Anticancer Drugs PDF

Anticancer drugs CHMI3427EL Winter 2024 Dr. S. Montaut 1 Cancer Healthy cells are under strict biochemical control for growth and differentiation. Cells divide and proliferate under the influence of various growth stimulators and are subject to arrested growth (senescence) and programmed cell death...

Anticancer drugs CHMI3427EL Winter 2024 Dr. S. Montaut 1 Cancer Healthy cells are under strict biochemical control for growth and differentiation. Cells divide and proliferate under the influence of various growth stimulators and are subject to arrested growth (senescence) and programmed cell death (apoptosis). In cancer, these regulatory processes have gone awry, and cells grow and divide uncontrollably, consuming energy and losing both structure and function due to an inability to adequately differentiate. Rampant cell division is accompanied by disabled cell death processes, leading first to cellular immortality and, eventually, to genetic instability. 2 Cancers can usually be classified as lymphatic, epithelial, nerve, or connective tissue–related, and tumor nomenclature is based on tissue of origin as follows: - carcinoma (epithelial origin), - sarcoma (muscle or connective tissue origin), - leukemia and lymphoma (lymphatic or hematologic origin), and - glioma (neural origin). The risk of receiving a cancer diagnosis commonly increases with age. The development of cancer occurs in four discrete steps or phases: initiation, promotion, transformation, and progression. 3 Exposure to a carcinogen initiates the neoplastic process (irreversible), which is promoted by environmental factors that favor proliferation of precancerous cells (reversible). It is at this promotion phase of carcinogenesis when healthy lifestyle modifications (including diet, exercise, vaccination, and smoking cessation) can have a positive impact on outcome. If protooncogenes transform over time (commonly 5-20 year) in a sequential multistep process to become oncogenes, cancer results. Oncogenes (e.g., myc and ras) can either overexpress or underexpress regulatory biochemicals, resulting in preferential and accelerated cellular growth. Concomitantly, tumor suppressor genes (e.g., antioncogenes like TP53 and Rb1) and DNA repair genes (e.g., PARP1) can be inhibited. 4 The cancer progresses as genetically fueled cellular proliferation leads to augmented invasion of the primary site and migration to new tissues through the metastatic highways of the bloodstream or lymphatic system. Many cancers have the ability to generate new blood vessels (angiogenesis) to ensure an ongoing supply of essential nutrients, and to provide an “escape hatch” for meandering cells to travel to distant sites and establish metastatic tumors. Vascular endothelial growth factor (VEGF) and plateletderived growth factor (PDGF) promote angiogenesis and serve as the molecular targets of some of the newer protein kinase inhibitor antineoplastic agents. There are also many opportunities within the metastatic cascade for the body to mount a successful immunological defense and destroy the would-be invaders. 5 6 7 In the tumor-node-metastasis (TNM) cancer staging classification system, the severity of solid tumor neoplastic growth is characterized by the size of the tumor mass (T1 to T4), the extent of lymph node involvement (N0 to N3), and whether distant metastasis has occurred (M0 or M1). The larger the subscripted number in each of these parameters, the more advanced and/or disseminated the disease. The TNM characteristics of a tumor can be translated into a comprehensive staging scale ranging from I (localized) to IV (metastatic). The intermediate disease severity stages indicate local (stage II) or regional (stage III) tissue invasion. 8 For cancers deemed “curable” (which excludes breast cancer and melanoma), cure equates to no evidence of disease for a minimum of 5 years. More commonly, the response category viewed as the pinnacle is complete response, in which the patient has no evidence of cancer, but where relapses are still possible. A partial response is claimed when tumor size has been reduced by 30% or more from baseline and there is no evidence of new lesions at the primary site or elsewhere. 9 The term clinical benefit has been used if this level of improvement is not reached, yet the patient has experienced significant attenuation of symptoms and/or enhancement of quality of life. A less optimistic response category is stable disease, in which tumor size has increased by less than 20% or decreased by less than 30%. Most dire is progression, a category that is characterized by tumor growth at the 20% or higher level relative to the lowest lesion diameter since beginning therapy and/or the formation of new lesions during treatment. 10 Surgery, radiation, and chemotherapy represent the triad of approaches to the treatment, and ideally eradication, of a diagnosed and potentially life-threatening cancer. Commonly given in combination, the order and timing of the interventions are dictated by many factors, including specific diagnosis, disease stage, and anticipated response based on clinical trials. 11 History In the 1940s: the glucocorticoids cortisone and prednisone were shown to induce tumor regression in a murine lymphosarcoma and in acute leukemia. In the 1940s: development of the cytotoxic nitrogen mustards for the treatment of lymphoma. Chemists used their understanding of mustard reactivity to design agents where N replaced S and conjugated aromatic rings attenuated lone pair nucleophilicity. These “kinder, gentler” nitrogen mustards were orally active agents and less systemically toxic. More recently, oxazaphosphorine-based prodrug mustards, activated predominantly by CYP2B6 (cyclophosphamide) and CYP3A4 (ifosfamide), have enjoyed widespread clinical use in the treatment of hematologic cancers, and testicular cancer and pediatric solid tumors, respectively. 12 In 1940 : p-aminobenzenesulfonamide was effective against streptococcal infections ushered in the era of antimetabolite chemotherapy. The development of antifolate antineoplastics, which were shown to be effective in combating childhood leukemias, got its start in the late 1940s. In the mid to late 1950s : development of antimetabolites based on the structures of endogenous purine and pyrimidine bases. Discovery that a very simple analogue of the endogenous pyrimidine uracil (5-fluorouracil) was a potent inhibitor of deoxythymidine monophosphate biosynthesis, and that inhibiting the production of this essential nucleotide produced positive results in patients suffering from colon, stomach, pancreatic, and breast cancers. 13 Late 1950s : Antimetabolites that target DNA polymerase (e.g., gemcitabine) were first conceptualized and synthesized and have found use in a variety of solid tumor and hematological cancers. In the 1960s: The antibiotic antineoplastics came into clinical use when the highly toxic actinomycin (discovered in the 1940s) was found to be effective in the treatment of human testicular cancer and uterine choriocarcinoma. In the 1960s: bleomycin (natural anticancer antibiotic) was found to be active against various hematologic cancers and solid tumors which led in more recent times to the development of semisynthetic analogues with both high potency and wider margins of safety. 14 In the 1960s: The antimitotic vinca alkaloids vincristine and vinblastine were shown to have activity against Hodgkin disease and acute lymphoblastic leukemia around the same time that the antibiotic antineoplastics were being developed. Early 1970s: introduction of organometallic platinum complexes to the antineoplastic arsenal and generation of cisplatin, the first organoplatinum complex to be marketed. Subsequent additions to the organoplatinum complex family were designed to either attenuate use-limiting toxicity (carboplatin) and/or overcome cisplatin resistance (oxaliplatin). 15 Early 1970s: the efficacy of sex hormones and hormone antagonists in fighting hormone-dependent cancers (e.g., estrogen receptor–positive breast cancer and prostate cancer) and the advent of therapeutic biologic response modifiers with direct antiproliferative effects (e.g., interferons) have added significantly to the therapeutic options available to providers and the cancer patients for whom they care. The later 1990s: introduction of the tyrosine kinase inhibitors (TKIs). Imatinib, the first rationally designed drug in the TKI class, was made available in 2001 and dramatically changed the treatment and clinical outcome of Ph+ leukemias. 16 Many molecules derived from plant or marine animal sources have demonstrated value either as cytotoxic chemotherapeutic agents themselves, or as synthetic springboards to anticancer drugs that can be readily produced in the laboratory. Examples of anticancer drugs or precursors found in plants include paclitaxel and its precursor 10-deacetylbaccatin III, vinca alkaloids (e.g., vincristine, vinblastine), podophyllotoxin (precursor to the epipodophyllotoxins etoposide and teniposide) and camptothecin (precursor to topotecan and irinotecan). Sea sponges have added cytarabine and eribulin. Nature undoubtedly holds many yet-to-be discovered therapies that could literally serve as a patient’s lifeline. 17 Resistance to Cancer Chemotherapy Some cancer cells acquire resistance to anticancer drugs by downregulating enzymes and carriers essential for drug transport or for the activation of antineoplastic prodrugs, or by upregulating enzymes involved in inactivating biotransformation. Other mechanisms of biochemical retaliation include downregulation of target enzymes or antigens, activation of kinase-associated biochemical pathways, altered drug uptake, inhibition of cellular repair proteins, apoptosis inhibition, and upregulated drug efflux mechanisms. 18 Efflux proteins involved in cancer chemotherapy resistance include classic multidrug resistance (MDR; e.g., P-glycoprotein) and multidrug resistance–associated (MRP) proteins. Both are classified as adenosine triphosphate (ATP)–binding cassette (ABC) transporters, but MRP (but not MDR) action depends on phase 2 biotransformations such as glucuronidation, sulfonation, and glutathione conjugation. It has been estimated that up to 90% of cancer-related deaths can be linked in some way to intrinsic (phenotype-related) or acquired resistance to therapy, and the identification and development of compounds that combat acquired resistance is an active area of research. 19 Kinase Inhibitors Mechanisms of action: Kinases are phosphorylating enzymes that are highly conserved in the three-dimensional structure of the catalytic (ATP-binding) domain. They regulate cell proliferation, differentiation, metabolism, and survival, and when functioning in a deregulated manner, can accelerate cell signaling cascades and cellular growth, induce tumors, and augment antiapoptotic processes. Aberrations in protein phosphorylation by kinases, stimulated by “‘mutation hotspots,” have been called “drivers” in neoplastic disease, underscoring the potential value of kinase inhibition in cancer chemotherapy. 20 Kinase inhibitors bind to the hydrophobic hinge region (the ATPbinding domain) that connects the N-terminal and C-terminal lobes of the kinase. A minimum of five potential binding pockets surround this site, which may help explain the otherwise surprising degree of selectivity exhibited by many anticancer kinase inhibitors. 21 Most marketed inhibitors bind reversibly to their residues, and do so in one of four distinct ways : - Type I inhibitors compete with ATP and bind to the enzyme in its active form. - Type II inhibitors bind to the inactive kinase conformer, which differs from the active form in the outward versus inward orientation, respectively, of a critical Asp residue that’s part of a DFG (Asp-Phe-Gly) residue trio (motif). - Type III molecules fit into a binding pocket adjacent to the active site (allosteric binding). - Type IV inhibitors seek out an allosteric binding site that is distant from the ATP-binding site. 22 SAR (Structure-activity Relationships): Type I inhibitors will contain a heteroaromatic ring that directly competes with ATP for access to the adenine-binding domain of the hinge region. Lipophilic and hydrogen-bonding functional groups attached to the heteroaromatic system insert into allosteric and hydrophobic pockets adjacent to the adenine pocket, enhancing affinity. Many contain polar functional groups that extend into the solvent interface. 23 Type II inhibitors access the same binding areas as type I, so many of the structural elements found on type I inhibitors are retained. Since type III inhibitors bind in allosteric pockets adjacent to the adenine-binding site and do not compete directly with ATP, hydrophobic, π-stacking, and hydrogen-bonding groups will be important to affinity and potency. With ATP located “next door,” electrostatic interactions between the inhibitor and the anionic terminal phosphate of the bound nucleotide, and/or with charged or polar residues lining the catalytic site, are often possible. Since they bind a distance from the adenine pocket, no such interactions are of importance to type IV inhibitors. 24 Four electrophilic kinase inhibitors contain a Michael alkylating moiety that allows them to act irreversibly through covalent bond formation with a nucleophilic Cys797 residue that flanks the ATPbinding site. 25 Resistance: Resistance to kinase inhibitors is multifaceted, with predominant “on target” resistance pathways mediated through affinity-attenuating mutations or conformational changes in the kinase enzyme. On-target resistance can also involve enhanced kinase affinity for the endogenous ATP ligand. Alternative resistance mechanisms include disruption of destructive processes that follow kinase inhibition (“on-pathway” resistance), upregulation of parallel pathways that accomplish the biochemical goals of the inhibited kinase, epigenetic modification of the tumor cell’s phenotype, and negative influences on inhibitor pharmacokinetics or metabolic stability. 26 The ability to screen tumor cells for operational resistance mechanisms is paving the way for the design of more tenacious therapies for use in mutation-nimble neoplasms. The use of combination kinase inhibitor therapy, selected to target-specific or parallel kinase pathways or crucial tumor cell properties, is another approach to delaying or overcoming resistance. 27 Ser/Thr Kinase Inhibitors Cyclin-dependent Kinase Inhibitors: Aberrant cyclin-dependent kinase (CDK) activity, particularly CDK2, CDK4, and CDK6 is associated with carcinogenic transformations in gene transcription and cellular regulatory mechanisms. The 20 known CDKs exist in active and inactive conformations, with action demanding complexation with a cyclin subunit and subsequent ATP-catalyzed phosphorylation of (most commonly) Thr. 28 CDKs 4 and 6, which contain the regulatory cyclin D1 and three subunits, respectively, have structural variances from the 18 other enzymes, which has proven valuable in the design of targeted inhibitors for use in therapy for advanced or metastatic hormone receptor positive (HR+)/HER2 negative breast cancer (the common indication of the three marketed CDK inhibitors). CDK4/6 has been shown to be intimately involved in breast cancer initiation and invasive progression, and it induces attenuation of retinoblastoma gene (Rb) phosphorylation and cell cycle arrest at G1. Patients who respond to these drugs must be Rb+. 29 Chemistry and SAR : Like all kinases, CDK proteins have N-and C-terminal lobes connected through a hinge region that serves as the ATP-binding catalytic site. Planar (aromatic) structures that mimic adenine, along with H-bonding moieties are critical to holding inhibitors to the catalytic site. The catalytic pocket is large and contains a Phe gatekeeper residue (Phe98 for CDK6) that can be targeted for inactivating interaction in the active conformation to which type I kinase inhibitors bind. Other key residues believed involved in inhibitor binding to CDK6 include Thr107 (Thr99 or Thr102 in CDK4), Asp104 and Asn150. A cationic Lys residue found on other CDKs decreases affinity for therapeutic inhibitors and promotes selectivity for CDK4/6. 30 A crystal structure of active CDK4/6-cyclin D bound to inhibitor is yet to be solved. SAR studies on selective CDK4/6 inhibitors demonstrated the importance of incorporating a cationic moiety (e.g., piperazine) positioned to form an affinity-enhancing ion-dipole bond with the CDK4/6 Thr residue while repelling the cationic Lys reside found on other CDKs (e.g., CDK1/2). The 2-aminopyrimidine group binds to the hinge region adenine site, as it has in many other kinase inhibitors. 31 Using palbociclib (Fig. next slide) as an example, N1 of the pyridopyrimidine nucleus may promote affinity through interaction with a unique His residue in the hinge region of CDK4 (His92) and CDK6 (His100). The affinity-enhancing piperazine terminus and pyridopyrimidine N8-cyclopentyl ring were declared important to potency, while the C5-CH3 and C6-acetyl moieties promoted CDK4 selectivity. The cationic piperazine should also enhance selectivity by repelling the cationic Lys residue found in other CDKs. Resistance to CDK4/6 inhibitors has been linked to Rb deficiency and the associated high levels of tumor suppressor protein p16INK4A and the deletion or inactivation of the CDKN2A gene. 32 Serine/threonine kinase inhibitors. 33 Specific drugs: all currently marketed CDK4/6 inhibitors are used in the treatment of HR+/HER2− metastatic breast cancer (MBC), and given in combination with the aromatase inhibitor letrozole. Two agents, palbociclib and abemaciclib, have found value in combination with the estrogen receptor antagonist fluvestrant in MBC that has progressed on endocrine therapy alone. Myelosuppression (potentially leading to infection), fatigue, and gastrointestinal distress, rash, and hair loss/thinning are common adverse effects. 34 All three CKD4/6 inhibitors are vulnerable to CYP3A4-catalyzed metabolism, and ribociclib and abemaciclib have clinically relevant active metabolites (Table 33.7 next slide). Fecal elimination predominates. The CYP-mediated metabolic properties of the marketed CDKs are provided in Table 33.7 and selected pharmacokinetic and therapeutic properties appear in Table 33.8 (slide 37). 35 36 37 38 Ribociclib: In a phase III placebo-controlled clinical trial with HR+/HER2− metastatic breast cancer patients, evaluation at 1.5 years documented that ribociclib in combination with letrozole provided significant increases in progression-free survival (63% vs. 42.2%) and overall response (52.7% vs. 37.1%). There may be a synergistic action when mTOR inhibitors are added to the ribociclib/aromatase inhibitor regimen. 39 Plasma concentrations of the main CYP3A4-generated active metabolite, known as LEQ803, represent approximately 10% of the dose, so the majority of the drug’s activity is attributed to the parent structure. Coadministration of strong CYP3A4 inhibitors or inducers should be avoided if at all possible. Minor metabolites are generated through oxidation and desaturation (M9) and oxygenation (M13) and there is no evidence of phase 2 sulfoconjugation. The drug inhibits CYP3A4 and possibly 1A2. 40 Hormone-Based Antineoplastic Agents (Representative) Structures that modulate the activity of estrogen and androgen receptors or influence levels of circulating hormones that can impact steroid-dependent tumors are available as antineoplastic agents. Antiestrogens : The antiestrogens used in the treatment of breast cancer often exhibit tissue-specific action at estrogen receptors, blocking hormone binding in breast and reproductive organs while displaying agonist actions in other tissues (e.g., uterus, bone). These dual-acting agents are more accurately referred to as selective estrogen receptor modulators or SERMs. 41 The three-fold structural requirements for estrogen receptor blockade by selective estrogen receptor modulators include: 1) 3 aromatic (phenyl) rings with a strictly maintained 3dimensional structure, 2) a cis ring that bears a potentially cationic amine moiety linked through an alkylether group, and 3) a phenol-bearing trans ring, where the OH (or isostere) is either a component of the parent drug or generated via metabolism. 42 Representative hormone-based antineoplastic agents. 43 Tamoxifen Citrate : It introduced the selective estrogen receptor modulator pharmacologic concept and ushered in the era of targeted breast cancer treatment and prevention therapy. Its indications range from disease prevention in high-risk patients to early-advanced breast cancer to ductal carcinoma in situ. Antiestrogens interfere with estrogen-dependent tumor growth by competing with estrogens for the receptor site and by turning off the normal processes of the genetic information within the nucleus. Although the full mechanism of action is not completely understood, the molecular basis for tissue-selectivity is apparent from crystallographic studies of the ligand binding domain. 44 The diethylstilbestrol-estrogen receptor α ligand binding domain co-crystal structure demonstrated that diethylstilbestrol binds analogous to 17β-estradiol (i.e., the ligand is well accommodated by the estrogen receptor α which completely encloses it within a ligand binding pocket) (Fig. next slide). Steroidal agonists and synthetic agonists such as diethylstilbestrol promote the formation of, and stabilize, the agonist conformation of estrogen receptor α in which the C-terminal helix 12 (H12) folds in toward the helical barrel structure of the ligand binding domain and encloses the ligand. In the agonist conformation, the activation function-2 (AF-2) located on the exterior of estrogen receptor α is able to bind to coactivators, recruit the preinitiation complex to estrogen response element sites, and activate transcription of estrogen-driven genes. 45 Agonist and antagonist conformations of estrogen receptor α (Erα). The left panel demonstrates the agonist conformation of the ERα ligand binding domain when bound to diethylstilbestrol, whereas the right panel demonstrates the antagonist conformation when bound to 4-hydroxytamoxifen. A key difference is the orientation of helix 12 (H12; indicated by red arrows). H12 is a short segment of helix located at the C-terminus of the ligand binding domain. In the agonist conformation, H12 folds toward the ligand and completely encloses the ligand within the barrel-shaped binding pocket. Externally, the activation function-2 of the agonist conformation forms a coactivator interaction site such that ERα recruits coactivators and activates ERα-dependent transcription. In the antagonist conformation, the basic amino side chain together with the cis aryl group is not accommodated by the receptor. Instead the ligand protrudes toward the exterior of the receptor. H12 is pushed into an alternate orientation which recruits corepressors in breast and uterine tissues, but still acts as an agonist in other 46 tissues such as bone. In contrast, for the antagonist conformations, as seen with 4hydroxytamoxifen (4-OH-TAM; active metabolite of tamoxifen), the entire ligand is not accommodated within the interior of the receptor (Fig. previous slide). Instead the basic amine side chain protrudes to the exterior of the estrogen receptor α helical barrel. The selective estrogen receptor modulator pushes H12 away from the coactivator binding site, preventing coactivator binding and activation function-2 function in some tissues; resulting instead in recruitment of corepressors in these tissues (breast and uterus). 47 Tamoxifen is a prodrug that is thought to require metabolic activation by CYP2D6 to produce the 4-hydroxytamoxifen (4-OH TAM) metabolite (Fig. next slide). Tamoxifen and 4-OH TAM are also metabolized by CYP3A4 to the relatively inactive N- desmethyl tamoxifen (NDM TAM; similar activity as tamoxifen) metabolite and the active 4-hydroxy N-desmethyl tamoxifen (4OH-NDM TAM; known as endoxifen in the literature) metabolite. These metabolites bind to the estrogen receptor with up to 30-fold greater affinity than tamoxifen, and this increased potency estrogen receptor binding translates into enhanced antiestrogen activity. Coadministration with CYP2D6 inhibitors can lead to persistent reduction in the plasma concentrations of the active metabolites. Concurrent chronic use of CYP2D6 inhibitors should be avoided if possible. 48 Metabolism of tamoxifen (TAM) and toremifene (TOR); NDM, Ndesmethyl. 49 Tamoxifen retains some intrinsic agonist activity, and therapy commonly results in tamoxifen resistance and the need to switch to other endocrine therapies. The emergence of tamoxifen resistance seems to be worse in obese patients where, even if gonadal estrogen synthesis is ablated, the fat cells produce estrogens using aromatase. 50 Immunomodulators. 51 Three closely related immunomodulatory drugs are used as maintenance therapy (lenalidomide) or in active treatment of newly diagnosed (thalidomide) or relapsed/refractory (pomalidomide) multiple myeloma, the latter two indications in combination with dexamethasone. Thalidomide was used in the late 1950s and early 1960s as an antiemetic and sedative in pregnant women, more than 10,000 European children were born with flipper-like arms and legs, a condition called phocomelia or “seal limb.” Although it is known that the teratogenic and sedative actions of chiral thalidomide are mediated by different stereoisomers, the in vivo inversion of the therapeutic R-isomer to its cytotoxic S-enantiomer prohibited the use of this drug in women who were or could become pregnant. 52 Thalidomide’s antiproliferative and proapoptotic actions allowed its very cautious use in multiple myeloma. In addition to arresting uncontrolled cell growth, thalidomide and its analogues deny multiple myeloma cells access to bone marrow stromal cells and various growth factors needed for tumor cell survival and augment circulating levels of natural killer cells, interleukin-2, and interferon-γ. Lenalidomide and pomalidomide, as more potent and less toxic immunomodulatory analogues, have effectively taken thalidomide’s therapeutic place, but all of these agents are contraindicated in pregnancy. 53 Boxed warnings mandate that female patients capable of becoming pregnant document they are not pregnant for two months prior contraceptive to beginning methods or therapy, opt for and total either use abstinence two from heterosexual intercourse during, and for one month following, therapy. During and for one month following therapy, male patients must use condoms during intercourse with childbearing-capable women. These drugs are only available through the appropriate restricted distribution program. 54 Topoisomerase Poisons Topoisomerases are enzymes that control the degree of DNA supercoiling and maintain proper DNA structure during replication and transcription to RNA. Topoisomerase IIα (topIIα) cleaves double-stranded DNA during the replication phase via a transesterification reaction involving a topoisomerase Tyr residue and a terminal 5′ phosphate but, through a reverse transesterification, repairs its own damage after replication is complete. 55 Topoisomerase I (topI) functions in essentially the same way, but cuts and religates a single DNA strand. Antineoplastic agents that function as topoisomerase poisons stimulate the DNA cleavage reaction but inhibit the DNA resealing activity of the enzymes, leaving the DNA irreversibly damaged and unable to replicate. Three chemically distinct classes of anticancer agents can be classified as topoisomerase poisons: - camptothecins, - epipodophyllotoxins, and - anthracyclines and related anthracenediones. 56 Camptothecins Camptothecin topoisomerase I (TopI) poisons. 57 Mechanism of Action: The biologic target of camptothecins is topI, rather than the topIIα enzyme that serves as the target for the epipodophyllotoxins and anthracyclines. However, the mechanism of antineoplastic action of both enzyme inhibitors is stabilization of a cleavable ternary (drug-enzyme-DNA) complex that does not permit the resealing of nicked DNA. Although the fragmented DNA is capable of resealing in the absence of drug, when DNA replication forks encounter the fragmented DNA, shear stress induces a double-stranded DNA break, killing the cell. Camptothecins are most toxic to cells undergoing active DNA replication and cell division (e.g., they are S-G2 phase specific). 58 The binding of camptothecins occurs in such a way as to stabilize a covalent DNA–topoisomerase bond at the point of single-strand breakage (Tyr723 on the human enzyme) but sterically keep a topI Lys532 residue from catalyzing the DNA religation reaction. The binding pocket, located within the DNA strand, is revealed only after the normal DNA nicking has occurred, explaining why these poisons preferentially bind to the enzyme–DNA complex rather than to unoccupied DNA or enzyme. The flat camptothecin ring system intercalates DNA at the site of cleavage, mimicking a DNA base pair. 59 The crystal structures of human ternary complexes involving the parent alkaloid (the water insoluble natural product camptothecin), and the semisynthetic analogue, topotecan have been solved, and important drug-protein interacting entities are noted in Table 33.11 (next slide). The C7, C9, and/or C10 substituents of the marketed compounds, which project into the major groove of DNA, do not hinder binding, and may actually enhance it through hydrophobic and/or H-bonding interactions. Position 7 is viewed as the most ripe for activity-enhancing modifications, as the topI region accommodating substituents at this position is spacious. 60 61 Proposed camptothecin resistance mechanisms are similar to those operational in many other classes of cytotoxic antineoplastic drugs, including downregulation or mutation of the target enzyme and cellular efflux. Breast cancer resistance protein and multidrug resistance-associated proteins microtubule-associated protein-2 and microtubule-associated protein-3 (rather than P-glycoprotein) have been suggested as resistance mediators, but a recent publication claims proteasomal destruction of topI may hold the key to resistance. TopI degradation in diseased cells is distinct from enzyme processing in healthy cells, and the extent of topI- Ser10 phosphorylation in tumors appears directly related to the rate of ubiquitin tagging, proteasome destruction and camptothecin resistance. 62 Chemistry: The parent camptothecin alkaloid, isolated from the bark of Camptotheca acuminata (the Chinese xi shu or “happy tree”), has antitumor activity, but its limited water solubility necessitated delivery as the sodium salt of the significantly less active hydrolyzed lactone. Lactonization of the hydroxy acid in acidic urine is significant, and elevated levels of active intact alkaloid in the kidney accounted for the hemorrhagic cystitis induced by this compound. For these reasons, development of this natural product as a chemotherapeutic agent was abandoned in the 1970s. 63 Irinotecan Hydrochloride : In combination with 5-fluorouracil (5FU) and leucovorin, this prodrug camptothecin is considered firstline therapy in the treatment of metastatic colorectal cancer, and can be used as second-line therapy in 5-FU resistant patients. Irinotecan is slowly bioactivated in the liver through hydrolysis of the C10-carbamate ester. The metabolite, known as SN-38, is 100-1000 times more active than the parent (Fig. next slide). Organic anion transporting protein 1B1 facilitates hepatic uptake of irinotecan, and the primary activating enzyme is the saturable carboxylesterase 2 (CES2). Butyrylcholinesterase can also activate irinotecan in plasma. 64 Irinotecan metabolism. 65 Levels of SN-38 are 50-100 times lower than the parent drug, but preferential protein binding of the lactone (95%) permits significant plasma levels of SN-38 compared to the less active hydroxy acid metabolite. There is significant individual variability in kinetics and clearance for irinotecan (30%) and SN-38 (80%), and a SN-38 terminal half-life of between 6 and 32 hours has been reported (compared to 5-18 hr for the prodrug parent). CYP3A4 cleaves the terminal piperidine ring through oxidation at the α-carbons, which is followed by hydrolysis of the resultant amides to inactive metabolites. Drug-induced toxicity can be exacerbated if strong CYP3A4 inhibitors are coadministered. 66 Prior to elimination, irinotecan is glucuronidated by UGT1A1 or sulfonated at the C10 phenol. Elimination of the parent drug and all metabolites except SN-38 glucuronide is predominantly fecal (66%), and biliary excretion is facilitated by ATP-binding cassette (ABC) transporting proteins. Diarrhea is a hallmark irinotecan adverse effect, potentially exacerbated by coadministered 5-fluorouracil. 67 Topotecan : This active camptothecin analogue is used by the intravenous route in the treatment of ovarian, cervical and small cell lung cancer that has either not responded to or relapsed after first-line therapy. An oral dosage form is available for use in relapsed small cell lung cancer. Lactone hydrolysis is rapid, reversible, and pH dependent, and binding to serum proteins is limited to approximately 35%. CYP3A4-mediated N-dealkylation to mono- and di-dealkylated metabolites occurs to a limited extent, and the O-glucuronides that form at multiple points along the metabolic path are excreted via the kidney (Fig. next slide). Because both topotecan and irinotecan are metabolized by CYP3A4, coadministration of strong CYP3A4 inducers and inhibitors should be avoided. 68 Topotecan metabolism. 69 Epipodophyllotoxins Epipodophyllotoxin topoisomerase IIα (TopIIα) poisons. Teniposide hydrochloride (Vumon) 70 The epipodophyllotoxins are semisynthetic glycosidic derivatives of podophyllotoxin, the major component of the resinous podophyllin isolated from the dried roots of the American mandrake or mayapple plant (Podophyllum peltatum). Although these compounds are capable of binding to tubulin and inhibiting mitosis, their primary mechanism of antineoplastic action is topIIα poisoning. TopIIα has two distinct DNA-independent binding sites for the epipodophyllotoxins, one within the catalytic domain and a second within the N-terminal ATP-binding domain. Once bound, the toxins stabilize the cleavable ternary complex, stimulating DNA ligation but inhibiting resealing. The DNAtopoisomerase fragments accumulate in the cell, ultimately resulting in apoptosis. The RNA transcription processes are also disrupted by the interaction of epipodophyllotoxins with topIIα. 71 The epipodophyllotoxin binding site has been probed with a carbenegenerating diazirine photoaffinity label, and a virtual library of 143 epipodophyllotoxin derivatives has been docked to a threedimensional human topIIα receptor model in order to identify key drug-enzyme interactions below. Proposed etoposide-TopIIα binding interactions. 72 An X-ray crystal of two etoposide molecules bound in a ternary complex with cleaved DNA and topIIβ (an isoform found in myocardium) has been solved, documenting that the area that binds the glycoside moiety of epipodophyllotoxins is unhindered. Epipodophyllotoxins are cell cycle specific and have their most devastating impact on cells in the S or early G2 phase. Resistance involves downregulation of topIIα, attenuation of enzymatic activity levels, development of novel DNA repair mechanisms, and P-glycoprotein–mediated cellular efflux. 73 Chemistry: A water-soluble phosphate ester analogue of etoposide can be administered in standard aqueous vehicles, permitting higher doses than the oil-modified formulations would allow. The phosphate ester is rapidly cleaved to the free alcohol in the bloodstream. 74 Metabolism: Epipodophyllotoxins are subject to metabolic transformation before renal and biliary elimination (Fig. next slide). Etoposide and teniposide undergo lactone hydrolysis to generate the inactive hydroxy acid as the major metabolite, but the parent drugs can also be transformed by CYP3A4-catalyzed O- demethylation and phase 2 glucuronide or sulfate conjugation. Phase 2 metabolism accounts for between 5% and 22% of the dose. CYP3A4 inhibitors can decrease clearance, leading to unwanted toxicity. 75 Epipodophyllotoxin metabolism 76 The catechol metabolite can oxidize to a reactive orthoquinone, and both have been proposed to promote topoisomerasemediated DNA cleavage, potentially enhancing the risk of the translocations that result in therapy-induced acute myeloid leukemia in children treated with these drugs. While the orthoquinone can be detoxified with reduced glutathione, endogenous supplies of this electrophile scavenger are limited. Etoposide and etoposide phosphate ester are used intravenously in combination with an organoplatinum agent (cisplatin, carboplatin) in the treatment of small cell lung cancer, and in combination with other agents in refractory testicular cancer. 77 Anthracyclines and Anthracenediones 78 Mechanism of Action: TopIIα, the topII isoform that predominates in rapidly dividing cells, is the molecular target for anthracycline anticancer agents. They stabilize the cleavable ternary drugenzyme-DNA complex, allowing DNA to be cut and covalently linked to the conserved Tyr residue. However, by inhibiting proper alignment of the cleaved DNA segments, they inhibit the resealing reaction. The aromatic portion of the anthracyclinone and the daunosamine sugar bind to DNA, with the anthracyclinone A ring bridging the gap between DNA and enzyme. The site of DNA cleavage contains an essential thymine-adenine (T-A) dinucleotide, and a small number of anthracycline-induced DNA breaks here results in a high level of cell death. 79 Chemistry: DNA intercalation by rings B, C and D of the anthracyclinone initiates antineoplastic action. The cyclic structure orients perpendicular to the long axis of DNA and the complex is stabilized through π-stacking and other affinity-enhancing interactions. A study which docked doxorubicin in a modeled DNA “postcleavage” intercalation site proposed highly efficacious Hbonds between topIIα Ser740 and the C5 quinone oxygen (ring C), Thr744 and the C4-OCH3 (ring D), and a DNA thymine base and the C9-OH (ring A). If present, a C14-OH should H-bond with the carbonyl oxygen of a DNA thymine base (Fig. next slide). Although the C4-OCH3 helps hold drug to enzyme, its removal increases planarity, facilitates intercalation, and directs daunosamine binding to better stabilize the ternary cleavable complex, thus increasing antineoplastic potency. 80 Proposed interaction between doxorubicin, DNA, and topoisomerase IIα. 81 Daunosamine binds in the DNA minor groove at the interface with topIIα and orchestrates DNA intercalation and the overall poisoning process. The molecular modeling study suggested that the cationic daunosamine 3′-amino binds with high affinity to the carbonyl oxygen of a DNA thymine base when in the naturally occurring α configuration. In the epimerized β configuration, the distance between these two moieties increases and unfavorable steric interactions with other DNA residues occur, yet cytotoxic potency increases. Antitumor activity of anthracyclines is thought to be related more to the proper positioning and stabilization of the drug within the cleavable ternary complex than to the actual drug affinity for DNA. The daunosamine 3′-amino group is in the natural α configuration in all marketed anthracycline antineoplastics. 82 Resistance to anthracycline chemotherapy can be intrinsic or acquired. Major resistance mechanisms include: (1) compromised drug transport across cell membranes, (2) active efflux via P-glycoprotein and multidrug resistance transporters, (3) changes in tumor cell responsiveness to apoptotic triggers, (4) alterations in topIIα expression and activity, and (5) augmented biochemical defenses against anthracycline-induced oxidative stress. Upregulation of the reductase enzymes that convert anthracyclines to their less active or inactive secondary alcohol (rubicinol) metabolites has been proposed as a potential mechanism of acquired resistance. 83 Chemical Mechanism of Cardiotoxicity - Acute toxicity : superoxide radical anion and hydroxyl radical (OH), both of which are formed via a one-electron reduction of the anthracyclinone quinone (ring C) to hydroquinone via a free radical semiquinone intermediate by NADPH/CYP450 reductase (Fig. next slide). Superoxide radical anions generate hydrogen peroxide (H2O2) in a Cu2+-dependent process that requires superoxide dismutase and proton. The fate of H2O2 dictates the degree of acute myotoxicity observed from the anthracycline. In the presence of catalase, H2O2 is rapidly converted to water and oxygen. However, in the absence of catalase and in the presence of ferrous ion (Fe2+), the highly damaging hydroxyl radical is generated via the Fenton reaction. 84 Anthracycline-mediated free radical formation. 85 Anthracyclines chelate strongly with di- and trivalent cations, including intracellular Fe3+, which can be reduced to Fe2+ enzymatically or via auto-reduction if the C13 substituent is CH2OH. The ready availability of the iron needed to generate hydroxyl radicals from H2O2 is essentially guaranteed. 86 Hydroxyl radicals promote single-strand breaks in DNA and could conceivably augment the cytotoxic action of anthracyclines; however, this is uncommon in tumor cells at standard antineoplastic doses. In contrast, hydroxyl radicals are readily generated in the myocardium because cardiac tissue does not contain significant amounts of catalase and other relevant cytoprotective enzymes. When H2O2 forms in the myocardium, it has no choice but to go down the Fenton pathway. Cardiac toxicity anthracyclines, is but the major use-limiting coadministration of side effect dexrazoxane of (an antioxidant and iron chelator) has been shown to lower its incidence when used with the C13-CH2OH substituted anthracyclines doxorubicin and epirubicin. 87 Chronic (Delayed) Cardiotoxicity: Rubicinol metabolites are believed responsible for the more life-threatening chronic cardiotoxicity that some patients experience. The C13-carbonyl is reduced via a two-electron mechanism to a usually less active or inactive rubicinol via cytosolic aldo-keto reductase (AKR1C3) and carbonyl reductase (CBR1) enzymes (Fig. next slide). Before excretion, anthracyclines can be further metabolized via hydrolytic or reductive deglycosidation to their 7-hydroxy or 7-deoxy aglycones, respectively, followed by O-dealkylation of the C4 methoxy ether (if present) and conjugation with either glucuronic acid or sulfate. The aglycones may also have cardiotoxic properties. 88 Anthracycline metabolism (AKR, aldo-keto reductase; CBR, carbonyl reductase). 89 The larger the C13-substituent the slower the AKR/CBR-catalyzed reduction reaction, and the longer the duration of cytotoxic activity. C13-substituents found on most marketed anthracyclines include CH3 (daunorubicin and idarubicin) and CH2OH (doxorubicin and epirubicin). Rubicinol metabolites concentrate in cardiomyocytes and induce profound increases in intracellular calcium concentrations. The synthesis of sarcomeric proteins essential to cardiomyocyte integrity is also impaired. As a result, myocardial contractility is compromised, and the myopathy presents as severe congestive heart failure. 90 TopIIβ, the predominant topII isoform in quiescent cells, is the only isoform found in the myocardium, and a critical role for this protein in anthracycline-induced cardiotoxicity has been proposed. Anthracycline binding to myocardial topIIβ disrupts mitochondrial function, initiates DNA double-strand breaks, stimulates p53induced apoptosis in cardiomyocytes, and may facilitate formation of reactive oxygen species. TopIIβ is currently viewed as a highly significant (and likely a primary) mediator of anthracycline-related cardiotoxicity. 91 Doxorubicin (Fig next slide) is used either alone or in combination therapy to treat a wide range of neoplastic disorders, including hematologic cancers and solid tumors in breast, ovary, stomach, bladder, and thyroid gland. Daunorubicin is administered for the treatment of lymphocytic leukemia and acute myeloid leukemia. 92 Anthracycline and related anticancer agents. 93 Mitosis Inhibitors The mitotic process depends on the structural and functional viability of microtubules, which are polymeric heterodimers consisting of isotypes of α- and β-tubulin. A γ-tubulin protein is found at the organizational center of the microtubule. The tubulin isotypes found in the microtubule are conserved throughout specific tissues within a given species and will impact the cell’s sensitivity to mitosis inhibitors. During cell division, tubulin undergoes alternating periods of structural growth and erosion known as “dynamic instability.” 94 The proteins alternatively polymerize and depolymerize through guanosine triphosphate (GTP)- and Ca2+-dependent processes, respectively. Polymerization involves the addition of tubulin dimers to either end of the tubule, although the faster-growing (+) end is more commonly involved. Polymerization results in tubular elongation, whereas depolymerization results in structural shortening. The frenetic alteration in structure, facilitated by microtubule-associated proteins (MAPs), ultimately allows for the formation of the mitotic spindle and the attachment of chromosomes that are prerequisites to cell division. Inhibiting the essential hyperdynamic changes in microtubular structure results in mitotic arrest and apoptosis. 95 Mitosis inhibitors. natural product natural product semisynthetic alkaloid derivative 96 Taxanes Mechanism of Action : Antineoplastic taxanes were originally isolated from the bark of the Pacific yew (Taxus brevifolia) but are now produced semisynthetically from an inactive natural precursor (10-deactetylbaccatin III) found in the leaves of the European yew (Taxus baccata), a renewable resource. Taxanes bind to polymerized β-tubulin at a specific hydrophobic receptor site comprised of the 31 N-terminal residues located deep within the tubular lumen. At standard therapeutic doses, binding promotes a stable tubulin conformation similar to that of the GTP-bound protein, rendering the microtubules prone to further polymerization and resistant to erosion. 97 Dynamic instability is disrupted, the mitotic spindle does not form, and microtubules collapse into dense aberrant structures known as asters. Mitosis stops and the cell dies. Cellular efflux by P-glycoprotein is a major mechanism of taxane resistance, as is β-tubulin mutation and overexpression of another isotype, βIII-tubulin. 98 Chemistry : Diterpenoid taxanes consist of a 15-membered tricyclic taxane ring system fused to an oxetane (D) ring, and contain an esterified β-phenylisoserine side chain at C13. The taxane ring system is conceptualized as having “northern” and “southern” halves. The southern segment is critical to receptor binding, while the northern section ensures the proper conformation of critical functional groups, including the C13isoserine side chain [with its C1′-carbonyl, free C2′-(R)-OH and C3′-(S)-benzamido or t-butoxycarboxamido groups], the benzoyloxy and acetoxy groups at C2 and C4, respectively, and the intact oxetane ring. The free C2′ hydroxyl in particular is essential to taxane antimitotic action. 99 Paclitaxel interacts in a folded (“T” or “butterfly”) conformation that places the C2-benzoyloxy and C3′-benzamido groups in close proximity. Their independent intermolecular engagement with a critical β-tubulin His residue perfectly positioned between them keeps them from interacting with one another. The oxetane ring, although capable of hydrogen bonding with receptor residues, is believed to serve a more critical role in properly orienting the C4acetoxy moiety for interaction within its hydrophobic binding pocket. The C1-OH also promotes conformational stability through intramolecular interaction with the carbonyl oxygen of the C2 benzoyloxy moiety. The areas of the paclitaxel structure where steric influences are most critical to receptor binding have been identified. See table next slide. 100 101 Metabolism : The taxanes are metabolized to significantly less cytotoxic metabolites by CYP enzymes (Fig. next slide). In humans, CYP2C8 converts paclitaxel to 6α-hydroxypaclitaxel, the major metabolite, which is 30-fold less active than the parent structure. CYP3A4 mediates the formation of additional minor phydroxylated metabolites of the benzamido and benzoyloxy moieties at C3′ and C2 respectively, and the 10-desacetyl metabolite has been documented in urine and plasma. 102 Taxane metabolism. 103 Docetaxel is oxidized exclusively by CYP3A4/5, with CYP3A4 having a 10-fold higher affinity for the drug than CYP3A5. The major metabolite, known as hydroxydocetaxel, is the hydroxymethyl derivative of the 3′-t-butoxycarboxamide Hydroxydocetaxel can be oxazolidinedione and further oxidized diasteriomeric side and chain. cyclized to hydroxyoxazolidinone metabolites prior to excretion. The oxazolidinedione has been linked to the edema and weight gain that are problematic adverse effects of docetaxel. 104 The C13 side chain is believed to guide the positioning of taxanes within the catalytic site of CYP enzymes. Specifically, the 3′-phenyl of paclitaxel has been proposed to properly orient C6 for hydroxylation through π-stacking interactions with CYP2C8 active site residues while decreasing affinity for CYP3A4 binding groups. The hydrophobic character of the 10-acetoxy group, found in paclitaxel, enhances CYP-mediated hydroxylation 2-5 fold by facilitating substrate binding and/or augmenting catalytic capability. Both isoforms are impacted by the presence of this ester, often to the same extent. 105 Paclitaxel: Paclitaxel conventional injection is indicated for intravenous use in combination with cisplatin as first-line therapy for advanced ovarian and non-small cell lung cancer. It is also used alone or in combination with the fluorouracil prodrug capecitabine in anthracycline-resistant metastatic breast cancer. Paclitaxel’s ability to upregulate thymidine phosphorylase, one of capecitabine’s activating enzymes, is the rationale behind the combination therapy. Paclitaxel resistance is mediated primarily through P-glycoprotein and βIII-tubulin overexpression. Regarding the latter, it is proposed that the exchange of Ala for Ser at position 277 in βIII-tubulin disrupts the critical H-bond with the C7-OH, resulting in a loss of affinity. 106 Docetaxel: The indications for docetaxel include cancers of the breast, prostate and head/neck, as well as non-small cell lung cancer and gastric adenocarcinoma. 107 Vinca Alkaloids Mechanism of Action : Several alkaloids found naturally in Catharanthus roseus (periwinkle) have potent antimitotic activity. Vinca alkaloids halt cell division by inhibiting polymerization. They bind to a single high-affinity β-tubulin site at the interface of two heterodimers within the tubular lumen near the GTP binding site on the (+) end of the tubules. Once bound, these alkaloids attenuate the uptake of the GTP essential to tubule elongation. Simultaneous binding to α- and β-tubulin results in protein crosslinking, which promotes a stabilized protofilament structure. Inhibition of microtubule elongation occurs at substoichiometric concentrations, at which alkaloid occupation of only 1%-2% of the total number of high-affinity sites can result in up to a 50% inhibition of microtubule assembly. 108 At high concentrations, when alkaloid binding to high-affinity sites becomes stoichiometric and lower-affinity binding sites on the tubule wall are also occupied, microtubular depolymerization is stimulated, leading to the exposure of additional alkaloidal binding sites and resulting in dramatic changes in microtubular conformation. Spiral aggregates, protofilaments, and highly structured crystals form, and the mitotic spindle ultimately disintegrates. The loss of the directing mitotic spindle promotes chromosome “clumping” in unnatural shapes (balls and stars), leading to cell death. Other nonmitotic toxicities related to the microtubule-disrupting action of vinca alkaloids include inhibition of axonal transport and secretory processes, and disturbances in platelet structure and function. 109 Chemistry : The vinca alkaloids are complex structures composed of two polycyclic segments, catharanthine and vindoline both of which are essential for high-affinity tubulin binding. Like paclitaxel and docetaxel, resistance is mediated, in part, through Pglycoprotein. Vinca alkaloids undergo O4-deacetylation to yield metabolites that are equipotent to or more active than the parent drug. They are also subject to extensive CYP3A4/5-mediated metabolism before biliary excretion. Vincristine alone is selectively metabolized by the 3A5 isoform. A representative hydrolyzed metabolite of vinblastine and the major CYP3A-generated metabolites of the other vinca alkaloids are shown in Figure next slide. 110 Major metabolites of vinca alkaloids. 111 As CYP metabolism is inactivating and CYP3A overexpression has been noted in human tumors, it has been proposed that CYP3A enzymes could contribute to vinca alkaloid resistance. Vincristine Sulfate: Vincristine is employed in many hematologic malignancies, and is a common choice in pediatric (as well as adult) acute lymphoblastic leukemia. It is also given in Wilms tumor (an almost exclusively pediatric cancer), rhabdomyosarcoma (in soft tissue) and neuroblastoma. 112 Vinblastine Sulfate: Vinblastine is used as palliative therapy in hematologic malignancies and has found utility in the treatment of advanced testicular carcinoma (often in combination with bleomycin), advanced mycosis fungoides (type of skin lymphoma), and Kaposi sarcoma. Vinorelbine Tartrate : Vinorelbine is used alone or in combination with cisplatin for first-line treatment of non-small cell lung cancer. 113 Antimetabolites Many antimetabolites stop de novo DNA biosynthesis by inhibiting the formation of the nucleotides that make up these life-sustaining polymers. The rate-limiting enzymes of nucleotide biosynthesis are the primary targets for the antimetabolites, since inhibition of these key enzymes is the most efficient way to shut down any biochemical reaction sequence. Other enzymes required in DNA biosynthesis can be inhibited, or chain elongation can be arrested through the incorporation of false nucleotides into the growing DNA strand. The antimetabolites serve as false substrates for critical nucleotide biosynthesis or polymerizing enzymes. 114 These enzyme inhibitors are structurally designed to look like the normal (endogenous) substrate. Through a form of chemical entrapment, they entice the enzymes to choose them over the endogenous substrate and, once they do, the antimetabolites bind them irreversibly or pseudoirreversibly. If the building block nucleotides cannot be synthesized or DNA polymerization is blocked, then DNA synthesis (and tumor growth) is stopped dead in its tracks. 115 Many antimetabolite antineoplastics are categorized by the class of nucleotide they inhibit. Purine antagonists inhibit the synthesis of adenosine monophosphate (AMP) and guanosine monophosphate (GMP), while pyrimidine antagonists stop the production of deoxythymidine monophosphate (dTMP). Antifolates shut down purine and pyrimidine biosynthesis pathways since folate cofactors are essential to both. DNA polymerase inhibitors are false nucleotides that, when added to a growing DNA chain, exert chemical pressure on further elongation of the chain. The structures of the anticancer antimetabolites are shown next slide. 116 Antimetabolites. 117 Pyrimidine Antagonists dTMP Biosynthesis : dTMP is produced via C5-methylation of deoxyuridine monophosphate (dUMP). The rate-limiting enzyme of the deoxythymidine monophosphate (dTMP) synthetic pathway is the sulfhydryl-containing thymidylate synthase, with 5,10- methylenetetrahydrofolate (5,10-methylene-THF) serving as the methyl-donating cofactor. All dTMP synthesis inhibitors will inhibit thymidylate synthase either directly or indirectly, and this will result in a “thymineless death” in actively dividing cells. Without dTMP and its deoxythymidine triphosphate metabolite, DNA will fragment and the cell will die. 118 With substrate and cofactor properly positioned, the nucleophilic sulfhydryl moiety of synthase Cys195 launches an intermolecular nucleophilic attack on the highly electrophilic C6 of dUMP, forming a new (albeit transient) covalent bond (step 1). The bond that breaks as a result is the 5,6-double bond of dUMP, and the released electrons are accepted by the perfectly positioned δ+ methylene group of the cofactor (step 2). When substrate C5 covalently joins with the cofactor methylene carbon, the imidazolidine ring breaks (step 3), releasing steric constraints on N10. Taken together, steps 1-3 generate a ternary complex of enzyme, substrate, and cofactor (Fig. next slide). 119 Synthesis of deoxythymidine monophosphate (dTMP). DHFR, dihydrofolate 120 reductase; SHMT, serine hydroxymethyltransferase; TS, thymidylate synthase. Liberated N10 then abstracts the C5-H of the cofactor (step 4) which, through a series of electronic rearrangements, releases the cofactor C6-hydrogen as hydride (H−). H− attacks the δ+ methylene moiety of the substrate, which restores the essential 5,6pyrimidine double bond of the product (dTMP) and regenerates thymidylate synthase. To complete the biochemical cycle, the released 7,8-DHF byproduct must be reduced to tetrahydrofolate (THF) via dihydrofolate reductase (DHFR) using NADPH. Finally, THF is converted to 5,10-methylene-THF through the action of serine hydroxymethyltransferase (SHMT) and vitamin B6. 121 With the enzyme and cofactor both regenerated, the cell is ready to synthesize another molecule of dTMP utilizing stored dUMP. This happens at a normal pace in healthy cells and at an uncontrolled rate in tumor cells. The thymidylate synthase enzyme is very large, and the active site binding motifs for both substrate and cofactor are highly conserved regardless of source. The human enzyme (hTS) has been crystallized, and key binding residues for substrate and cofactor have been identified. Many of the residues identified in the figure next slide have been confirmed in a binding study with mutant (R163K) human thymidylate synthase and dUMP, as well as the high affinity false substrate 5-F-dUMP. 122 dUMP and 5,10-methylene-THF binding to thymidylate synthase. 123 Fluorouracil : Pyrimidine Analogue This fluorinated pyrimidine prodrug must be converted to its deoxyribonucleotide before it will be recognized by thymidylate synthase. Activation of fluorouracil and floxuridine. 124 The active form differs from the endogenous substrate only by the presence of the 5-F group, which holds the key to the DNA biosynthesis inhibiting action of this drug. The C6 position of the false substrate is significantly more δ+ than normal due to the strong electron-withdrawing effect of the adjacent C5 fluorine. This greatly increases the rate of attack by Cys195, resulting in a very fast formation of a fluorinated ternary complex (Fig. next slide). The small size of the fluorine atom assures no steric hindrance to the formation of this false complex. The next step in the pathway requires the abstraction of the C5-H (as proton) by the lone pair of electrons on N10 of the cofactor (kept primarily in unionized form by a resonance-facilitated low pKa), but this is no longer possible. 125 Mechanism of action of fluorouracil. 126 Not only is the C5-fluorine bond stable to cleavage, the δ− fluorine atom and unionized N10 would repel one another because they are both electron rich. The false ternary complex cannot break down, no thymidine-based product is formed, no folate cofactor is released, and most importantly, the rate-limiting thymidylate synthase enzyme is not regenerated. With thymidylate synthase directly and irreversibly inhibited, dTMP can no longer be synthesized, DNA will be irreversibly damaged, and the cell will die. Importantly, a secondary mechanism of therapeutic fluorouracil toxicity is the incorporation of the false triphosphorylated ribonucleotide (5-F-UTP) into RNA, a chemical deal breaker for RNA viability. Therefore, fluorinated uracil antimetabolites inhibit the biosynthesis of both DNA and RNA. 127 Fluorouracil is administered in the palliative treatment of colorectal, breast, stomach, and pancreatic cancers. It is rapidly cleared from the bloodstream, and although up to 20% of a dose is excreted unchanged in the urine, most undergoes hepatic catabolism via a series of enzymes that includes the polymorphic dihydropyrimidine dehydrogenase (DPD) (Fig. next slide). Major toxicities are related stomatitis/esophagopharyngitis, to and bone marrow potential depression, gastrointestinal ulceration. Nausea and vomiting are common. 128 Fluorouracil metabolism. 129 Methotrexate : Antifolate Methotrexate is a folic acid antagonist structurally designed to compete successfully with 7,8-dihydrofolate (7,8-DHF) for the dihydrofolate reductase (DHFR) enzyme. The direct inhibition of DHFR causes cellular levels of 7,8-DHF to rise, which in turn results in feedback (indirect) inhibition of thymidylate synthase. Methotrexate also inhibits glycinamide ribonucleotide (GAR) formyltransferase, a key enzyme in the synthesis of purine nucleotides. 130 131 Methotrexate’s C4-NH2 substituent and the fully aromatic pteridine ring system hold the key to its dihydrofolate reductase (DHFR)inhibiting action. It has been proposed that the N5 position of endogenous dihydrofolate (DHF) is protonated by Glu30 of DHFR and, in cationic form, binds to DHFR Asp27 (Fig. next slide). N5 is the strongest base in the DHF structure, in part due to the attenuating impact of the C4 carbonyl on N1 electron density. Additional affinity-enhancing interactions between enzyme and substrate have also been identified. Once bound, the substrate’s 5,6-double bond is positioned close to the NADPH cofactor so that the transfer of hydride can proceed. 132 Misorientation of methotrexate at dihydrofolate reductase. 133 In contrast, the C4-NH2 substituent of methotrexate enriches electron density at N1 through π-electron donation, increasing its basic character between 10- and 1,000-fold and promoting protonation by Glu30 at the expense of N5. Because N1 and N5 are across the pteridine ring from one another, the interaction of N1 with the dihydrofolate reductase (DHFR) Asp27 will effectively stand the false substrate “on its head” relative to the orientation of 7,8-dihydrofolate (Fig. previous slide). With the 5,6-double bond of methotrexate 180° away from the bound NADPH cofactor, and stabilized by the fully aromatic pteridine ring, the possibility for reduction is eliminated. 134 The dihydrofolate reductase (DHFR) enzyme will be pseudoirreversibly bound to a molecule it cannot reduce, which ties up the DHFR enzyme and prevents the conversion of dihydrofolate to tetrahydrofolate (THF). In turn, this halts the synthesis of the 5,10-methylene-THF cofactor required for deoxythymidine monophosphate (dTMP) biosynthesis and causes feedback inhibition of the thymidylate synthase enzyme. The cell will die a “thymineless death.” Methotrexate is given orally in the treatment of breast, head and neck, and various lung cancers as well as in non-Hodgkin lymphoma. 135 The monoglutamate tail of methotrexate permits active transport into cells via a reduced folate carrier (RFC1, a transporter extensively expressed on tumor cell membranes), where it subsequently undergoes intracellular folylpolyglutamate synthase (FPGS)-catalyzed polyglutamation. This process adds several anionic carboxylate groups to the molecule and traps the drug at the site of action. Polyglutamation is more efficient in tumor cells than in healthy cells, which promotes the selective toxicity of this drug. The polyglutamated drug will be hydrolyzed back to the parent structure by γ-glutamyl hydrolase before renal elimination. Up to 90% of an administered dose is excreted unchanged in the urine within 24 hours. 136 Cancer cells can become resistant to methotrexate over time. Acquired resistance mechanisms include increased of expression of dihydrofolate reductase and other enzyme targets, impaired reduced folate carrier 1-mediated transport, active cellular efflux by P-glycoprotein, and/or attenuated intracellular polyglutamation. 137 DNA Alkylating and Crosslinking Agents The primary target of DNA alkylating and cross-linking agents is the actively dividing DNA molecule. The DNA cross-linkers are extremely reactive electrophilic structures. The nucleophilic groups on various DNA bases (particularly, but not exclusively, the N7 of guanine) readily attack the electrophilic drug, resulting in irreversible alkylation or complexation of the DNA base. Some DNA alkylating agents, such as the nitrogen mustards and nitrosoureas, are bifunctional, meaning that one molecule of the drug can bind two distinct DNA bases. Most commonly, the alkylated bases are on different DNA molecules, and interstrand DNA cross-linking through two guanine N7 atoms results. 138 Cell cycle arrest and apoptosis follow as the cell tries unsuccessfully to repair the chemical insult. The DNA alkylating antineoplastics are not cell cycle specific, but they are more toxic to cells in the late G1 or S phases of the cycle. This is the time when DNA is unwinding and exposing its nucleotides, increasing the chance that vulnerable DNA functional groups will encounter the electrophilic antineoplastic drug and launch the nucleophilic attack that leads to its own destruction. The DNA alkylators have a great capacity for inducing both mutagenesis and carcinogenesis. 139 Organoplatinum complexes also cross-link DNA, and many do so by binding to adjacent guanine nucleotides (called diguanosine dinucleotides) on a single strand of DNA. This leads to intrastrand DNA cross-linking. The anionic phosphate group on a second strand of DNA stabilizes the drug-DNA complex and makes the damage to DNA replication irreversible. Some organoplatinum agents also damage DNA through interstrand cross-linking. 140 * DNA alkylating and cross-linking agents. 141 Nitrogen Mustards and Aziridine-Mediated Alkylators Nitrogen mustards are bis(β-haloalkyl)amines. The term “bis” means two, and the “halo” (short for halogen) in the nomenclature is invariably chlorine. The two chlorine atoms dramatically decrease the basic strength of the amino nitrogen through a strong negative inductive effect. As a result, the unionized conjugate of these drugs predominates at physiologic pH. This is intentional because it is the lone pair of electrons on the unionized amine that allows for the formation of the highly electrophilic aziridinium ion, which is the reactive DNA-destroying intermediate generated by all true mustards. 142 Mechanism of Action : - step 1 (Fig. next slide): the lone pair of electrons on the unionized amine conducts an intramolecular nucleophilic attack at the βcarbon of the mustard, displacing chloride anion and forming the highly electrophilic aziridinium ion intermediate. The carbon atoms of this strained quaternary amine are highly electrophilic due to the strong negative inductive effect of the cationic nitrogen. - step 2 : a DNA nucleophile conducts an intermolecular nucleophilic attack, which breaks the aziridine ring and alkylates DNA. Although guanine is the preferred nucleic acid base involved, the less nucleophilic adenine is also known to react. Of critical importance is the fact that the lone pair of electrons on the mustard nitrogen is regenerated when the aziridine ring cleaves. 143 DNA destruction through nitrogen mustard-mediated alkylation. 144 - Steps 3 and 4 : repetitions of steps 1 and 2, respectively, involving the second arm of the mustard and a second molecule of DNA. Ultimately, two molecules of DNA will be cross-linked through the carbon atoms of what was once the nitrogen mustard. Tethered together, the DNA strands cannot separate nor replicate, so transcription of DNA to RNA is halted. In an attempt to liberate the DNA from the mustard’s covalent stranglehold, hydrolytic depurination (step 5) cleaves the bound guanine residues from the DNA strand. However, the DNA released from this mustard trap is damaged and unable to replicate, and cell death is the inevitable result. If this is happening in a tumor cell, the therapeutic goal has been accomplished. If it is happening in a healthy cell, particularly one with a short half-life, the patient may experience adverse effects that can be use-limiting. 145 Chemistry : An aliphatic nitrogen substituent R (e.g., CH3) will release electrons to the amine through σ bonds. This electronic enrichment enhances the nucleophilic character of the lone pair of electrons and increases the speed at which the δ+ β-carbon of the mustard will be attacked. Whether in a tumor cell or a healthy cell, as soon as the aziridinium ion forms, it will react with unpaired DNA and/or other cell nucleophiles, such as electron-rich SH, OH and NH groups of amino acids on enzymes or membrane-bound receptors. The body’s water can also react with (and inactivate) the aziridinium ion. 146 An aromatic nitrogen substituent R (e.g., phenyl) conjugated with the mustard nitrogen will stabilize the lone pair of electrons through resonance. Resonance delocalization significantly slows the rate of intramolecular nucleophilic attack, aziridinium ion formation, and DNA alkylation. Nitrogen mustards can decompose in aqueous media through formation of the inactive dehalogenated diol shown next slide. Both the mustard nitrogen (pathway a) and the oxygen of water (pathway b) can act as nucleophiles to advance this degradative process. The decomposition reactions can be inhibited if the nucleophilic character of these atoms is eliminated through protonation, so buffering solutions to an acidic pH (e.g., 3-5) helps to enhance stability in aqueous solution. 147 Aqueous decomposition of nitrogen mustards. 148 Chlorambucil : Chlorambucil is one of three aromatic nitrogen mustards with a resonance-stabilized amine. The drug is active intact and also undergoes β-oxidation to provide an active phenylacetic acid mustard metabolite which is responsible for some of the observed antineoplastic activity. 149 It is also mutagenic, teratogenic and carcinogenic; it can induce nonlymphocytic leukemia, although secondary malignancies are considered rare events. It is administered for the management of chronic lymphocytic leukemia, non-Hodgkin lymphoma and Hodgkin lymphoma. Elimination of inactive mono- and dihydroxy spontaneous hydrolysis products (structure previous slide) of both the parent drug and phenylacetic acid metabolite is primarily via the urine. 150 A structurally similar aromatic mustard, melphalan, is used primarily in the treatment of multiple myeloma. l-Phe was purposefully incorporated into this antineoplastic to promote active transport into tumor cells, however studies indicate that melphalan enters cells through facilitated diffusion rather than by active transport. 151 Cyclophosphamide : Cyclophosphamide is a chiral prodrug antineoplastic agent requiring activation by metabolic and nonenzymatic processes (Fig. next slide). The initial metabolic step is mediated primarily by CYP2B6 (and, to a lesser extent, by CYP3A4 and CYP2C isoforms) and involves regioselective hydroxylation at C4 of the oxazaphosphorine ring to generate a carbinolamine. This hydroxylation reaction must occur before the molecule will be transported into cells, and approximately 90% of an administered dose will be appropriately converted. 152 Cyclophosphamide metabolism. 153 CYP3A4 and CYP2B6 stereospecifically catalyze an inactivating N-dechloroethylation reaction on the R and S isomers, respectively, which yields highly nephrotoxic and neurotoxic chloroacetaldehyde. Chloroacetaldehyde toxicity is accompanied by glutathione depletion, indicating that this electrophilic byproduct alkylates Cys residues of critical cell proteins. Alkylation of Lys, adenosine, and cytidine residues is also possible. The CYP-generated carbinolamine undergoes nonenzymatic, reversible cleavage to provide the aldophosphamide tautomer, either in the bloodstream or inside cells. Acrolein, a highly reactive α,β-unsaturated aldehyde, is cleaved from aldophosphamide via intracellular spontaneous β-elimination, generating phosphoramide mustard. With a pKa of 4.75, the mustard will be persistently anionic at intracellular pH and trapped inside the cell. 154 The fate of phosphoramide mustard is varied. Most cyclizes to the quaternary aziridinium ion, which alkylates DNA in the manner of all mustards. Some decomposes, losing phosphoric acid and ammonia. This leaves the secondary bis(β-chloroethyl)amine mustard, which cyclizes to form a tertiary aziridine (rather than a quaternary aziridinium) species. The free tertiary aziridine will protonate at intracellular pH to provide the cationic conjugate acid, and the carbon atoms of both conjugates are still sufficiently δ+ to attract DNA nucleophiles, albeit less vigorously than the permanently cationic quaternary amine. The net result is DNA alkylation and cell death. Oxidation of oxazaphosphorine intermediates along the metabolic pathway by cytosolic alcohol or aldehyde dehydrogenase is inactivating. 155 The need for metabolic activation in the liver means lowered gastrointestinal toxicity and less nonspecific toxicity for cyclophosphamide compared with other DNA alkylating agents, but cyclophosphamide is not without its toxic effects. Acrolein, generated during the decomposition of aldophosphamide, is a very electrophilic and highly reactive species, and it causes extensive damage to cells of the kidney and bladder. While acrolein can be produced in kidney via CYP3A4-mediated metabolism, it is predominantly generated in liver, where it readily conjugates with glutathione (Fig. next slide). 156 GSH conjugation with acrolein. 157 When the acrolein-glutathione or mercapturic acid (N- acetylcysteine) conjugate is delivered to the bladder for excretion, the conjugate can cause direct toxicity or cleave to release electrophilic acrolein to the cells. Without additional glutathione to re-scavenge liberated toxin, the acrolein will be attacked at its δ+ terminus by the nucleophilic SH of bladder cell Cys residues (Fig. previous slide). Mesna is available as adjuvant therapy in case of overt toxicity or as a prophylactic protectant. A sulfhydryl reagent, mesna is transported in the bloodstream as the inactive disulfide (dimesna) and reduced by glutathione dehydrogenase to the reactive sulfhydryl in the proximal tubules. After delivery to the bladder, the SH group competes with Cys residues for the alkylating acrolein, as shown in Figure next slide. 158 Acrolein detoxification by mesna. 159 Cyclophosphamide is most commonly used in combination with other antineoplastic agents to treat a wide range of neoplasms, including leukemias, lymphomas, multiple myeloma, ovarian adenocarcinoma, and breast cancer. 160 Organoplatinum Complexes Mechanism of Action: Organoplatinum antineoplastic agents contain an electron-deficient metal atom that acts as a magnet for electron-rich DNA nucleophiles. Like nitrogen mustards, organoplatinum complexes are bifunctional and can accept electrons from two DNA nucleophiles. Intrastrand cross-links most frequently occur between adjacent guanine residues referred to as diguanosine dinucleotides (60%-65%) or adjacent guanine and adenine residues (25%-30%). Interstrand cross-linking, which occurs much less frequently (1%-3%), usually involves guanine and adenine bases. 161 Chemistry: All currently marketed organoplatinum anticancer agents are Pt(II) complexes with square planar geometry. Platinum is inherently electron deficient, but the net charge on the organometallic complex is zero due to the contribution of electrons by two of the four ligands bound to the parent structure. Most commonly, the electron-donating ligand is chloride. Before reacting with DNA, the electron-donating ligands are displaced through nucleophilic attack by cellular water. When the displaced ligands are chloride anions (e.g., cisplatin), the chloridepoor environment of the tumor cell facilitates the process, driving the generation of the active, cytotoxic hydrated forms (Fig. next slide). 162 Cisplatin activation and DNA cross-linking. 163 Since the original ligands leave the metal with their electrons, the hydrated organoplatinum molecule has a net positive charge. The hydrated Pt analogues are readily attacked by DNA nucleophiles (e.g., the N7 of adjacent guanine residues) due to the net positive charge that has been regained on the Pt atom (Fig. previous slide). The DNA bases coordinate with the Pt, and in the cis configuration, DNA repair mechanisms are unable to permanently correct the damage. The net result is a major change in DNA conformation such that base pairs that normally engage in hydrogen bond formation are not permitted to interact. The two amine ligands of the complex are bound irreversibly to the Pt atom through very strong coordinate covalent bonds. 164 They cannot be displaced by DNA nucleophiles, but they do stabilize the cross-linked DNA-Pt complex by forming strong ion-dipole bonds with the anionic phosphate residues on DNA. The DNA distortion prompts a futile cycle of damage recognition and repair before succumbing to cell cycle arrest and apoptosis. Cisplatin The simplest of the organometallic antineoplastic agents, cisplatin is used in the treatment of metastatic testicular and ovarian cancer and advanced bladder cancer. It is highly nephrotoxic. To proactively protect against kidney damage, patients should be aggressively hydrated with chloride-containing solutions and magnesium supplementation should be initiated. 165 Mannitol diuretics can be administered to promote continuous excretion of the drug and its hydrated analogues. Sodium thiosulfate, which accumulates in the renal tubules, has also been used to neutralize active drug in the kidneys in an effort to avoid nephrotoxicity (see below) Cisplatin inactivation by sodium thiosulfate. 166 Amifostine, a thiol-generating prodrug, has been evaluated as a means to decrease risk of serious hearing loss secondary to cisplatin therapy. It is activated by alkaline phosphatase, an enzyme with higher activity in normal tissue compared to tumor cells. It is proposed that activated amifostine interacts with cisplatin to reduce the number of drug-DNA adducts, perhaps via the mechanism shown in Figure next slide. 167 Amifostine activation and reaction with cisplatin. 168 Resistance to cisplatin therapy can be intrinsic in colorectal, prostate, and lung cancer, or acquired after multiple courses of therapy (e.g., in ovarian cancer). Resistance is mediated through several distinct mechanisms, including: (1) compromised carrier-mediated cellular transport via the copper transporting protein CTR1; (2) enhanced intracellular inactivation through drug trapping in vesicles; (3) drug inactivation through conjugation to Cys and/or Metcontaining GSH and metallothionein proteins; (4) uncontrolled expression of noncoding RNA (lncRNA), (5) cellular efflux by P-type ATP 7A/7B, multidrug resistance, and (possibly) P-glycoprotein proteins, and 169 (6) increased DNA repair and/or tolerance to cisplatin-induced DNA damage. Cisplatin damage can be successfully repaired by nucleotide excision repair proteins, which remove platinum- damaged segments from the DNA, and these proteins are often upregulated in cisplatin-resistant tumors. Cisplatin-DNA (and carboplatin-DNA) adducts are also recognized by mismatch repair (major molecular response) proteins. The downregulation of major molecular response in cisplatin and carboplatin-treated cancer cells induces resistance through the loss of an apoptotic response that normally follows several ill-fated attempts to repair organoplatinuminduced damage. Testicular tumors are particularly responsive to cisplatin due to their inherent deficiency in DNA repair processes. 170 Carboplatin Carboplatin forms the same cytotoxic hydrated intermediate as cisplatin but does so at a 10-fold slower rate, making it a 20- to 40- fold less potent chemotherapeutic agent. Suppression of platelets and white blood cells is the most significant toxic reaction, and nonhematologic toxicities (e.g., emesis, nephrotoxicity, and ototoxicity) are rare. Carboplatin is only approved for use in the treatment of advanced ovarian cancer. 171 Miscellaneous Antineoplastic Agents The anticancer agents included in Table 33.13 (slides 173 and 174) do not fit neatly into other antineoplastic drug classes. 172 173 174 Bleomycin Sulfate : it is a mixture of naturally occurring glycopeptides, predominantly bleomycin A2. Through DNA intercalation in guanine-rich regions, the aromatic bithiazole ring system (in partnership with the pyrimidine ring on the opposite side of the structure) positions bleomycin for DNA destruction via cytotoxic free radicals. The disaccharide, polyamine, imidazole, and pyrimidine structures are very electron rich and readily chelate intracellular Fe2+. Once chelated, Fe2+ is oxidized to Fe3+ with a concomitant reduction of bound oxygen and the release of the highly reactive and cytotoxic hydroxyl radical. The ferric hydroperoxide bleomycin complex is considered the cytotoxic form responsible for both single- and double-stranded DNA breaks. 175 Through the direct abstraction of a hydrogen atom from 4′ of a deoxyribose, a free radical is generated that subsequently decomposes to a DNA-destroying 4′-hydroperoxide. A highly electrophilic pyrimidine base propenal that inactivates essential cellular proteins via Cys alkylation is also produced (Figure next slide). Reduced glutathione is proposed to serve a protective role by acting as propenal scavenger and, until depleted, saves cellular proteins from alkylation. 176 Mechanism of bleomycin-induced damage of DNA and proteins. 177 The action of bleomycin is terminated through the action of bleomycin hydrolase, a cytosolic aminopeptidase that cleaves the terminal amide moiety to form the inactive carboxylate metabolite (Fig. next slide). The metabolic conversion of the electron- withdrawing amide to an electron-donating carboxylate increases the pKa of the α-amino group, which normally interacts with DNA in the unionized conjugate form. After hydrolysis, the ratio of ionized to unionized amine increases approximately 126-fold, destroying DNA affinity and leading to the loss of therapeutic action. Drug destruction via the bleomycin hydrolase pathway is rapid, and tumors will be resistant to bleomycin if they contain high concentrations of the enzyme. Conversely, tumors that are poor in bleomycin hydrolase (e.g., squamous cell carcinoma) respond well to this agent. 178 Bleomycin hydrolase–mediated inactivation of bleomycin. 179 Bleomycin is used in the palliative treatment of squamous cell head and neck cancers, testicular and other genital carcinomas, and Hodgkin lymphoma. 180 Drugs Used for the Treatment of Hormone-Dependent Cancers (Breast, Prostate) Hormone-Dependent Breast Cancer Tamoxifen (slides 41-50) Treatment of Prostate Cancer Treatment of Advanced or Metastatic Castration-Sensitive Prostate Cancer Gonadotropin-Releasing Hormone Therapy : Gonadotropin- releasing hormone (GnRH) Agonists 181 Modern androgen deprivation therapy involves the use of analogs of gonadotropin-releasing hormone (GnRH) which possess greater potency than the natural GnRH hormone. These agonists bind to GnRH receptors (GnRH-Rs) in the pituitary and cause an initial increase in luteinizing hormone (LH) and follicule-stimulating hormone, and, subsequently, increased testosterone synthesis in testes. Continuous overstimulation of GnRH-Rs desensitizes the gonadotroph cells and downregulates GnRH-R expression, resulting in decreased LH synthesis and nearly abrogating testicular steroidogenesis. Gonadal androgen synthesis is suppressed via feedback inhibition of the hypothalamus pituitary gonada axis. 182 The gonadotropin-releasing hormone (GnRH) agonists (Fig. next slide) are administered in a continuous, nonpulsatile manner, which is in contrast to the pulsatile release of the endogenous hormone GnRH. Further, they bind with higher affinity to gonadotropinreleasing hormone receptor in the pituitary than does the endogenous hormone, cumulatively causing a durable effect in the majority of patients upon constant or intermittent dosing. 183 Goserelin GnRH agonists used for the treatment of prostate cancer. The molecular graph of GnRH decapeptide is shown at the top of the figure with amino acid notation underneath the molecular graph. Enzymatic cleavage sites within GnRH are indicated therein. Amino acid notation of the structures of the GnRH superagonists leuprolide, goserelin, and triptorelin are below the GnRH 184 structure. Modifications at positions 6 and 10 are highlighted in red. Gonadotropin-releasing hormone (GnRH) agonists are peptidic agents that mimic the natural hormone GnRH and consist of an approximately 10-amino-acid segment of GnRH modified to stabilize the N- and C-terminus. Further, modifications at the 6 and 10 positions of the decapeptide block enzyme cleavage sites and increase agonist activity (Fig. previous slide). For example, the first site can be blocked by replacing glycine 6 (Gly6) with a hydrophobic d-amino acid such as d-leucine in leuprolide, d-serine modified with a t-Bu group in goserelin, or d-tryptophan in triptorelin. Another enzyme cleavage site can be blocked by eliminating Gly10, as is seen in both leuprolide and goserelin. Changes in GnRH structure at other amino acid positions decrease activity. 185 Triptorelin : Triptorelin pamoate is a synthetic decapeptide: 5-oxo-lprolyl-l-histidyl-l-tryptophyl-l-seryl-l-tyrosyl-d-tryptophyl-l-leucyl-l- arginyl-l-prolylglycine amide. Metabolic pathways are unknown as no metabolites of triptorelin have been identified, presumably due to complete degradation. It is eliminated by both liver and kidneys with 42% excreted in the urine as intact peptide, which increased to 62.3% in patients with liver disease. 186 Castration-Resistant Prostate Cancer (CRPC) Antiandrogens (Competitive androgen receptor antagonists) : Secondary hormonal therapy to suppress the reactivated androgen receptor (AR) axis in CRPC patients can be achieved by antiandrogens that compete with endogenous androgens for binding and antagonize the AR. 187 First and second generation nonsteroidal antiandrogens. 188 Flutamide : it is completely absorbed from the gastrointestinal tract and undergoes extensive first-pass metabolism by CYP1A2 to its major metabolite, 2-hydroxyflutamide, and its hydrolysis product, 3trifluoromethyl-4-nitroaniline. 2-Hydroxyflutamide is a more powerful antiandrogen in vivo, with higher affinity for the receptor than that of flutamide. 2-Hydroxyflutamide inhibits the metabolism of flutamide and both 2- and 4-hydroxylation of 17β-estradiol. Flutamide is a pure antagonist, whereas 2-hydroxyflutamide is a more potent androgen receptor (AR) antagonist but also can activate AR escape mutants at higher concentrations. These findings raise the possibility that increased conversion of flutamide to 2-hydroxyflutamide or accumulation of 2-hydroxyflutamide in cells may contribute to the anomalous responses to flutamide that are observed in some advanced prostate cancers. 189 Nilutamide : One of the methyl groups attached to the hydantoin ring is stereoselectively hydroxylated to a chiral metabolite, which subsequently is oxidized to its carboxylic acid metabolite. Less than 2% of nilutamide is excreted unchanged in the urine. In vitro, the nitro group of nilutamide was reduced to the amine and hydroxylamine moieties by nitric oxide (NO) synthases, a flavin monooxygenase system. This reduction proceeds via formation of a nitro anion free radical or via its reduction to its hydroxylamino derivative, which could explain some of the poorly investigated toxic effects of this drug which overshadow the therapeutic effects of nilutamide. 190 191 Drugs Used for Benign Prostatic Hyperplasia α1-Adrenergic Antagonists Often the first-line treatment for lower urinary tract symptoms and benign prostatic hyperplasia (BPH), the α1-adrenergic antagonists treat the increased adrenergic tone of the sympathetic nervous system by relaxing the muscles at the neck of the bladder and in the prostate, thereby reducing the pressure on the urethra and increasing the flow of urine. They do not cure BPH but, rather, help to alleviate some of the symptoms. For moderate symptoms of BPH, α1-adrenergic antagonists are often used due to their faster onset of symptom relief as compared with 5-reductase inhibitors. 192 They can also be used in combination with 5-reductase inhibitors when prostate volume is large as α1-adrenergic antagonists do not decrease prostate volume. Alfuzosin, tamsulosin, and silodosin (Fig. next slide) are used exclusively as first-line α1-adrenergic antagonists for the treatment of benign prostatic hyperplasia (BPH), while doxazosin and terazosin have also been used to treat high blood pressure. Prazosin, another α1-antagonist, is not indicated for the treatment of BPH. Tamsulosin, alfuzosin, and silodosin are uroselective α1-adrenergic antagonists developed specifically to treat BPH. When BPH and erectile dysfunction are present, α1-adrenergic antagonists are used in combination with a phosphodiesterase inhibitor. 193 α1-Adrenergic antagonists for treatment of benign prostatic hyperplasia. 194 The most common adverse effects for α1-adrenergic antagonists are related to abnormal ejaculation and orthostatic hypotension, with vasodilation, dizziness, headache, and tachycardia. 195 Tamsulosin (catecholamine-sulfonamide) exhibits uroselectivity for the α1A-adrenergic receptor and is a first-line drug for the treatment of benign prostatic hyperplasia, with no utility for treating hypertension due to lower affinity for vascular α1B-adrenergic receptor. Tamsulosin is O-deethylated by CYP3A4 to phenolic metabolites that are conjugated to glucuronide or sulfate before renal excretion and by O-demethylation and 3′-hydroxylation to catechol metabolites that also are conjugated with glucuronide and sulfate. Tamsulosin should be avoided in patients with severe sulfa allergies due to the presence of the aryl sulfonamide. 196 197 5α-Reductase Inhibitors The 5-reductase inhibitors work by suppressing the production of intraprostatic 5-dihydrotestoterone, thereby reducing the size of the prostate. When the prostate volume is large, finasteride and dutasteride are the most commonly used drugs used either alone or in combination with α1-adrenergic antagonists. Unlike α1antagonists, 5-reductase inhibitors are able to reverse benign prostatic hyperplasia to some extent and so may delay the need for surgery. Mechanism of Action : Inhibitors of 5-dihydrotestoterone (DHT) biosynthesis can result in a decrease in both circulating and target-tissue (prostate and skin) DHT concentrations, thus blocking its androgenic action in these tissues. 198 The enzyme targeted for 5-dihydrotestoterone (DHT) biosynthesis inhibition is 5-reductase (5AR), which converts testosterone into the more active metabolite DHT (Fig. next slide). Three isozymes of 5AR have been reported: 5α-reductase type 1 (5AR1), type 2 (5AR2), and type 3 (5AR3). 5AR1 is predominantly expressed in the skin, liver, brain, and prostate. 5AR2 is present in prostate, seminal vesicles, skin, liver, and hair follicles. The mechanism for testosterone reduction to DHT involves a H- transfer from NADPH to the 5α-position of testosterone, leading to an enol intermediate which can undergo enzyme-mediated tautomerization to form the products of DHT and NADP+. 199 Conversion of testosterone into 5α-dihydrotestosterone (DHT) by 5α-reductase. 200 Two azasteroid-17-amide derivatives of medrogestone have been developed as potent competitive inhibitors of 5-reductase (5AR) and approved for the treatment of benign prostatic hyperplasia : finasteride, a selective inhibitor of 5AR2 and dutasteride, a nonselective inhibitor of 5AR1 and 5AR2 (Fig. next slide). Thus, the inhibition of 5AR2 suppresses the metabolism of testosterone to 5dihydrotestoterone (DHT), resulting in significant decreases in plasma and intraprostatic DHT concentrations. 201 5α-Reductase inhibitors. 202 Finasteride and dutasteride are competitive inhibitors of 5-reductase (5AR) that also have the ability to work as mechanism-based inhibitors to inactivate 5AR through an apparent irreversible modification of the enzyme. They exhibit differing selectivity for 5AR. The inhibition constants (median inhibitory concentrations [IC50]), in Table 24.15, suggest that finasteride is 30 times more selective for 5AR2, whereas dutasteride appears to be approximately two times more potent as an inhibitor of 5AR2 as compared to 5AR1. 203 Finasteride binds to 5-reductase where testosterone normally binds and accepts a H- transfer from NADPH to the 1α-position of the A-ring of finasteride forming an enol intermediate (Fig. next slide). Due to the presence of the 4-aza group, the enol does not tautomerize to the keto group, but rather is postulated to form an enolate that can attack the electrophilic NADP+ still present to form an enzyme-bound, dihydrofinasteride-NADP adduct that slowly releases dihydrofinasteride. Finasteride and dutasteride are 4-aza steroids with a 1-ene and trans A-B ring junction and are thought to mimic the pathway of testosterone reduction to 5- dihydrotestoterone. It was found that lipophilic groups at the 17β position improve biological activity. The mechanism-based inhibition explains the potency and specificity of finasteride and dutasteride in the treatment of benign prostatic hyperplasia. 204 Mechanism-based inactivation of 5-reductase by finasteride. 205 Finasteride is extensively metabolized in the liver, primarily via CYP3A4, to two major metabolites: monohydroxylation of the t-butyl side chain, which is further metabolized via an aldehyde intermediate to the second metabolite, a monocarboxylic acid (Fig. next slide). The metabolites show approximately 20% the inhibition of finasteride for 5-reductase. 206 Metabolites of finasteride and dutasteride. 207 Dutasteride is extensively metabolized in humans by CYP3A4 to three major metabolites: 4′-hydroxydutasteride and 1,2- dihydrodutasteride, which are less potent than parent drug, and 6′hydroxydutasteride, which is comparable to the parent drug as an inhibitor of both 5-reductase 1 (5AR1) and 5-reductase 2 (5AR2) (Fig. previous slide). Dutasteride and its metabolites are excreted mainly (40%) in feces as dutasteride-related metabolites. Because finasteride and dutasteride are metabolized primarily by CYP3A4, the CYP3A4 inhibitors may increase these drugs’ blood levels and, possibly, cause drug-drug interactions. 208 Phytotherapy Extracts of the saw palmetto berry (Serenoa repens) are widely used for the treatment of benign prostatic hyperplasia (BPH), often as an alternative to pharmaceutical agents. It has been thought that 160 mg twice-daily dose of saw palmetto extract was approximately as effective as 5-reductase inhibitors over time. However, increasing doses of a saw palmetto fruit extract did not reduce lower urinary tract symptoms more than placebo in clinical trials. Other Western herbs that have been investigated for the treatment of BPH include pumpkin seeds (Cucurbita pepo), nettle root (Urtica dioica or Urtica urens), bee pollen (particularly that from the rye plant), African potato (tubers of Hypoxis rooperi), and the African tree Pygeum africanum, also known as Prunus africanum. 209