Alkylating agents Platinating agents Antimetabolites Topoisomerase inhibitors

1. Interaction of RT with CT and other AgentsBill McBrideDept. Radiation OncologyDavid Geffen School MedicineUCLA, Los Angeles, Ca.wmcbride@mednet.ucla.edu

2. Classes of Chemotherapy Agents • Alkylating agents • Platinating agents • Antimetabolites • Topoisomerase inhibitors • Anti-microtubular agents • Miscellaneous

3. Alkylating agents • Nitrogen mustard derivatives: cyclophosphamide, chlorambucil, melphalan, ifosfamide, mechlorethamine • Ethylenimines: Thiotepa and Hexamethylmelamine • Nitrosoureas: BCNU (carmustine), CCNU (lomustine), Streptozocin • Alkylsulfonates:  Busulfan • Hydrazines and Triazines: altretamine, procarbazine, dacarbazine, temodar • Highly reactive alkyl groups (e.g. —CH2Cl) covalently bind to intracellular macromolecule, such as DNA • Bifunctional crosslink are more effective (interstrand DNA crosslinks) • Limited cell cycle specificity, carcinogenic Classes of Agents

4. Classes of Agents • Platinating agents • Cisplatin, Carboplatin, oxiplatin • Exist in 2+ oxidation state with 4 groups that interact with DNA (95% intrastrand 5% interstrand cross-linkages) • Nausea, vomiting, kidney toxicity, less myelosuppression than with alkylating agents

5. Classes of Agents • Antimetabolites • Purine/pyrimidine analogs • 5-FU, cytosine arabinoside, gemcitabine, iododeoxyuridine • Antifolates • Methotrexate • interfere with normal cell function (e.g. DNA synthesis) • Cell cycle specific, tend to cause DNA damage and block repair, less carcinogenic than alkylating agents

6. Classes of Agents • Topoisomerase inhibitors • Topo I inhibitors • Camptothecin derivatives such as topotecan, irinotecan (CPT11) • Topo II inhibitors • Epipodophyllotoxins such as etoposide, teniposide • Topo II inhibitors plus other effects • Anthracyclines such as daunorubicin, doxorubicin (Adriamycin), idarubicin, epirubicin • Topoisomerases relax dsDNA to allow replication/transcription by single (I) or double (II) strand nick. DSBs form when the replication fork meets the DNA/topo cleavable complex - in S phase

7. Classes of Agents • Antimicrotubular agents • Vinca alkaloids • Taxanes: paclitaxel (Taxol), docetaxel (Taxotere) • Bind to tubulins (different site) and inhibit microtubular disassembly • Cause G2M arrest

8. Others • Proteasome Inhibitors • Bortezumib, Velcade, PS-341 • Boronic acid dipeptide • Inhibits proteasome core chymotryptic activity reversibly • Effective in drug refractory multiple myeloma • Causes • Cell cycle arrest • Apoptosis of cancer cells • Immunosuppression • Anti-inflammatory • Anti-angiogenesis • Downregulation of NF-B (and many other signal transduction molecules) • Radiosensitizer and chemosensitizer

9. Chemotherapeutic Considerations • Pharmacokinetics • Concentration of metabolites over time • Absorption, Distribution, Metabolism, Elimination • Pharmacodynamics • Cellular response to drug

10. Pharmacokinetics • Concentration of metabolites over time • Normally measured by the area under the concentration/time curve • However, maintaining a certain level may be more important for some drugs than others • Continuous delivery better than bolus • Topo I inhibitors, anti-metabolites, taxanes

11. Pharmacokinetics • Absorption, Distribution, Metabolism, Elimination • Absorption • Depends on route of administration • Intravenous route preferred for pharmacokinetic reasons • Oral is best for some eg Temozolomide • Regional delivery may be more effective (glioma) • Influenced by physical form and barriers to penetration/absorption e.g. blood-brain barrier • Distribution • Requires blood/fluid flow to organs/tissues/tumor • Diffusion kinetics • Size and chemical form • Protein and tissue binding, lipid solubility, pH, etc.

12. Pharmacokinetics • Metabolism • Phase 1 active metabolites often produced in liver • Phase II inactive metabolites produced by conjugation • Some drugs requires activation (cyclophosphamide) • Influenced by genetic polymorphisms (5-FU - dihydropyrimidine dehydrogenase deficiency is toxic and high thymidylate synthase levels decrease efficacy) • Liver function affects metabolism • Excretion • Primarily in kidney or biliary tract • Phase I active metabolites (carboplatin) or Phase II metabolites (doxorubicin) can give toxicity • Kidney function affects clearance • Influenced by protein and tissue binding, lipid solubility, pH, etc. • Doxorubicin slow release is due to high lipid solubility

13. Pharmacodynamics • Cellular response to drug depends on • Microenvironment • Cell cycle phase • Drug resistance mechanisms • Intracellular metabolism • Sensitivity to cell death/survival pathways • Difficult to get predictors from in vitro survival data

14. Microenvironment 10 Mitomycin C, misonidazole, metronidazole, etanidazole, tirapazamine, doxorubicin 10 Bleomycin, procarbazine,dactinomycin 1 1 Aerated 0.1 Surviving fraction 0.1 Surviving fraction Hypoxic less sensitive more sensitive 0.01 0.01 Aerated Hypoxic 0.001 0.001 0 50 100 150 200 250 0 2 4 6 8 10 µg/ml bleomycin µM mitomycin

15. Cell Cycle M Phase Alkylating Agents G2 phase Paclitaxel Bleomycin G0 - quiescence S phase Docetaxel Methotrexate Ara-C, 6TG, Hydroxyurea Vinblastine Doxorubicin G1/S phase Alkylating Agents, Cisplatin

16. Mechanisms of Drug Resistance Mechanism Decreased uptake Increased efflux Decrease in drug activation Increase in drug catabolism Increase or decrease in levels of target molecule Alterations in target molecule Inactivation by binding to sulfhydryls (e.g. glutathione) Increased DNA repair Decreased ability to undergo apoptosis Drugs Methotrexate, melphalan, cisplatin Anthracyclines, vinca alkaloids, etoposide, taxanes Many antimetabolites Many antimetabolites Methotrexate, topoisomerase inhibitors Methotrexate, other antimetabolites, topoisomerase inhibitors, Gleevec Alkylating agents, cisplatin, anthracyclines Alkylating agents, cisplatin, anthracyclines, etoposide Alkylating agents, cisplatin, anthracyclines, etoposide

17. Mechanisms of Drug Resistance • Impaired drug influx • passive diffusion • energy & temperature independent • facilitated diffusion • transport carrier on membrane • energy & temperature independent • active transport • carrier-mediated process • energy & temperature dependent • reduced folate carrier - mutation? • Melphalan  binding affinity for drug and  number of transport sites / slower carrier mobility

18. Mechanisms of Drug Resistance • Increased drug efflux • Many natural drugs/derivatives (taxanes, vinca alkaloids, anthracyclines) have shared mechanisms of resistance, e.g. substrates for membrane-based ATPase-dependent proteins (pumps) • P-glycoprotein (mdr1) • High levels in kidney & adrenals; intermediate in lung, liver, colon and rectum • Co-specificity with proteasome enzymes • Inhibitors • Calcium channel blockers (verapamil) • Cyclosporin A • Tariquidar, zosuquidar (phase 1/2)

19. Summary of Drug Therapy • Plethora of cytotoxic agents • Selective (not exclusive) targets - proliferating cells • Major problem: drug resistance • Principal mechanisms • altered membrane transport (P-glycoprotein); • altered target enzyme (mutated topoisomerase II) • decreased drug activation • increased drug degradation (e.g. altered expression of drug-metabolizing enzyme) • drug inactivation (conjugation with glutathione) • drug interactions • enhanced DNA repair; failure to apoptose (e.g. mutation of p53)

20. How Effective is CT - and how is it best combined with RT? • Responses are often referred to as PR or CR. • Defined by the endpoint - pathology/imaging/clinical • If a tumor has 1010 cells, a PR may decrease this to 2x109, which is not much of an improvement. • Patients in complete remission can have anywhere between 0-109 cells as a tumor burden. If 10yr relapse-free survival is 30% without and 40-45% with adjuvant chemotherapy, as is the case in early breast cancer regimens, Withers calculated that this represents about 2 logs of tumor cell kill – easy to achieve with RT. • Neoadjuvant chemotherapy may cause accelerated tumor repopulation! Concomitant delivery of drugs with RT is often better than sequential delivery.

21. Therapeutic Index • Need to increase therapeutic index • Bone marrow major toxicity • Normally treat to MTD, except for palliative cases (5-FU for advanced colorectal Ca) • Some tumors are drug “resistant” others are “sensitive” but recur - are the cancer stem cells being killed? Many seem to have enhanced drug efflux pumps…..

22. Combination Therapies • CT combinations • Different classes of agents with minimally overlapping toxicities • RT plus CT • Adjuvant therapy (P-glycoprotein inhibitors) • Biological targeting + CT/RT

23. Chemotherapy and Radiation • Combination of chemotherapy and radiation can increase cure rate, but also the potential for normal tissue toxicity • Dose Enhancement Ratio (DER) • Dose of radiation alone to produce an effect divided by dose of radiation to give same effect in combination with drug • Therapeutic Gain Factor • Ratio of DER for tumor to DER of dose-limiting normal tissue

24. 1 10-1 DB 10-2 10-3 Synergy vs. Additivity Drug B Drug A Drug A + B 1 1 SA DA ? DA 10-1 10-1 SB X S1 10-2 10-2 DB 10-3 10-3 0 1 2 3 4 5 6 0 1 2 3 4 5 6 0 1 2 3 4 5 6 Claims for synergy are often made if S1 = SA x SB but this is only true if there is no shoulder. Tannock et al: The Basic Science of Oncology 4th Ed.

25. Isobologram Analysis Subadditive Protective Antagonistic Dose of Agent A Envelope of Additivity Supra-additive Synergistic Dose of Agent B After Steel and Peckham, 1959

27. ChemoRT • Meta-analyses have shown that chemotherapy (concomitant, neoadjuvant, adjuvant) improves survival in non-metastatic HNSCC (other than NPC) by 4.4% at 5yrs. • Bourhis et al PASCO 22:488, 2004 • Concomitant gives an absolute benefit of 6.5-8% at 5yrs, irrespective of fractionation scheme although altered fractionation gives a survival advantage, and is better than neoadjuvant. Platinum-based regimens best. It comes with an increase in early and late toxicity. • Pignon et al IJROBP 69: S112, 2007

28. Randomized Phase III Trial of Sequential Chemoradiotherapy Compared With Concurrent Chemoradiotherapy in Locally Advanced Nonsmall-Cell Lung Cancer • Fournel et al Journal of Clinical Oncology, 23; 5910-5917, 2005. • Two hundred five patients were randomly assigned. Pretreatmentcharacteristics were well balanced between the two arms. Therewere six toxic deaths in the sequential arm and 10 in the concurrentarm. Median survival was 14.5 months in the sequential arm and16.3 months in the concurrent arm (log-rank test P = .24). Two-,3-, and 4-year survival rates were better in the concurrentarm (39%, 25%, and 21%, respectively) than in the sequentialarm (26%, 19%, and 14%, respectively). Esophageal toxicity wassignificantly more frequent in the concurrent arm than in thesequential arm (32% v 3%). CONCLUSION: Although not statistically significant, clinically importantdifferences in the median, 2-, 3-, and 4-year survival rateswere observed, with a trend in favor of concurrent chemoradiationtherapy, suggesting that is the optimal strategy for patientswith locally advanced NSCLC.

29. Chemoradiation in the management of esophageal cancer. • Kleinberg L, Forastiere AA. J Clin Oncol. 2007 Sep 10;25(26):4110-7 • The combination of chemotherapy, fluorouracil and cisplatin, and radiation has improved outcome for patients with esophageal cancer. A randomized controlled trial confirmed a long-term survival benefit when this chemotherapy was added to radiotherapy for squamous cell carcinoma, but the approach has not been definitively assessed in patients with adenocarcinoma. Preoperative chemoradiotherapy has been tested in numerous phase II studies and underpowered or flawed phase III studies. Nevertheless, collectively, the evidence strongly suggests that preoperative chemoradiotherapy improves outcome, and thus, this strategy has become a standard treatment option. Attempts to improve outcome by intensifying conventional cytotoxic drugs or increasing the radiation dose have not been successful. Camptothecin and taxane-based regimens combined with radiation have altered the toxicity profile, but substantial improvement in survival outcomes has yet to be demonstrated. Future improvements will likely require the incorporation of targeted agents that add minimally to existing toxicity, the use of molecular predictors of response to individualize selection of the chemotherapeutic regimen, and early identification of responders such that therapy might be altered dynamically.

30. Phase III Study of Concurrent Chemoradiotherapy Versus Radiotherapy Alone for Advanced Nasopharyngeal Carcinoma: Positive Effect on Overall and Progression-Free Survival • Lin et al. Journal of Clinical Oncology 21: 631-637, 2003 • Two cycles of concurrent chemotherapywith cisplatin 20 mg/m2/dy plus fluorouracil 400 mg/m2/d by 96-hourcontinuous infusion during the weeks 1 and 5 of RT. Median follow-up of 65 months, 26.2% (37of 141) and 46.2% (66 of 143) of patients developed tumor relapsein the CCRT and RT-alone groups, respectively. The 5-year overallsurvival rates were 72.3% for the CCRT arm and 54.2% for theRT-only arm (P = .0022). The 5-year progression-free survivalrates were 71.6% for the CCRT group compared with 53.0% forthe RT-only group (P = .0012). Although significantly more toxicitywas noted in the CCRT arm, including leukopenia and emesis,compliance with the combined treatment was good. The secondcycle of concurrent chemotherapy was refused by nine patientsand was delayed for 1 week for another nine patients.

31. ASTRO 2007: Temozolomide (Temodar) Offers Long-Term Survival for Glioblastoma • Mirimanoff • 10.9% at two years for patients getting radiation alone, compared with 27.2% for those getting radiation and the medication. At three years, the corresponding rates were 4.4% and 16.4%. At four years, the rates were 3% and 12.1%.The differences were significant at P<0.0001. • Patients (48% of total) with a methylated methylguanine methyl transferase (MGMT) promoter, …. had a four-year survival of 22.1% if they had the combination therapy, compared to 5.2% for radiation alone. The difference was significant at P=0.04.

32. Hedgehog signal activation in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy. • Br J Cancer. 2008 May 20;98(10):1670-4. Epub 2008 May 13. • Yoshikawa R, Nakano Y, Tao L, Koishi K, Matsumoto T, Sasako M, Tsujimura T, Hashimoto-Tamaoki T, Fujiwara Y. • The zinc finger protein glioma-associated oncogene homologue 1 (Gli-1) is a critical component of the Hedgehog (Hh) signalling pathway, which is essential for morphogenesis and stem-cell renewal, and is dysregulated in many cancer types. As data were not available on the role of Gli-1 expression in oesophageal cancer progression, we analysed whether it could be used to predict disease progression and prognosis in oesophageal cancer patients undergoing neoadjuvant chemoradiotherapy (CRT). Among 69 patients with histologically confirmed oesophageal squamous cell carcinomas (ESCCs), 25 showed a pathological complete response after preoperative CRT. Overall survival (OS) was significantly associated with lymph-node metastasis, distant metastasis, and CRT, and was further correlated with the absence of both Gli-1 nuclear expression and residual tumour. All patients with Gli-1 nuclear expression (10.1%) had distant or lymph-node metastasis, and six out of seven died within 13 months. Furthermore, patients with Gli-1 nuclear-positive cancers showed significantly poorer prognoses than those without (disease-free survival: mean DFS time 250 vs 1738 months, 2-year DFS 0 vs 54.9%, P=0.009; OS: mean OS time 386 vs 1742 months, 2-year OS 16.7 vs 54.9%, P=0.001). Our study provides the first evidence that Gli-1 nuclear expression is a strong and independent predictor of early relapse and poor prognosis in ESCC after CRT. These findings suggest that Hh signal activation might promote cancer regrowth and progression after CRT.

33. Radiation Sensitizers • Halogenated pyrimidines • 5-iododeoxyuridine (IudR), 5-bromo-deoxyuridine (BrdU) • Activity is dependent on amount of incorporation into DNA • Blocks DNA repair and sensitize to RT • Limited clinical usefulness due to toxicity

34. Exploiting Low Tumor Oxygenation with Hypoxic Cytotoxins

35. Hypoxic Cytotoxin Radiation / Chem. Drug . Surviving Fraction Combined Distance from Capillary (µm) 0 50 100 Hypoxia Causes Resistance to Radiation and Anticancer Drugs Capillary O2 O2 O2 Hypoxic cytotoxins should have an at least additive effect with RT 150

36. Radiosensitization by Targeting Hypoxia • Anemia has a -ve effect on RT outcome • Blood transfusions • EPO potentiates tumor growth!!!! • Hyperbaric oxygen • Pure oxygen at 3 atmospheres • Small patient numbers, unconventional fx • Perfluorocarbon emulsions •  oxygen carrying capacity of blood • Efaproxiral: synthetic modifier of hemaglobin • ARCON • AR = accelerated radiation for proliferation; CO = carbogen (95% O2; 5% CO2) for chronic hypoxia; N = nicotinamide (vitamin B3 analogue) for acute hypoxia

37. Randomized HBO Studies Medical Research Council

38. Radiosensitizers • Radiosensitizers such as nitroimidazoles can “mimic” oxygen and fix damage • Associated with some toxicity and there were only rarely efforts to determine if the tumors were hypoxic in advance of treatment • However there have been positive trials…… • DAHANCA 5 trial using nimorazole in treatment of advanced squamous cell carcinoma of the head and neck

39. Radiation Sensitizers • Hypoxia meta-analysis (Overgaard) • 10,602 patients, 82 trials, HBO vs sensitizers vs carbogen vs blood transfusions • Greatest benefit in head & neck (largest group?) • Hypoxia problem  in squamous cell carcinomas, in adenocarcinomas • Improvement in local control = 5%; survival = 3%;  complication rate = 0.6% (NS) Selection for patients with hypoxic tumors would help!!!!

40. And a meta-analysis by Jens Overgaard has shown significantly improved survival and loco-regional control Journal of Clinical Oncology, 25: pp. 4066-4074, 2007

41. Radiation Protectors • Produce vasoconstriction or alter metabolism causing reduction in oxygen concentration in tissue/organ • sodium cyanide, carbon monoxide, epinephrine, histamine, serotonin • Scavenge free radicals • sulfhydryl compounds • dimethyl sulfoxide (DMSO), superoxide dismutase enzymes (SODs) • Hydrogen atom donation to facilitate direct repair to a radical site on DNA • glutathione, cysteine, WR compounds

42. Radiation Protectors • WR compounds • WR-638 (cystaphos) • Oral tablets carried by Soviet troops • Requires intravenous or intraperitoneal administration • WR-1607 • Effective radioprotector: dose of 10 mg/kg • Cardiotoxicity • Marketed as d-CON (rat poison) • WR-2721 (amifostine) • Phosphorothioate -- prodrug • Dephosphorylation (alkaline phosphatase) WR-1065 • Differential protection in normal tissues (bone marrow, gut, salivary glands > lungs > brain) • For complete benefit, need to increase radiation dose? • Clinical trials: some benefit -- RTOG phase III for xerostomia • Toxicity still an issue, as is fear of protecting tumor NH2CH2CH2SPO3HNa CH3(CH2)9NHCH2SSO3H

43. Radioprotectors • Thiols can protect either by • scavenging free radicals RSH + .OH RS . H2O • hydrogen atom donation to radicals in target molecules (chemical repair) X . + RSH XH + RS .RS. Is recycled by the glutathione redox cycle

44. Radioprotectors Radioprotectors such as WR-2721 (Ethyol:Amifostine), which contain thiol/sulfhydryl/SH groups work in experimental systems, but clinically are associated with side-effects if given systemically. May be useful if given locally or to prevent second cancers, which seems to need a lower dose than for radioprotection CHO cells Cysteamine 10mmol WR-151326 DTT 25mmol control

45. Questions:Interaction of Radiotherapy with other Agents

46. 151. Temozolomide (Temodar) is an • Alkylating agent • Platinating agent • Antimetabolite • Topoisomerase inhibitor • Anti-microtubular agent • #1 – It is most effective if the O-6-methylguanine transferase (MGMT) DNA repair enzyme is silenced by methylation.

47. 152. 5-Fu is an • Alkylating agent • Platinating agent • Antimetabolite • Topoisomerase inhibitor • Anti-microtubular agent • #3 – 5-fluorouracil is a pyrimidine analog

48. 153. Which of the following has its action adversely affected by hypoxia • Bleomycin • Procarbazine • Dactinomycin • Doxorubicin • #4 – hypoxia causes resistance to doxorubicin but together they can result in severe cardiotoxicity

49. 154. Which of the following is true for the multiple drug resistance protein mdr1 • It blocks drug influx into tumor cells • It is expressed only in tumor cells • It increases drug eflux from a cell • It increases drug half-life • #3 – it is the prototypical ATP-binding cassette (ABC) transporter that pump transport of various substances out of cells, including many chemotherapeutic agents

50. 155. The dose enhancement ratio is • The dose of drug that is needed to enhance the effect of RT • The dose of radiation that is needed with the drug to that without the drug for a given isoeffect • The dose of radiation alone to the dose of radiation with drug that is needed for a given isoeffect • The tumor control probability with drug plus radiation divided by that for radiation alone • #3 – with radiation we work with isoeffective levels where possible