Presenter:Shafhan Dustur, dr.

Brain Tumor Talk:

Penggunaan Temozolomide padaTatalaksana CNS Tumor

Pembimbing:Dr. Joni Wahyuhadi, dr., Sp.BS (K)Dr. Rahadian I. Susilo, dr., Sp.BS (K)Irwan Barlian I.H., dr., Sp.BS(K)

2Overview

qApa itu temozolomide (TMZ)?

qBagaimana cara kerjanya?

qBagaimana peran TMZ dalam pengobatan high grade glioma?

qBagaimana peran TMZ dalam pengobatan CNS Tumor?

qApa itu MGMT ? Bagaimana hubungan TMZ dengan MGMT ?

3

qSintesis tahun 1984qKelas imidotetrazinoneqLipophilicqAntineoplasma

Introduction

qBerat molekul yang kecil (194 Da) àefektif menembus BBB

qDapat diberikan secara oral tanpabatasan diet

q100% dari dosis oral memasuki alirandarah

qEfek samping berat yang rendah

Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 103

O6-MeG mispairs with thymine (not cytosine) during DNA replication, alerting DNA mismatch repair (MMR) [15-16]. MMR exclusively recognises the mispaired thymine on the daughter strand and excises it, yet O6-MeG persists in the template strand. Therefore, futile cycles of thymine re-insertion and excision result in persistent DNA strand breaks, causing replication fork collapse [17]. G2/M cell cycle arrest is triggered, occurring in the second cell cycle following treatment [18-20] via ATR/CHK1-dependent signalling [21]; ultimately, apoptosis ensues [22] (Figs. 3 and 4). A good response to TMZ therefore requires functional MMR and low levels of MGMT.

The quantitatively more abundant N7-MeG and N3-MeA lesions are rapidly repaired by DNA base excision repair (BER; Figs. 4 and 5). N7-MeG appears not to be markedly cytotoxic: in contrast, N3-MeA lesions are lethal if not intercepted [23].

Therefore, the most important DNA repair systems impacting the mechanism of action and cytotoxicty of TMZ are MGMT (direct repair), MMR and BER (Figs. 4 and 5).

DNA REPAIR MECHANISMS CONTRIBUTING TO TEMOZOLOMIDE RESISTANCE

Direct Repair

MGMT (O6-Alkylguanine-DNA alkyltransferase; AGT) repairs O6-alkylguanine adducts in a single step, independently of any other protein or cofactors (Fig. 2). It is a small protein (22 kDa) present in both the cytoplasm and nucleus. Upon DNA alkylation, a shift towards more nuclear localisation may facilitate the repair process [24]. MGMT is able to repair not only O6-MeG, but also guanine residues with longer O6-alkyl adducts such as ethyl, chloroethyl, hydroxyethyl, n-propyl, n-butyl, and more bulky cyclic lesions conferred by benzyl or pyridyloxobutyl groups, but with diminishing efficiency as adduct size increases [25-27]. The O6-alkyl group is transferred from guanine to the active site cysteine residue (Cys 145) of MGMT in a stoichiometric, auto-inactivating reaction, thereby repairing DNA and inactivating MGMT [28]. MGMT binds damaged substrate DNA in the minor groove, the target base is then

N N

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Fig. (1). Structure and activation route of prodrug temozolomide.

Fig. (2). Repair of O6-methylguanine adducts by O6-methylguanine-DNA methyltransferase.

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4Farmakokinetik TMZ

Koukourakis, G. V., Kouloulias, V., Zacharias, G., Papadimitriou, C., Pantelakos, P., Maravelis, G., Fotineas, A., Beli, I., Chaldeopoulos, D., & Kouvaris, J. (2009). Temozolomide with radiation therapy in high grade brain gliomas: Pharmaceuticals considerations and efficacy; a review article. Molecules, 14(4), 1561–1577. https://doi.org/10.3390/molecules14041561

5Farmakokinetik TMZ

qKonsentrasi plasma setelahpemberian temozolomide

Koukourakis, G. V., Kouloulias, V., Zacharias, G., Papadimitriou, C., Pantelakos, P., Maravelis, G., Fotineas, A., Beli, I., Chaldeopoulos, D., & Kouvaris, J. (2009). Temozolomide with radiation therapy in high grade brain gliomas: Pharmaceuticals considerations and efficacy; a review article. Molecules, 14(4), 1561–1577. https://doi.org/10.3390/molecules14041561

6Metabolisme Temozolomide

5-aminoimidazole-4-carboxamide

(AIC)

monometil triazen 5- (3-methyltriazen-1-yl) - imidazole-4-carboxamide (MTIC)

diazoimidazole

Temozolomide

kationmetildiazonium

7

qTemozolomide bekerja dengan memodifikasi DNA atau RNA melaluipenambahan gugus metil (alkilasi) ke guanin di situs N7 dan O6 sertaadenin di situs N3

Mekanisme Kerja TMZ

PHARMACOLOGYVIGNETTE Temozolomide (Temodar)

J.R. WesolowskiP. Rajdev

S.K. Mukherji

SUMMARY: Temozolomide, an oral alkylating agent, is a commonly used medicine in the treatment ofanaplastic astrocytoma and glioblastoma multiforme. This paper will present the mechanism of actionas well as the clinical role for this chemotherapeutic drug.

ABBREVIATIONS: FDA ! US Food and Drug Administration; MTIC ! methyl triazeno imidazolecarboxamide

Temozolomide (Temodar) is an oral alkylating agent ap-proved by the FDA for use in the first-line treatment of

glioblastoma multiforme as well as for recurrent anaplasticastrocytoma.1 Alkylating agents are some of the oldest drugs inthe chemotherapy arsenal and were originally developed aschemical weapons during the early part of the twentieth cen-tury. Their anti-neoplastic aspects were only further investi-gated after World War II.2 Traditional alkylating agents act byproducing DNA cross-linkages, thus inhibiting DNA and cel-lular replication. Other similarly acting drugs, such as temo-zolomide, work by methylating DNA, which also results ininhibited DNA and cellular replication.2 All such agents actnonspecifically and affect both cancerous and normal cellsalike. However, cancer cells divide more rapidly than normaltissue and thus should be more sensitive to these effects.

Proposed Mechanism of ActionTemozolomide is an oral alkylating agent, first developed in theearly 1980s at Aston University in Great Britain.3 The proposedmechanism of action is based on the ability of its metabolites to

deposit methyl groups on DNA guanine bases. After oral admin-istration, the prodrug temozolomide is readily absorbed in thesmall intestine, with good penetration of the blood-brain barrierdue to its small size (194 Da). It then undergoes spontaneousintracellular conversion via hydrolysis into a potent methylatingagent, MTIC.4 MTIC methylates a number of nucleobases, mostimportant, the guanine base. This results in the formation ofnicks in the DNA, followed by apoptosis, because cellular repairmechanisms are unable to adjust to the methylated base (Fig 1).5

Clinical IndicationsTemozolomide was granted FDA approval in the treatment ofrecurrent anaplastic astrocytoma in 1999, with subsequent ap-proval for the first-line therapy of glioblastoma multiforme(Fig 2).1 The agent has also shown some activity in patientswith metastatic melanoma.6,7

Administration and EffectsTemozolomide is an oral prescription-only drug (though anintravenous form is available) administered once daily. The

Fig 1. Schematic illustration of the proposed mechanism of temozolomide. Temozolomide is converted intracellularly into MTIC, which methylates DNA. Cellular repair mechanisms cannotadjust, resulting in DNA nicks and ultimately apoptosis.

Received and accepted April 7, 2010.

From the University of Michigan Medical Center, Ann Arbor, Michigan.

Please address correspondence to Jeffrey R. Wesolowski, MD, University of MichiganMedical Center, 1500 East Medical Center Dr, Ann Arbor, MI 48109; e-mail:jefwesol@med.umich.edu

DOI 10.3174/ajnr.A2170

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AJNR Am J Neuroradiol 31:1383– 84 ! Sep 2010 ! www.ajnr.org 1383

8Mekanisme Kerja TMZ

q MGMT (O6-methylguanine-DNA methyltransferase)

q MMR (Mismatch Repair)q BER (Base Excision Repair)

Schreck, K. C., & Grossman, S. A. (2018). Role of Temozolomide in the Treatment of Cancers Involving the Central Nervous System. Oncology (Williston Park, N.Y.), 32(11), 555-560,569.

9Apa itu MGMT ?

q O6-methylguanine-DNA methyltransferase (MGMT) à enzimperbaikan DNA

q Gen MGMT terletak pada kromosom10q26.3

q Ekspresi gen MGMT dipengaruhi olehmetilasi promotor MGMT di CPG Island

q Metilasi promotor MGMT merupakanpenanda prediktif yang baik untukrespon terhadap kemoterapi agenalkilasi.

q Metilasi, dimediasi oleh 5`-methylcytosine methyltransferase, berlangsung di sitosin CpG Island

Yu, W., Zhang, L., Wei, Q., & Shao, A. (2020). O6-Methylguanine-DNA Methyltransferase (MGMT): Challenges and New Opportunities in Glioma Chemotherapy. Frontiers in Oncology, 9(January), 1–11. https://doi.org/10.3389/fonc.2019.01547

10Mekanisme Resistensi TMZ

Zhang, J., F.G. Stevens, M., & D. Bradshaw, T. (2012). Temozolomide: Mechanisms of Action, Repair and Resistance. Current Molecular Pharmacology, 5(1), 102–114. https://doi.org/10.2174/1874-470211205010102

11

104 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al.

Fig. (3). A) Effect of temozolomide on glioblastoma multiforme cell growth. Cells were seeded at a density of 650 per well. After 24 h, temozolomide was introduced. At the time of drug addition and following 7 days incubation, MTT assays were performed to determine cell growth. B) Expression of methylguaninemethyltransferase protein in SNB19M and U373M cells. Western blot assays were performed following separation of protein from whole cell lysates. C) Effect of temozolomide on SNB19V cell cycle.

Fig. (4). Key DNA repair mechanisms influencing cellular response to temozolomide.

flipped out of the helix and bound to MGMT, altering the conformation of the DNA binding domain allowing alkylated MGMT to be detached from DNA and degraded through the ubiquitin/proteasomal system [15, 29].

MGMT protects cells from carcinogens; however, it is also able to protect cancer cells from chemotherapeutic

alkylating agents such as TMZ. Tissue expression is variable, with high protein expression in liver and lower expression in haematopoietic tissues and brain [30-31]. Tumour MGMT expression is immensely variable, highest levels are found in breast, ovarian and lung tumours, whereas lowest activity is observed in gliomas, pancreatic

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Mekanisme Resistensi TMZ

Zhang, J., F.G. Stevens, M., & D. Bradshaw, T. (2012). Temozolomide: Mechanisms of Action, Repair and Resistance. Current Molecular Pharmacology, 5(1), 102–114. https://doi.org/10.2174/1874-470211205010102

12Pengukuran Kadar MGMT

Promotor Metilasi

Nonquantitative MSP

Quantitative MSP (qMSP)

Pyrosequencing

Methylation-sensitive multiplex ligation-dependent probe amplification (MS-MPLA)

Infinium Methylation EPIC BeadChip Array

mRNA expression

Quantitative real-time PCR (qRT-PCR)

Protein expression

IHC

Protein Activity

Enzymatic assays

Butler, M., Pongor, L., Su, Y. T., Xi, L., Raffeld, M., Quezado, M., Trepel, J., Aldape, K., Pommier, Y., & Wu, J. (2020). MGMT Status as a Clinical Biomarker in Glioblastoma. Trends in Cancer, 6(5), 380–391. https://doi.org/10.1016/j.trecan.2020.02.010

13Temozolomide pada High Grade Glioma

q Efektivitas dan keamanan RT + TMZ vs. RT saja

q OS yang lebih baik pada RT+TMZ dibandingkan RT saja

q Efek samping hematologi grade 3 dan 4: lebih tinggi pada RT+TMZ

q RT + TMZ dikaitkan dengan peningkatanOS PFS secara signifikan dibandingkan RT saja

14

Analisa Kaplan-Meier overall survival (OS) dan progression free survival (PFS) berdasarkan status promotor MGMT (unmethylated [Un-meth] vs methylated [Meth]) dan radiotherapy (RT) + temozolomide (TMZ) vs RT saja

Hegi, M. E., Liu, L., Herman, J. G., Stupp, R., Wick, W., Weller, M., Mehta, M. P., & Gilbert, M. R. (2008). Correlation of O6-methylguanine methyltransferase (MGMT) promoter methylation with clinical outcomes in glioblastoma and clinical strategies to modulate MGMT activity. Journal of Clinical Oncology, 26(25), 4189–4199. https://doi.org/10.1200/JCO.2007.11.5964

Temozolomide pada High Grade Glioma

15Temozolomide pada High Grade Glioma

16Temozolomide pada High Grade Glioma

17Temozolomide pada High Grade Glioma

Stupp, R., Hegi, M. E., Gilbert, M. R., & Chakravarti, A. (2007). Chemoradiotherapy in malignant glioma: Standard of care and future directions. Journal of Clinical Oncology, 25(26), 4127–4136. https://doi.org/10.1200/JCO.2007.11.8554

18Efek Samping TMZ

Bae, S. H., Park, M. J., Lee, M. M., Kim, T. M., Lee, S. H., Cho, S. Y., Kim, Y. H., Kim, Y. J., Park, C. K., & Kim, C. Y. (2014). Toxicity profile of temozolomide in the treatment of 300 malignant glioma patients in Korea. Journal of Korean Medical Science, 29(7), 980–984. https://doi.org/10.3346/jkms.2014.29.7.980

19Oligodendroglioma

q Adanya : 1p/19q codeletion

q RT + Kemoterapi PCV q Grade 2 and 3 Oligodendrogliomas : RT + PCV

vs RT Saja à ↑ OS 7 Tahun (Schreck & Grossman, 2018)

q Uji coba fase III secara acakq 274 pasien dengan AOD, AOA, dan AAq FRT (60 Gy hingga 33 fraksi)q 4 siklus PCV vs. 8 siklus TMZ

q Tidak ada perbedaan dalam PFS dan OS antara duarejimen agen kemoterapi tersebut

q TMZ dapat diberikan pada kasus gagal terapi PCV atau risiko hematotoksitas ↑

20Ependymoma

q Standar untuk terapi ependymoma saat ini: maximal safe surgical resection lalu diikuti RT pada beberapa kasus

q Kemoterapi à cegah irradiasi dini pada anak

q TMZ pada ependymoma berulang à respon sangatterbatas

q Soffietty et.al. à pola respon, hasil dan korelasidengan metilasi promotor MGMT

q Respon yang baik pada ependymoma rekurennamun respon lambat, dan berlaku hanya padayang belum pernah kemoterapi

q Promotor metilasi MGMT tidak mempengaruhirespon maupun OS

q Buccoliero et al : metilasi promotor MGMT dan ekspresiprotein MGMT pada ependymoma à 75% ekspresiprotein MGMT yang tinggi

21Primary CNS Lymphomaq PCNSL sangat sensitif terhadap metotreksat dosis tinggi

(HD-MTX) à standard regimen (overall response rate of 74%)

q Regimen kemoterapi lain : rituximab, cytarabine, temozolomide,

atau procarbazine/vincristine

q Penggunaan terapi TMZ dan status metilasi dari gen

MGMT pada 17 pasien PCNSL yang refrakter atau

relaps

q Peningkatan media overall survival

q Hanya 1 pasien yang mengalami trombositopenia

dan neutropenia grade 3

q Tidak ada perbedaan secara statistik status MGMT

dengan median overall survival pasien

q Adanya toksisitas moderate berupa trombositopenia

grade 3 dan 4 akibat penggunaan TMZ

q TMZ dipertimbangkan pada pasien yang kontraindikasi

pemberian HD-MTX

22Pituitary adenomas/carcinomas

q Studi terbatasq Publikasi pertama: tahun 2006q Bengtsson D, et.al (2015) à 24 pasien: respon

rate terhadap monotherapy TMZ pada pituitary adenoma agresif: 37-41%.

q Temozolomide adalah pengobatan yang efektif pada kasustumor hipofisis yang agresif

q Tingkat keberhasilan 69%

q The European Society of Endocrinology (ESE) (2017) merekomendasikan TMZ sebagai kemoterapi linipertama untuk pasien APT dan PC

q Evaluasi pada 3 bulan pertama dengan dosis standar 150-200mg/m2 selama 5 hari setiap 28 hari

23Brain Metastase

q Zhao et al (2016): efektivitas TMZ+RT vs RT saja

q tidak didapatkan adanya keuntungan padapeningkatan OS dan PFS

q Peningkatan efek samping: trombositopenia dangangguan GI tract

q Ma et al (2016): keamanan dan efektivitas TMZ+RT vs RT saja

q Total 19 RCT

q tidak ada perbedaan secara statistik baik PFS maupun OS antara kelompok radioterapi plus temozolomide dan radioterapi saja

24Meduloblastoma

q ORR (objective response rate) 42.5%, PFS pada bulanke 6 dan 12 adalah 30% and 7.5%.

q Tidak ada extrahematological efek mayor berbahayaq Efek samping : thrombocytopenia (17.5%),

neutropenia (7.5%), dan anemia (2.5%)

q Wang, et.al (2009): penggunaan temozolomide padaanak-anak dengan tumor otak embrional rekurenyang gagal pada terapi lini pertama.

q Respon yang baik pada setengah kasus, efek sampingyang ringan, supresi sumsum tulang ringan.

q Toleransi yang baik, kepuasan terhadap kualitashidup

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