Alkylating Agents

1. Alkylating Agents Largest class of antineoplastics General Properties/Mechanism: • All are electrophilic molecules that covalently modify nucleophilic molecules in cells • DNA most important adduct (N7 and O6 of Guanine) for anticancer properties General Types of Alkylating Agents: • Monofunctional • Cause single strand DNA breaks • Bifunctional • Inhibit DNA replication and transcription by crosslinking DNA Subtypes: • Nitrogen mustards • Nitrosoureas • Platinum Compounds

2. Nitrogen Mustards Compound • Mechlorethamine (Mustargen) • Hodgekin’s/other lymphomas • and chronic leukemias. • MOPP regimen MOPP (Mechlorethamine, Vincristine (Oncovin), Procarbazine, Prednisone) • Chlorambucil (Leukeran) • Chronic lymphocytic leukemia, • malignant lymphomas • Reactivity: >Alkeran, <Mustargen • Less alopecia and nausea • Cumulative bone marrow toxicity

3. Mechlorethamine (Mustargan) • Mustargan serves as a prototype for the mustards (and other alkylating agents) because of its spectrum of activity, its toxicities and its mechanism of action. It is highly unstable and reactive – still in use in combination therapy, MOPP, 80% response, >50% cure • Toxicity: Nausea and vomiting (N&V). Dose limiting toxicity (DLT) is bone marrow suppression, white blood cells, esp. granulocytes. Max suppression at 10-12 days, recovery around 42 days. • ADME: t1/2 less than 30 min., admin in free flowing catheter • Analogs: Chlorambucil (Leukeran), Chronic Lymphocytic Leukemia; Melphalan (Alkeran), Multiple Myeloma (longer half-lives, p.o.); Busulfan (Busulfex), CML

4. Most Common Nitrogen Mustards • Cyclophosphamide (Cytoxan) • Malingnant lymphomas, various carcinomas. • Activated by P450 enzymes • Most versatile nitrogen mustard • Better bone marrow recovery than Mustargen Ifosphamide (Isophosphamide, Ifex) Lung, breast, ovarian, lymphomas

5. Cyclophosphamide (Cytoxan) • Use: Most widely used alkylating agent. Non-Hodgkin's lymphomas (CHOP), Breast (CMF increases 7 yr survival form 31% to 49 %) • Toxicity: N&V, anaphalactoid rxns. DLT, bone marrow suppression, max 10-12 days, recovery by 21 days (relatively platelet sparing); alopecia, hemorrhagic cystitis (minimized by diuresis), etc. • ADME: May be administered parenterally, in a large range of doses. Resistant cells may have aldehyde oxidase (like the liver) • Analog: Isofosfamide (Ifex)

6. Nitrosoureas Compound Chloroethylcyclohexyl nitrosourea (Lomustine, CCNU) Bischloronitrosourea (Carmustine, BCNU) • BCNU and CCNUs:Brain tumors, Hodgkin’s disease, melanoma. • Lipid soluble: (CNS active) • O6 of Guanine preferred alkylation site • Prolonged cummulative myelosuppression • Teratogenic

7. BCNU (Carmustin) • Use: Primary glioblastoma (with surgery and radiation can increase live span from 20 weeks to 50 weeks) • Toxicity: N&V. DLT bone marrow suppression, max 28 days; recovery by 42 days. • ADME: Administered by injection. Unique among antineoplastics for its high oil-to-water partition coefficient (very lipophillic) and low ionization at physiological pH – risk of hematological toxicity persist due to storage in the liver & adipose tissue • Analog: CCNU (lomustine), may be administered orally. Temozolomide (Temodar)is a new non-nitrosourea used frequently in glioblastoma

8. Platinum compounds • Cisplatin (Platinol) • testicular, ovarian, head and neck, lung, • bladder cancer • Curative in combination for metastatic testicular cancer • Carboplatin (Paraplatin) • ovarian, non-small cell and small cell lung cancers • less toxic (~45×) than cisplatin but is generally less active • Oxaliplatin (Eloxatin) • broad anticancer activity: colorectal, ovarian, pancreatic, non-Hodgkins lym, breast, lung, prostate, etc • Lacks cross-resistance with other platinum compounds • Generally less toxic than other platinum compounds

9. Cisplatin (Platinol) Use: Most used in combination therapy - testicular tumors (PEB ~ 85% curative) Toxicity: DLT – nephrotoxicity, can be treated with Amifostine (a thiophosphate) Major N&V, hearing loss at high end. ADME : Administered i.v. after 1-2 liters saline and mannitol (chloride diuresis)  Analog: Carboplatin (Paraplatin), Oxaliplatin (Eloxatin) is now a first line drug for combination therapy of colon cancer

10. Other Alkylators • Streptozocin (Zanosar), a nitrosourea • Use: Malignant pancreatic insulinoma, pancreatic carcinoid, doubles 1 yr survival rates (Nausea and vomiting notable, renal toxicity in 2/3 cases – bone marrow suppression in ~ 20%) • Procarbazine (Mutalane) • Use: Hodgkin’s Disease (MOPP protocol) • Few reports of cross resistance, minor toxicities (bone marrow suppression), psychic disturbances, nausea, vomiting, Weak MAO inhibitor - available orally • Dacarbazine (DTIC) • Temozolomide & DTIC are metabolized to same active compound: 5-amino-imidizole-4 carboxamide - thought to be the active alkylating species • DTIC Use: Melanoma, Hodgkin’s disease (ABVD), dose i.v. • Temozolomide Use: malignant gliomas

11. Alkylating Agents - Summary • Largest class of antineoplastic drugs • Nitrogen Mustards • Nitrosoureas • Platinum Compounds • All are electrophilic compounds that react with DNA • Bifunctional: Nitrogen mustards & platinum compounds • Monofuctional: Nitrosoureas • Toxicities vary depending on the particular compound • Many have severe bone marrow suppression • Platinum compounds tend to show more renal toxicities

12. Metabolites and their Antimetabolites General Properties/Mechanism: • Antimetabolites resemble cellular metabolites and act to interfere with DNA synthesis or the synthesis of DNA precursors. • These are classical cell cycle specific anti-cancer agents • Most are prodrugs that must be activated through incorporation into the normal biosynthetic pathways • Toxicities: only partially selective to tumor cell – toxic to all rapidly dividing normal cells • Bone marrow and intestinal epithelium are particularly sensitive • General Drug classes: • Antifolates • Antinucleotide analogs

13. Folate Antimetabolites • Methotrexate, Amethopterin • Trophoblastic choriocarcinoma, lymphoblastic leukemia, Burkitt’s • lymphoma • Class: Folate antagonist • Mechanism: Dihydrofolate Reductase inhibitor: inhibits dTMP synthesis. Kills in S-phase (G1 and S RNA and protein synthesis) • Toxicity: bone marrow, GI, renal, alopecia, teratogen • Resistance: increased/altered DHFR, decreased uptake

14. Nucleotide Analogs • 5-Fluorouracil; 5-FU (Efudex) • Colorectal, breast, gastric, pancreatic colon cancers (35% decrease in recurrence). Used topically for premalignant skin lesions. • Class: Pyrimidine analog • Mechanism: irreversible inhibition of thymidylate synthase (TS). Incorporation into DNA/RNA. Kills in G1 and S-phases. Penetrates CNS. • Toxicity: N&V, GI toxicity. DLT bone marrow suppression • Resistance: increased/altered TS • F-UMP: incorporated into RNA affecting processing/function • F-dUMP: Inhibits Thymidylate Synthase • ADME: Administered i.v., half-life in the blood of ~10-20 min • Analog: Floxuridine (FUDR), often infused into the hepatic artery for liver cancer

15. Modulation of 5-FU & MTX with Leucovorin Rescue • Leucovorin (reduced form of folic acid) is commonly used to rescue cells exposed to folate antagonists (i.e., methotrexate) - does not function as a cytotoxic chemotherapy agent • High-dose methotrexate with leucovorin rescue (HDM-L) is a component of the standard regimen for adjuvant therapy of osteosarcoma, produces a high complete response rate in CNS lymphomas, and is a part of standard curative therapy for childhood ALL • Leucovorin promotes 5-FU inhibition of Thymidylate Synthase – used in colon cancer

16. More nucleotide analogs… • Cytosine arabinoside; araC; Cytarabine(Cytosar) • Acute myelogenous leukemia • Class: Nucleoside sugar analog • Mechanism: activated to ara-CTP (DNA polymerase inhibitor) by dCMP kinase. Kills in S-phase • Toxicity: severe bone marrow hypoplasia, GI • Resistance: polymerase alteration, decreased activation, increased deactivation (dCMP deaminase) • Seen as a 2'-deoxynucleoside • Phosphorylated and competes with dCTP for incorporation into DNA • 2'-OH in  position: Steric hindrance with base affects base stacking interactions in DNA helix • 1 AraC per 2000 bases reduces growth rate by 50%

17. More nucleotide analogs… • 6-Mercaptopurine; 6-MP (Purinethol) • Acute leukemias (40% remission in children) • Class: Purine analog • Mechanism: HPRTase: Hypoxanthine-guanine phosphoribosyltransferase makes the nucleoside phosphate from the free base - inhibition of purine synthesis; incorporation into RNA/DNA unclear. Kills in S-phase. • Toxicity: bone marrow, nausea, vomiting • Resistance: decreased HPRTase activity (needed to incorporate 6-MP into RNA/DNA) 6-Thioguanine (Thioguanine) Same as 6-MP Used against granulocytic Leukemia with cytarabine

18. Antimetabolites - Summary • Classical cell-cycle dependent agents • Require bioactivation • Classes • Antifolates • Antinucleotide analogs • Inhibit enzymes required for DNA synthesis • Toxicities extend to tissues with higher rates of cellular turnover

19. Antibiotics General classes of carcinolytic antibiotics: • Cyclic pentapeptides • Anthracyclines • Complex glycopeptides General Properties/Mechanism: • All bind to DNA or DNA associated proteins. • All inhibit RNA synthesis (transcription) • Most are Isolated from Steptomyces broths • Some induce DNA strand breaks by free radical production and/or inhibition of Topoisomerase II (Topo-II) Pure Topoisomerase Inhibitors: • Two Classes: Topo I and Topo II • Inhibit topoisomerase enzymes resulting in DNA breaks

20. Actinomycin D (Cosmogen) • Wilm’s tumor (kidney, in children), Ewing’s and Kaposi’s sarcomas, MTX-resistant choriocarcinoma. • Class: Cyclic pentapeptide • Mechanism: DNA intercalation; inhibits transcription. Kills in the G1 and S phases. • Toxicity: Myelosuppression, GI, skin. • RNA polymerase much more sensitive than DNA polymerase. • Causes single-strand DNA breaks. • Free radical formation • Topo II inhibition One of the most potent anticancer drugs per mole

21. Anthracyclines • Doxorubicin (Adriamycin, ADM) • Breast, head, neck, thyroid, bladder, testes, prostate, ovary, Wilm’s and Ewing’s sarcomas, leukemias and lymphomas • Class: Anthracycline • Mechanism: DNA intercalator, DNA breaks by free radical damage and Topo-II interference. Kills in S phase. • Toxicity: DLT - cardiotoxicity (total dose, often irreversible), myelosuppression • Resistance: decreased/altered Topo-II, P-glycoprotein activity (decreased accumulation) Doxorubicin-polymer conjugates have reduces toxicity, increases targeting

22. Doxorubicin (Adriamycin) Mech: Intercalation and blockage of topoisomerase II, oxygen radical production because of quinone structure. One of the most useful cancer drugs – new formulations extending uses. Toxicity: Myelosuppression max in week 2; recovery in week 4. Radiation recall. Toxicity limiting total dose is a cardiomyopathy, a unique toxicity in 1/3 of patients if the total dose exceeds 550 mg/m2. Dexrazoxane (Zinecard) an antioxidant metal chelator can reduce anthracycline cardiotoxicity. ADME: Administered i.v., cleared by the liver. Analogs: Daunomycin (Daunorubicin), doxorubicin, about ½ as potent, Mitoxantrone (Novantrone), a synthetic doxorubicin with reduced cardiotoxicity; Idarubacin (Idamycin).

23. Bleomycin A2 and B2 Complex Glycopeptides • Bleomycin (Blenoxane, BLM; mixture of A2 and B2) • Head, neck, testes (Curative with vinblastine and cisplatin), uterine, cervix, Hodgkin’s and non-Hodgkin’s lymphomas. • Class: Complex glycopeptide • Mechanism: DNA binding region. DNA breaks by free radical damage. Kills in G2 phase. • Toxicity: Cardiotoxicity, edema, lung and skin fibrosis - Minimal myelo- or immunosuppression • Resistance: Increased DNA repair

24. Bleomycin (Blenoxane) • Mech.: Has DNA binding region & metal binding region (Cu2+ or Fe2+)Transfers e- from M2+ to O2 to form activated oxygen species. Causes double strand breaks in DNA - Cytotoxicity correlates with DNA fragmentation • Use: Testicular tumors - curative in combination with cisplatin and etoposide (PEB) • Toxicity: N&V, skin toxicity. DLT - pulmonary fibrosis at total dose of 400 U. • ADME: Bleomycin is excreted primarily by the kidneys Pulmonary toxicity is probably enhanced when dosage is not adjusted for renal dysfunction. • Resistance: Bleomycin appears to undergo extensive metabolism by a specific cysteine proteinase, bleomycin hydrolase – low levels in the lungs and skin.

25. Pure Topo-II Inhibitor • Etoposide (VePesid, VP-16) • Testicular, small cell lung • Mechanism: Stabilizes Topo-II-DNA complex. Causes double strand break. Kills in G2 and S phases. • Toxicity: DLT - bone marrow, nausea, diarrhea (GI), UNCERTAIN effect on secondary neoplasia • ADME: Administered p.o. and i.v, largely cleared by kidney • Resistance: P-glycoprotein activity (decreased accumulation) Source: mandrake plant (podophyllum peltatum) • Podophyllotoxin binds microtubules and inhibits assembly. • Etoposide does not inhibit assembly of microtubules

26. CPT 11, Irinotecan (Camptosar) Mech: “Pure” Topoisomerase I poison - source: derivatives of camptothecin (CPT), a cytotoxic plant alkaloid isolated from the Chinese tree Camptotheca acuminata Use: Colorectal cancer, and small cell and non-small cell lung cancer - has also shown positive responses in pancreatic cancer, refractory lymphoma, malignant gliomas, and gynecologic cancers Toxicity: GI toxicity, severe diarrhea. DLT - bone marrow suppression ADME: Administered i.v., cleared by kidney Analogs: topotecan (Hycamtin) Pure Topo-I Inhibitor

27. Taxanes • Paclitaxel (Taxol) • Breast, ovary, lung, head and neck • Mechanism: Stabilizes microtubule assembly “mitotic spindle poison” G2/M arrest. • Toxicity: DLT - Myelosuppression, peripheral neuropathy, allergic reactions to injection are a problem • ADME: Administered i.v., cleared by the liver, susceptible to MDR • Resistance: Altered tubulin, P-glycoprotein activity (decreased accumulation) • Analog: docetaxel – semisynthetic, similar to paclitaxel • Taxol source: Taxus brevifolia • Limited supply • Now made semisynthetically • From needles of the Himalayan Yew (Taxus bacatta)

28. Vinca alkaloids • Vincristine (Oncovin) • Hodgkin’s disease, childhood leukemia • Mechanism: Destabilizes microtubule assembly “mitotic spindle poison” G2/M arrest. • Toxicity: DLT - peripheral neuropathy • Resistance: Altered tubulin, P-glycoprotein activity (decreased accumulation) • Vinblastin (Velban) • Testicular (cisplatin, bleomycin) • Mechanism: Destabilizes microtubule assembly “mitotic spindle poison” G2/M arrest. • Toxicity: DLT – bone marrow suppression • Resistance: Altered tubulin, P-glycoprotein activity (decreased accumulation) Source: periwinkle plant (Vinca rosea) Despite similar structures, incomplete cross-resistance Administered i.v., susceptible to MDR, cleared largely by the liver

29. Misc. agents • Hydroxyurea • Use: AML or the blastic phase of CML • Because of its radiosensitizing effects, it is used with radiation therapy to treat head and neck carcinoma • Mechanism: Hydroxyurea blocks ribonucleotide reductase, the rate-limiting enzyme of DNA synthesis. • Hydroxyurea inhibits ribonucleotide reductase by binding to the M2 subunit and disrupting the iron complex. • This enzyme is responsible for converting ribonucleotides to deoxyribonucleotide triphosphates (dNTPs), which are critical to DNA synthesis and repair. • Toxicity: Hematopoietic depression is the major toxic effect; UNCERTAIN risk of secondary leukemia; teratogen • Resistance: alterations in sensitivity of ribonucleotide reductase

30. Misc. Agents cont. – Hormonal • Tamoxifen (Nolvadex) • Estrogen Receptor (ER) positive breast cancer • Class: Antiestrogen SERMS (synthetic estrogen receptor modulators) • Mechanism: Block estrogen response by competitive inhibition of ER. Inhibits G1 to S transition. • Toxicity: rarely severe adverse reactions to tamoxifen include vasomotor symptoms (hot flushes), N&V & vaginal bleeding . Tamoxifen also increases the incidence of endometrial cancer by two- to threefold • Anastrozole (Arimidex) • Fourth generation of aromatase inhibitor - effective against ER positive breast cancer • Class: Aromatase inhibitor • Mechanism: Anastrozole significantly suppresses serum estradiol levels Anastrozole inhibits aromatase (CYP19), the enzyme that catalyzes the final step in estrogen production. • Toxicity: rarely severe adverse reactions – some musculoskeletal toxicity

31. Other Hormone/hormone-related • Prednisone • Hodgkin's disease, acute lymphocytic leukemia (ALL), lymphomas • Class: Corticosteroid • Mechanism: Antianabolic effects • Toxicity: Cushingoid symptoms • Diethylstibestrol, DES • Prostate cancer • Class: Estrogenic (non-steroid) • Mechanism: Block androgen dependence • Toxicity: developmental mutagen/teratogen

32. Leuprolide (Lupron), GnRH peptide, used with Flutamide Mech.: Acts on pituitary to inhibit FSH and LH release Constant GnRH Causes Testosterone or Estrogen Levels to Fall to Castration Levels Use : Prostate cancer, and breast cancer prevention Toxicity: Less than DES, N&V, hot flashes Analog: Goserelin (Zoladex) Hormones-related therapy cont.

33. Antiandrogens • Flutamide (Eulixen) • Oral nonsteroidal antiandrogen - metastatic prostatic carcinoma • Mechanism: Inhibits the uptake and/or nuclear binding of testosterone and dihydrotestosterone by prostatic tissue - most effective when combined with LHRH agonists • Toxicity: rarely severe adverse • Bicalutamide (Casodex) • oral nonsteroidal antiandrogen structurally related to flutamide but has a long plasma half-life (once daily dosing) • Mechanism: Inhibits the uptake and/or nuclear binding of testosterone and dihydrotestosterone by prostatic tissue - most effective when combined with LHRH agonists - bicalutamide is more selective for the peripheral androgen receptor and has less activity at the central androgen receptor • Toxicity: rarely severe adverse

34. Hormone-related - Summary • Breast and Prostate cancers frequently demonstrate hormone-dependent growth (at least in early stage disease) • Classes: • GnRH agonists • Antiestrogens (includine for prevention) • Aromatase inhibitors • Antiandrogens • Corticosteroids • Toxicities tend to be more mild

35. Newer Molecularly Targeted Agents (1) • Tyrosine Kinase Inhibitors • imatinib (Gleevec) – Bcr-Abl/c-kit kinase inhibitor (CML, GIST) • Toxicity: generally mild – N&V, edema & muscle cramps, Neutropenia and thrombocytopenia • gefitinib (Iressa) - EGFR tyrosine kinase inhibitor (non-small cell lung cancer) Antibodies • Trastuzumab (Herceptin) – anti-HER2/neu monoclonal (breast cancer) • Bevacizumab (Avastin) - anti-VEGF monoclonal (colon cancer) • Cetuximab (Erbitux) - anti-EGFR monoclonal antibody (colon cancer) • Others – including antibody conjugates to drug/toxin

36. Newer Molecularly Targeted Agents (2) • Differentiation Inducers • Retinoic Acid derivatives • Tretinoin, ATRA (Avita) • Uses: acute promyelocytic leukemia (APL) and undergoing phase III investigation in the treatment of Kaposi's sarcoma. Frequently given in combination with an anthracycline • Toxicities: Generally mild - cause dry skin, cheilitis, reversible hepatic enzyme abnormalities, bone tenderness, and hyperlipidemia • Others - proteasome inhibitor • Bortezomib (Velcade) – • Uses: Multiple Myeloma, in trials for other leukemias • dose-limiting toxicities in the following organ systems: gastrointestinal, hematopoietic, lymphatic, and renal

37. Important Cytokines and Biological Response Modifiers • Biologic Agents Used to bolster immune response: • IL-2 (interleukin-2) T cell stimulation, highly toxic, used in metastatic melanoma • Interferons (Inf 2a,b) used in hairy cell leukemia • Filgrastim (Neupogen) G-CSF • Sargramostim (Leukine) GM-CSF • BCG (Bacillus Calmette-Guerin Live- Mycobacterium bovis ) used in bladder cancer

38. Combination Chemotherapy Considerations • Most cancers are/become refractory to treatment by a single agent, combinations of anticancer drugs are often used. The following rules apply to combining drugs in cancer chemotherapy: • The drugs each must have some activity against the cancer. • The drugs should act via different mechanisms. • The drugs should have minimal overlapping toxicity. • Cellular resistance to each drug should occur by different mechanisms. •  The first drug combination, made famous by Dr. V. DeVita and others at NIH, is called MOPP (Mechlorethamine, oncovonin, procarbazine, and prednisone). MOPP is a curative treatment for Hodgkin's disease and its development was a major step forward.