Advertisement

Investigational New Drugs

, Volume 26, Issue 1, pp 25–34 | Cite as

Pharmacological evaluation of C-3 modified Betulinic acid derivatives with potent anticancer activity

  • Praveen Rajendran
  • Manu JaggiEmail author
  • Manoj K. Singh
  • Rama Mukherjee
  • Anand C. Burman
Preclinical Studies

Summary

In vitro and in vivo pharmacological screening of Betulinic acid (BA) and five dihydro-BA derivatives modified at C-3 position [4-nitrobenzyl-oximino (1), 2-4-difluoro-benzoyloxy (2), 2-4-difluoro-benzylidene-amino (3), benzoyl-hydrazono (4), and 4-fluorophenyl-hydrazono (5)], having potent in vitro anti-cancer activity was carried out using ADME, animal PK and tumor studies. We found that BA and the derivatives had poor aqueous solubility (<0.1 μg/ml), low to moderate permeability (log Pe < −5.0) and high plasma protein binding (>70%). Although BA and 5 were metabolized by human liver microsomes, derivatives 1, 2, 3 and 4 possessed good in vitro metabolic stability. Except 3 which inhibited CYP1A2 isoform by more than 50% none of the other compounds inhibited key cytochrome P450 enzyme isoforms (CYP1A2, CYP2C9, CYP2D6 and CYP3A4) at 10 μM. Based on in vitro results one derivative 1 was tested in rodent PK and tumor studies. We found that 1 exhibited favorable pharmacokinetic characteristics of a systemically administered drug and showed better in vivo anti-tumor efficacy as compared to BA in a human colon cancer xenograft model. Our results show that BA derivatives are potential anti-cancer compounds which need to be explored in detail.

Keywords

Betulinic acid Derivatives Cancer ADME In vitro In vivo 

Abbreviations

BA

betulinic acid

PAMPA

parallel artificial membrane permeability assay

References

  1. 1.
    Cichewicz RH, Kouzi SA (2004) Chemistry, biological activity, and chemotherapeutic potential of betulinic acid for the prevention and treatment of cancer and HIV infection. Med Res Rev 24:90–114PubMedCrossRefGoogle Scholar
  2. 2.
    Pisha E, Chai H, Lee IS, Chagwedera TE, Farnsworth NR, Cordell AC et al (1995) Discovery of Betulinic acid as a selective inhibitor of human melanoma that functions by induction of apoptosis. Nat Med 1:1046–1051PubMedCrossRefGoogle Scholar
  3. 3.
    Fulda S, Friesen C, Los M, Scaffidi C, Mier W, Benedict M, Nunez G, Krammer PH, Peter ME, Debatin KM (1997) Betulinic acid triggers CD95 (APO-1/Fas)- and p53-independent apoptosis via activation of caspases in neuroectodermal tumors. Cancer Res 57:4956–4964PubMedGoogle Scholar
  4. 4.
    Schmidt ML, Kuzmanoff KL, Ling-Indeck L, Pezzuto JM (1997) Betulinic acid induces apoptosis in human neuroblastoma cell lines. Eur J Cancer 33:2007–2010PubMedCrossRefGoogle Scholar
  5. 5.
    Fulda S, Jeremias I, Steiner HH, Pietsch T, Debatin KM (1999) Betulinic acid: a new cytotoxic agent against malignant brain-tumor cells. Int J Cancer 82:435–441PubMedCrossRefGoogle Scholar
  6. 6.
    Thurnher D, Turhani D, Pelzmann M, Wannemacher B, Knerer B, Formanek M, Wacheck V, Selzer E (2003) Betulinic acid: a new cytotoxic compound against malignant head and neck cancer cells. Head Neck 25:732–740PubMedCrossRefGoogle Scholar
  7. 7.
    Sami A, Taru M, Salme K, Jari YK (2006) Pharmacological properties of the ubiquitous natural product betulin. Eur J Pharm Sci 29:1–13CrossRefGoogle Scholar
  8. 8.
    Mukherjee R, Jaggi M, Rajendran P, Siddiqui MJ, Srivastava SK, Vardhan A, Burman AC (2004a) Betulinic acid and its derivatives as anti-angiogenic agents. Bioorg Med Chem Lett 14:2181–2184PubMedCrossRefGoogle Scholar
  9. 9.
    Mukherjee R, Jaggi M, Rajendran P, Srivastava SK, Siddiqui MJ, Vardhan A, Burman AC (2004b) Synthesis of 3-O-acyl/3-benzylidene/3-hydrazone/3-hydrazine/17-carboxyacryloyl ester derivatives of Betulinic acid as anti-angiogenic agents. Bioorg Med Chem Lett 14:3169–3172PubMedCrossRefGoogle Scholar
  10. 10.
    Mukherjee R, Jaggi M, Siddiqui MJ, Srivastava SK, Rajendran P, Vardhan A, Burman AC (2004c) Synthesis and cytotoxic activity of 3-O-acyl/3-hydrazine /2-bromo/20,29-dibromo Betulinic acid derivatives. Bioorg Med Chem Lett 14:4087–4091PubMedCrossRefGoogle Scholar
  11. 11.
    Chatterjee P, Kouzi SA, Pezzuto JM, Hamann MT (2000) Biotransformation of the antimelanoma agent Betulinic acid by Bacillus megaterium ATCC 13368. Appl Environ Microbiol 66:3850–3855PubMedCrossRefGoogle Scholar
  12. 12.
    Kouzi SA, Chatterjee P, Pezzuto JM, Hamann MT (2000) Microbial transformations of the antimelanoma agent Betulinic acid. J Nat Prod 63:1653–1657PubMedCrossRefGoogle Scholar
  13. 13.
    Akihisa T, Takamine Y, Yoshizumi K, Tokuda H, Kimura Y, Ukiya M, Nakahara T, Yokochi T, Ichiishi E, Nishino H (2002) Microbial transformations of two lupane-type triterpenes and anti-tumor-promoting effects of the transformation products. J Nat Prod 65:278–282PubMedCrossRefGoogle Scholar
  14. 14.
    Lewis DFV, Dickins M, Weaver RJ, Eddershaw PJ, Goldfarb PS, Tarbit MH (1998) Molecular modeling of human CYP2C subfamily enzymes CYP2C9 and CYP2C19: Rationalization of substrate specificity and sitedirected mutagenesis experiments in the CYP2C subfamily. Xenobiotica 28:235–268PubMedCrossRefGoogle Scholar
  15. 15.
    Wen Z, Martin D, Bullock P, Lee KH, Smith PC (2006) Glucuronidation of anti-HIV drug candidate bevirimat: Identification of human UDP-glucuronosyltransferases and species differences. Drug Metab Dispos 35(3):440–448PubMedCrossRefGoogle Scholar
  16. 16.
    Udeani GO, Zhao G-M, Shin YG, Cooke BP, Graham J, Beecher CWW, Kinghorn AD, Pezzuto JM (1999) Pharmacokinetics and tissue distribution of Betulinic acid in CD-1 mice. Biopharm Drug Dispos 20:379–383PubMedCrossRefGoogle Scholar
  17. 17.
    Shin YG, Cho KH, Chung SM, Graham J, Gupta TKD, Pezzuto JM (1999) Determination of betulinic acid in mouse blood, tumor, and tissue homogenates by liquid chromatography-electrospray mass spectrometry. J Chromatogr B Biomed Sci Appl 732:331–336PubMedCrossRefGoogle Scholar
  18. 18.
    Yasukawa K, Takido M, Matsumoto T, Takeuchi M, Nakagawa S (1991) Sterol and triterpene derivatives from plants inhibit the effects of a tumor promoter, and sitosterol and betulinic acid inhibit tumor promotion in mouse skin two-stage carcinogenesis. Oncology 48:72–76PubMedGoogle Scholar
  19. 19.
    Yasukawa K, Yu SY, Yamanouchi S, Takido M, Akihisa T, Tamura T (1995) Some lupane-type triterpenes inhibit tumor promotion by 12-O-tetradecanoylphorbol-13-acetate in two-stage carcinogenesis in mouse skin. Phytomedicine 4:309–313Google Scholar
  20. 20.
    Zuco V, Supino R, Righetti SC, Cleris K, Marchesi E, Gambacorti-Passerini C, Formelli F (2002) Selective cytotoxicity of betulinic acid on tumor cell lines, but not normal cells. Cancer Lett 175:17–25PubMedCrossRefGoogle Scholar
  21. 21.
    Mukherjee R, Kumar V, Srivastava SK, Agarwal SK, Burman AC (2006) Betulinic acid derivatives as anticancer agents: structure activity relationship. Anticancer Agents Med Chem 6:271–279PubMedCrossRefGoogle Scholar
  22. 22.
    Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63PubMedCrossRefGoogle Scholar
  23. 23.
    Kansy M, Fischer H, Kratzat K, Senner F, Wagner B, Parrilla I (2001) High-throughput artificial membrane permeability studies in early lead discovery and development. Pharmacokinetic Optim Drug Res 12:448–464Google Scholar
  24. 24.
    Sugano K, Hamada H, Machida M, Ushio H (2001) High throughput prediction of oral absorption: improvement of the composition of the lipid solution used in parallel artificial membrane permeation assay. J Biomol Screen 6:189–196PubMedCrossRefGoogle Scholar
  25. 25.
    Rodrigues AD (1994) Use of in vitro human metabolism studies in drug development. Biochem Pharmacol 48:2147–2156PubMedCrossRefGoogle Scholar
  26. 26.
    Tassaneeyakul W, Birkett DJ, Veronese ME, McManus ME, Tukey RH, Quattrochi LC, Gelboin HV, Miners JO (1993) Specificity of substrate and inhibitor probes for human cytochromes P450 1A1 and 1A2. J Pharmacol Exp Ther 265:401–407PubMedGoogle Scholar
  27. 27.
    Schmitz G, Lepper H, Estler CJ (1993) High-performance liquid chromatographic method for the routine determination of diclofenac and its hydroxy and methoxy metabolites from in vitro systems. J Chromatogr 620:158–163PubMedCrossRefGoogle Scholar
  28. 28.
    Kronbach T, Mathys D, Gut J, Catin T, Meyer UA (1987) High-performance liquid chromatographic assays for bufuralol 1-hydroxylase, debrisoquine 4-hydroxylase, and dextromethorphan O-demethylase in microsomes and purified cytochrome P-450 isozymes of human liver. Anal Biochem 162:24–32PubMedCrossRefGoogle Scholar
  29. 29.
    Buters JT, Korzekwa KR, Kunze KL, Omata Y, Hardwick JP, Gonzalez FJ (1994) cDNA-directed expression of human cytochrome P450 CYP3A4 using baculovirus. Drug Metab Dispos 22:688–692PubMedGoogle Scholar
  30. 30.
    Cheng X, Shin YG, Levine BS, Smith AC, Tomaszewski JE, van Breemen RB (2003) Quantitative analysis of betulinic acid in mouse, rat and dog plasma using electrospray liquid chromatography/mass spectrometry. Rapid Commun Mass Spectrom 17:2089–2092PubMedCrossRefGoogle Scholar
  31. 31.
    Friedman MA, Woodcock J, Lumpkin MM, Shuren JE, Hass AE, Thompson LJ (1999) The safety of newly approved medicines: do recent market removals mean there is a problem. JAMA 281:1728–1734PubMedCrossRefGoogle Scholar
  32. 32.
    Lasser KE, Allen PD, Woolhandler SJ, Himmelstein DU, Wolfe SM, Bor DH (2002) Timing of new black box warnings and withdrawals for prescription medications. JAMA 287:2215–2220PubMedCrossRefGoogle Scholar
  33. 33.
    World Health Organization (2006) Cancer. Retrieved on 2007-05-24Google Scholar
  34. 34.
    Stoeltzing O, Liu W, Reinmuth N, Parikh A, Ahmad SA, Jung YD, Fan F, Ellis LM (2003) Angiogenesis and antiangiogenic therapy of colon cancer liver metastasis. Ann Surg Oncol 10:722–733PubMedCrossRefGoogle Scholar
  35. 35.
    Benson AB III (2006) New approaches to the adjuvant therapy of colon cancer. Oncologist 11:973–980PubMedCrossRefGoogle Scholar
  36. 36.
    Diez-Fernandez R, Salinas Hernandez P, Giron-Duch C (2006) A review of chemotherapy for metastatic colon cancer. Farm Hosp 30:359–369PubMedCrossRefGoogle Scholar
  37. 37.
    Chintharlapalli S, Papineni S, Ramaiah SK, Safe S (2007) Betulinic acid inhibits prostate cancer growth through inhibition of specificity protein transcription factors. Cancer Res 67:2816–2823PubMedCrossRefGoogle Scholar
  38. 38.
    Jaggi M, Mukherjee R (1995) Establishment of tumorigenic cell lines from biopsies of human colon adenocarcinomas. J Basic App Biomed 3:27–35Google Scholar
  39. 39.
    Britten CD, Hilsenbeck SG, Eckhardt G, Marty J, Mangold G, MacDonald JR, Rowinsky EK, Von Hoff DD, Weitman S (1999) Enhanced antitumor activity of 6-hyroxymethylacylfulvene in combination with Irinotecan and 5-Fluorouracil in the HT29 colon tumor xenograft model. Cancer Res 59:1049–1053PubMedGoogle Scholar
  40. 40.
    Bolin SJ, Zhao H, Hunter K, Szanto AK, Ruggeri B (2006) The effects of the oral, pan-VEGF-R kinase inhibitor CEP-7055 and chemotherapy in orthotopic models of glioblastoma and colon carcinoma in mice. Mol Cancer Ther 5:1744–1753CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Praveen Rajendran
    • 1
  • Manu Jaggi
    • 1
    Email author
  • Manoj K. Singh
    • 1
  • Rama Mukherjee
    • 1
  • Anand C. Burman
    • 1
  1. 1.Experimental Oncology LabDabur Research FoundationGhaziabadIndia

Personalised recommendations