5. Kalinoglou AS, Deli C, Kitas GD, Jamurtas AZ. Muscle wasting in rheumatoid arthritis: the role of oxidative stress. World J Rheumatol. 2014;4(3):44–53.

    Article  Google Scholar 

  6. Al-Youzbaki WB, Fatehi HIA, Yassen AT. Oxidant and antioxidant status in patients with rheumatoid arthritis treated by methotrexate. Iraqi J Comm Med. 2013;1:63–7.

    Google Scholar 

  7. Singh JA, Saag KG, Bridges SL Jr, Akl EA, Bannuru RR, Sullivan MC. American College of Rheumatology Guideline for the treatment of rheumatoid arthritis. Arthritis Rheumatol. 2016;68(1):1–26.

    Article  PubMed  Google Scholar 

  8. Nimitha VV, Parvathy S, Nair SC, Viswanad V. Leflunomide loaded solid lipid nanoparticle in rheumatoid arthritis. Int J Pharm Techno. 2017;9(2):29681–706.

    CAS  Google Scholar 

  9. Tian H, Cronstein BN. Understanding the mechanisms of action of methotrexate implications for the treatment of rheumatoid arthritis. Bull NYU Hosp Jt Dis. 2007;65(3):168–73.

    PubMed  Google Scholar 

  10. Zhang Y, Cun D, Kong X, Fang L. Design and evaluation of a novel transdermal patch containing diclofenac and teriflunomide for rheumatoid arthritis therapy Asian J. Pharm Sci. 2014;9:251–9.

    Google Scholar 

  11. Van RP. Leflunomide improves the clinical response in patients with active rheumatoid arthritis treated with methotrexate. Clin Exp Rheumatol. 2003;21(6):695–6.

    Google Scholar 

  12. Mottonen T, Hannonen P, Leirisalo-Repo M, Nissila M, Kautiainen H, Korpela M, et al. Comparison of combination therapy with single-drug therapy in early rheumatoid arthritis: a randomised trial. FIN-RACo trial group. Lancet. 1999;353:1568–73.

    Article  CAS  PubMed  Google Scholar 

  13. Weinblatt ME, Kremer JM, Coblyn JS, Maier AL, Helfgott SM, Morrell M, et al. Pharmacokinetics, safety and efficacy of combination treatment with methotrexate and leflunomide in patients with active rheumatoid arthritis. Arthritis Rheum. 1999;42(7):1322–8.

    Article  CAS  PubMed  Google Scholar 

  14. Jafari S, Maleki-Dizaji N, Barar J, Barzegar-Jalali M, Rameshrad M, Adibkia K. Methylprednisolone acetate-loaded hydroxyapatite nanoparticles as a potential drug delivery system for treatment of rheumatoid arthritis: in vitro and in vivo evaluations. Eur J Pharm Sci. 2016;91:225–35.

    Article  CAS  PubMed  Google Scholar 

  15. Bohner M, Tadier S, van Garderen N, de Gasparo A, Döbelin N, Baroud G. Synthesis of spherical calcium phosphate particles for dental and orthopedic applications. Biomatter. 2013; 3(2). pii: e25103.

  16. Manda MG, da Silva LP, Cerqueira MT, Pereira DR, Oliveira MB, Mano JF, et al. Gellan gum-hydroxyapatite composite spongy-like hydrogels for bone tissue engineering. J Biomed Mater Res A. 2018;106(2):479–90.

    Article  CAS  PubMed  Google Scholar 

  17. Bhowmick A, Banerjee SL, Pramanik N, Jana P, Mitra T, Gnanamani A, et al. Organically modified clay supported chitosan/hydroxyapatite-zinc oxide nanocomposites with enhanced mechanical and biological properties for the application in bone tissue engineering. Int J Biol Macromol. 2018;106:11–9.

    Article  CAS  PubMed  Google Scholar 

  18. Uskokovic V, Uskokovic DP. Nanosized hydroxyapatite and other calcium phosphates: chemistry of formation and application as drug and gene delivery agents. J Biomed Mater Res B Appl Biomater. 2011;96(1):152–91.

    Article  PubMed  Google Scholar 

  19. Sarkar C, Chowdhuri AR, Kumar A, Laha D, Garai S, Chakraborty J, et al. One pot synthesis of carbon dots decorated carboxymethyl cellulose- hydroxyapatite nanocomposite for drug delivery, tissue engineering and Fe3+ ion sensing. Carbohydr Polym. 2018;181:710–8.

    Article  CAS  PubMed  Google Scholar 

  20. Zhang Y, Zhang L, Ban Q, Li J, Li CH, Guan YQ. Preparation and characterization of hydroxyapatite nanoparticles carrying insulin and gallic acid for insulin oral delivery. Nanomedicine. 2017;14(2):353–64.

    Article  PubMed  Google Scholar 

  21. Chen L, Chen T, Cao J, Liu B, Shao C, Zhou K, et al. Effect of Tb/Mg doping on composition and physical properties of hydroxyapatite nanoparticles for gene vector application. Trans Nonferrous Met Soc China. 2018;28:125–36.

    Article  CAS  Google Scholar 

  22. Leung TK, Chen CH, Lai CH, Lee CM, Chen CC, Yang JC, et al. Bone and joint protection ability of ceramic material with biological effects. Chin J Physiol. 2012;55(1):47–54.

    Article  CAS  PubMed  Google Scholar 

  23. Chakraborty S, Vimalnath KV, Rajeswari A, Shinto A, Sarma HD, Kamaleshwaran K, et al. Preparation, evaluation, and first clinical use of 177 Lu-labeled hydroxyapatite (HA) particles in the treatment of rheumatoid arthritis: utility of cold kits for convenient dose formulation at hospital radiopharmacy. J Labelled Comp Radiopharm. 2014;57:453–62.

    Article  CAS  PubMed  Google Scholar 

  24. Habibovic P, de Groot K. Osteoinductive biomaterials--properties and relevance in bone repair. J Tissue Eng Regen Med. 2007;1:25–32.

    Article  CAS  PubMed  Google Scholar 

  25. Barradas AM, Yuan H, van Blitterswijk CA, Habibovic P. Osteoinductive biomaterials: current knowledge of properties, experimental models and biological mechanisms. Eur Cell Mater. 2011;21:407–29.

    Article  CAS  PubMed  Google Scholar 

  26. Mukesh U, Kulkarni V, Tushar R, Murthy RS. Methotrexate loaded self-stabilized calcium phosphate nanoparticles: a novel inorganic carrier for intracellular drug delivery. J Biomed Nanotechnol. 2009;5(1):99–105.

    Article  CAS  PubMed  Google Scholar 

  27. Żuber Z, Turowska-Heydel D, Sobczyk M, Banach-Górnicka M, Rusnak K, Piszczek A, et al. Methotrexate efficacy and tolerability after switching from oral to subcutaneous route of administration in juvenile idiopathic arthritis. Reumatologia. 2016;54(1):19–23.

    Article  PubMed  PubMed Central  Google Scholar 

  28. Branco JC, Barcelos A, de Araujo FP, Sequeira G, Cunha I, Patto JV, et al. Utilization of subcutaneous methotrexate in rheumatoid arthritis patients after failure or intolerance to oral methotrexate: a multicenter cohort study. Adv Ther. 2016;33(1):46–57.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Jahromi LP, Mohammadi-Samani S, Ashrafi H, Azadi A. A reversed-phase high performance liquid chromatography (HPLC) method for bio-analysis of methotrexate. TIPS. 2016;2(2):101–8.

    CAS  Google Scholar 

  30. van Roon EN, Yska JP, Raemaekers J, Jansen TL, van Wanrooy M, Brouwers JR. A rapid and simple determination of A77 1726 in human serum by high-performance liquid chromatography and its application for optimization of leflunomide therapy. J Pharm Biomed Anal. 2004;36(1):17–22.

    Article  PubMed  Google Scholar 

  31. Uskokovic V, Desai TA. Phase composition control of calcium phosphate NPs for tunable drug delivery kinetics and treatment of osteomyelitis. I. Preparation and drug release. J Biomed Mater Res A. 2013;101(5):1416–26.

    Article  PubMed  Google Scholar 

  32. Lett JA. Influence of surfactant on synthesis of HAp by hydrothermal route. J Chem Pharm Res. 2015;7(3):187–90.

    CAS  Google Scholar 

  33. Anwar M, Akhter S, Mallick N, Mohapatra S, Zafar S, Rizvi MM, et al. Enhanced anti-tumor efficacy of paclitaxel with PEGylated lipidic nanocapsules in presence of curcumin and poloxamer:In-vitro and In-vivo studies. Pharmacol Res. 2016;113:146–65.

    Article  CAS  PubMed  Google Scholar 

  34. Rawat P, Manglani K, Gupta S, Kalam A, Vohora D, Talegaonkar S, et al. Design and development of bioceramic based functionalized PLGA nanoparticles of Risedronate for bone targeting: in-vitro characterization and Pharmacodynamic evaluation. Pharm Res. 2015;32(10):3149–58.

    Article  CAS  PubMed  Google Scholar 

  35. Tariq M, Alam MA, Singh AT, Iqbal Z, Panda AK, Talegaonkar S. Biodegradable polymeric NPs for oral delivery of epirubicin: in vitro, ex vivo, and in vivo investigations. Colloids Surf B Biointerfaces. 2015;128:448–56.

    Article  CAS  PubMed  Google Scholar 

  36. Devanand Venkatasubbu G, Ramasamy S, Ramakrishnan V, Kumar J. Nanocrystalline hydroxyapatite and zinc-doped hydroxyapatite as carrier material for controlled delivery of ciprofloxacin. 3 Biotech. 2011; 1:173–186.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Shi XL, Wang LP, Feng X, Fan DD, Zang WJ, Wang B, et al. Inhibition of adjuvant-induced arthritis by nasal administration of novel synthetic peptides from heat shock protein 65. BMC Musculoskelet Disord. 2014;15(1):253.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Marry RL, Geetha T, Varalakshmi P. Effect of Vernonia Cinerea less flower extract in adjuvant-induced arthritis. Gen Pharmacol. 1998;31:601–6.

    Article  Google Scholar 

  39. Gomaa A, Elshenawy M, Afifi N, Mohammed E, Thabit R. Enhancement of the anti-inflammatory and anti-arthritic effects of theophylline by a low dose of a nitric oxide donor or non-specific nitric oxide synthase inhibitor. Br J Pharmacol. 2009;158:1835–47.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Arora R, Kuhad A, Kaur IP, Chopra K. Curcumin loaded solid lipid NPs ameliorate adjuvant-induced arthritis in rats. Eur J Pain. 2015;19(7):940–52.

    Article  CAS  PubMed  Google Scholar 

  41. Mossiat C, Laroche D, Prati C, Pozzo T, Demougeot C, Marie C. Association between arthritis score at the onset of the disease and long-term locomotor outcome in adjuvant-induced arthritis in rats. Arthritis Res Ther. 2015;17:184.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Cheng BC, Yu H, Guo H, Su T, Fu XQ, Li T, et al. A herbal formula comprising Rosae Multiflorae Fructus and Lonicerae Japonicae Flos, attenuates collagen-induced arthritis and inhibits TLR4 signalling in rats. Sci. Rep. 2016;10(6):20042.

    Article  Google Scholar 

  43. Habig WH, Pabst MJ, Jakoby WB. Glutathione S-transferases. J Biol Chem. 1974;249:7130–9.

    CAS  PubMed  Google Scholar 

  44. Mannervik B. Measurement of glutathione reductase activity: In:Maines MD. editor-in-chief. Current protocols in toxicology. 2001; Chapter 7: Unit7.2, John Wiley and Sons, Inc.

  45. Bergmeyer HU, Bernt E. Lactate-dehydrogenase, UV-assay with pyruvate and NADH. In: Bergmeyer HU, editor. Methods of enzymatic analysis, vol. 2. New York: Academic Press; 1974. p. 574–9.

    Chapter  Google Scholar 

  46. Reitman S, Frankel S. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. Amer J Clin Pathol. 1957;28:56–63.

    Article  CAS  Google Scholar 

  47. Rodgerson DO, Osberg IM. Sources of error in spectrophotometric measurement of aspartate aminotransferase and alanine aminotransferase activities in serum. ClinChem. 1974;20:43–50.

    CAS  Google Scholar 

  48. Comar JF, Babeto de Sá-Nakanishi A, de Oliveira AL, Marques Nogueira Wendt M, Bersani Amado CA, et al. Oxidative state of the liver of rats with adjuvant-induced arthritis. Free Radic Biol Med. 2013;58:144–53.

    Article  CAS  PubMed  Google Scholar 

  49. Sanwlani S, Rawat K, Pal M, Himadri B, Bohidar VAK. Cellular uptake induced biotoxicity of surface-modified CdSe quantum dots. J Nanopart Res. 2014;16:2382.

    Article  Google Scholar 

  50. Moron M, Deoierre J, Mannervik B. Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver. Biochim Biophys Acta. 1979;582(1):67–78.

    Article  CAS  PubMed  Google Scholar 

  51. Lykkesfeldt J. Malonaldehyde as biomarker of oxidative damage to lipids caused by smoking. Clinic ChimActa. 2007;380:50–8.

    Article  CAS  Google Scholar 

  52. Ferraz MP, Monteiro FJ, Manuel CM. Hydroxyapatite nanoparticles: a review of preparation methodologies. J Appl Biomater Biomech. 2004;2:74–80.

    CAS  PubMed  Google Scholar 

  53. Thomas SC, Harshita MPK, Talegaonkar S. Ceramic nanoparticles: fabrication methods and applications in drug delivery. Curr Pharm Des. 2015;21(42):6165–88.

    Article  CAS  PubMed  Google Scholar 

  54. Ginebra MP, Espanol CCM, Pastorino D, Montufar EB. Calcium phosphate cements as drug delivery materials. Adv Drug Deliv Rev. 2012;64:1090–110.

    Article  CAS  PubMed  Google Scholar 

  55. Zhou S, Zheng X, Yu X, Wang J, Weng J, Li X, et al. Hydrogen Bonding Interaction of Poly (D,L-Lactide)/hydroxyapatite Nanocomposites. Chem Mater. 2007;19:247–53.

    Article  CAS  Google Scholar 

  56. Verma G, Barick KC, Shetake NG, Pandey BN, Hassan PA. Citrate-functionalized hydroxyapatite NPs for pH-responsive drug delivery. RSC Adv. 2016;6:77968–76.

    Article  CAS  Google Scholar 

  57. Kumar V, Leekha A, Tyagi A, Kaul A, Mishra AK, Verma AK. Preparation and evaluation of biopolymeric NPs as drug delivery system in effective treatment of rheumatoid arthritis. Pharm Res. 2017;34(3):654–67.

    Article  CAS  PubMed  Google Scholar 

Download references

ACKNOWLEDGMENTS and DISCLOSURES

Authors are indebted to Intas Pharmaceuticals and Fresenius Kabi Ltd., India for providing gift samples of Teriflunomide and Methotrexate respectively. We are also thankful to Dr. Ambrish Kumar Tiwari, Veterinary officer, Jamia Hamdard, New Delhi and Dr. Purnima Rawat, Jamia Hamdard, New Delhi for their inputs regarding animal studies. We appreciate the contribution of AIIMS, New Delhi India to carry out TEM analysis. All authors declare no conflict of interest with any of the subject matter or materials discussed in the manuscript.

Funding

This work was financially supported by Indian Council of Medical Research (BMS/FW/Nano/2015–20580/March15/14/govt), New Delhi, India, by providing Senior Research Fellowship.

Author information

Author notes

  1. Shweta Pandey and Vijay Kumar contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, 110 062, India

    Shweta Pandey, Farhan Jalees Ahmad & Sushama Talegaonkar

  2. Nano Biotech Lab, Department of Zoology, Kirori Mal College, University of Delhi, Delhi, 110 007, India

    Vijay Kumar, Ankita Leekha & Anita Kamra Verma

  3. Department of Pharmacology, Vallabhbhai Patel Chest Institute, University of Delhi, Delhi, 110 007, India

    Nishant Rai

  4. Department of Pharmaceutics, Delhi Pharmaceutical Sciences & Research University, Pushp Vihar, Sector 3, New Delhi, 110017, India

    Sushama Talegaonkar

Authors
  1. Shweta Pandey
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Vijay Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ankita Leekha
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nishant Rai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Farhan Jalees Ahmad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anita Kamra Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Sushama Talegaonkar
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Sushama Talegaonkar.

Electronic supplementary material

ESM 1

(DOCX 298 kb)

ESM 2

(DOCX 170 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Pandey, S., Kumar, V., Leekha, A. et al. Co-Delivery of Teriflunomide and Methotrexate from Hydroxyapatite Nanoparticles for the Treatment of Rheumatoid Arthritis: In Vitro Characterization, Pharmacodynamic and Biochemical Investigations. Pharm Res 35, 201 (2018). https://doi.org/10.1007/s11095-018-2478-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s11095-018-2478-2

KEY WORDS

Search

Navigation