M. Wasif Saif and Edward Chu
ANTIFOLATES
Reduced folates play a key role in one-carbon metabolism, and they are essential for the biosynthesis of purines, thymidylate, and protein biosynthesis. Aminopterin was the first antimetabolite with documented clinical activity in the treatment of children with acute leukemia in the 1940s. This antifolate analog was subsequently replaced by methotrexate (MTX), the 4-amino, 10-methyl analog of folic acid, which remains the most widely used antifolate analog, with activity against a wide range of cancers (Table 19.1), including hematologic malignancies (acute lymphoblastic leukemia and non-Hodgkin’s lymphoma) and many solid tumors (breast cancer, head and neck cancer, osteogenic sarcoma, bladder cancer, and gestational trophoblastic cancer).
Pemetrexed is a pyrrolopyrimidine, multitargeted antifolate analog that targets multiple enzymes involved in folate metabolism, including thymidylate synthase (TS), dihydrofolate reductase (DHFR), glycinamide ribonucleotide (GAR) formyltransferase, and aminoimidazole carboxamide (AICAR) formyltransferase.1,2 This agent has broad-spectrum activity against solid tumors, including malignant mesothelioma and breast, pancreatic, head and neck, non–small-cell lung, colon, gastric, cervical, and bladder cancers.3–5
The third antifolate compound to have entered clinical practice is pralatrexate (10-propargyl-10-deazaaminopterin), a 10-deazaaminopterin antifolate that was rationally designed to bind with higher affinity to the reduced folate carrier (RFC)-1 transport protein, when compared with MTX, leading to enhanced membrane transport into tumor cells. It is also an improved substrate for the enzyme folylpolyglutamyl synthetase (FPGS), resulting in enhanced formation of cytotoxic polyglutamate metabolites.6,7 When compared with MTX, this analog is a more potent inhibitor of multiple enzymes involved in folate metabolism, including TS, DHFR, and GAR and AICAR formyltransferases. This agent is presently approved for the treatment of relapsed or refractory peripheral T-cell lymphomas.8
Mechanism of Action
The antifolate compounds are tight-binding inhibitors of DHFR, a key enzyme in folate metabolism.1 DHFR plays a pivotal role in maintaining the intracellular folate pools in their fully reduced form as tetrahydrofolates, and these compounds serve as one-carbon carriers required for the synthesis of thymidylate, purine nucleotides, and certain amino acids.
The cytotoxic effects of MTX, pemetrexed, and pralatrexate are mediated by their respective polyglutamate metabolites, with up to 5 to 7 glutamyl groups in a γ-peptide linkage. These polyglutamate metabolites exhibit prolonged intracellular half-lives, thereby allowing for prolonged drug action in tumor cells. Moreover, these polyglutamate metabolites are potent, direct inhibitors of several folate-dependent enzymes, including DHFR, TS, AICAR formyltransferase, and GAR formyltransferase.1
Mechanisms of Resistance
The development of cellular resistance to antifolates remains a major obstacle to its clinical efficacy.9,10 In experimental systems, resistance to antifolates arises from several mechanisms, including an alteration in antifolate transport because of either a defect in the reduced folate carrier or folate receptor systems, decreased capacity to polyglutamate the antifolate parent compound through either decreased expression of FPGS or increased expression of the catabolic enzyme γ-glutamyl hydrolase, and alterations in the target enzymes DHFR and/or TS through increased expression of wild-type protein or overexpression of a mutant protein with reduced binding affinity for the antifolate. Gene amplification is a common resistance mechanism observed in various experimental systems, including tumor samples from patients. In in vitro and in vivo experimental model systems, the levels of DHFR and/or TS protein acutely increase after exposure to MTX and other antifolate compounds. This acute induction of target protein in response to drug exposure is mediated, in part, by a translational regulatory mechanism, which may represent a clinically relevant mechanism for the acute development of cellular drug resistance.
Clinical Pharmacology
The oral bioavailability of MTX is saturable and erratic at doses greater than 25 mg/m2. MTX is completely absorbed from parenteral routes of administration, and peak serum levels are achieved within 30 to 60 minutes of administration.
The distribution of MTX into third-space fluid collections, such as pleural effusions and ascitic fluid, can substantially alter MTX pharmacokinetics. The slow release of accumulated MTX from these third spaces over time prolongs the terminal half-life of the drug, leading to potentially increased clinical toxicity. It is advisable to evacuate these fluid collections before treatment and monitor plasma drug concentrations closely.
Renal excretion is the main route of drug elimination, and this process is mediated by glomerular filtration and tubular secretion. About 80% to 90% of an administered dose is eliminated unchanged in the urine. Doses of MTX, therefore, should be reduced in proportion to reductions in creatinine clearance. Renal excretion of MTX is inhibited by probenecid, penicillins, cephalosporins, aspirin, and nonsteroidal anti-inflammatory drugs.
Pemetrexed enters the cell via the RFC system and, to a lesser extent, by the folate receptor protein. As with MTX, it undergoes polyglutamation within the cell to the pentaglutamate form, which is at least 60-fold more potent than the parent compound. This agent is mainly cleared by renal excretion, and in the setting of renal dysfunction, the terminal drug half-life is significantly prolonged to up to 20 hours. Pemetrexed, therefore, should be used with caution in patients with renal dysfunction. In addition, renal excretion is inhibited in the presence of other agents including probenecid, penicillins, cephalosporins, aspirin, and nonsteroidal anti-inflammatory drugs.
As with other antifolate analogs, pralatrexate is transported into the cell by the RFC carrier protein and then metabolized by FPGS to form longer chain polyglutamates, with up to four additional glutamate residues attached to the parent molecule. About 34% of the parent drug is cleared in the urine during the first 24 hours after drug administration. As such, caution is advised when using pralatrexate in patients with renal dysfunction. As with MTX and pemetrexed, the concomitant administration of other agents such as probenecid, penicillins, cephalosporins, aspirin, and nonsteroidal anti-inflammatory drugs, may inhibit renal clearance.
Toxicity
The main side effects of MTX are myelosuppression and gastrointestinal (GI) toxicity, which are usually completely reversed within 14 days, unless drug-elimination mechanisms are impaired. In patients with compromised renal function, even small doses of MTX may result in serious toxicity. MTX-induced nephrotoxicity is thought to result from the intratubular precipitation of MTX and its metabolites in acidic urine. Antifolates may also exert a direct toxic effect on the renal tubules. Vigorous hydration and urinary alkalinization have greatly reduced the incidence of renal failure in patients on high-dose regimens. Acute elevations in hepatic enzyme levels and hyperbilirubinemia are often observed during high-dose therapy, but these levels usually return to normal within 10 days. Methotrexate given concomitantly with radiotherapy may increase the risk of soft tissue necrosis and osteonecrosis.
The original rationale for high-dose MTX therapy was based on the concept of selective rescue of normal tissues by the reduced folate leucovorin (LV). However, recent data suggest that high-dose MTX may also overcome resistance mechanisms caused by impaired active transport, decreased affinity of DHFR for MTX, increased levels of DHFR resulting from gene amplification, and/or decreased polyglutamation of MTX.
The main toxicities of pemetrexed and pralatrexate include dose-limiting myelosuppression, mucositis, and skin rash, usually in the form of the hand-foot syndrome (HFS). Other toxicities include reversible transaminasemia, anorexia and fatigue syndrome, and GI toxicity. These side effects are reduced by supplementation with folic acid (350 μg orally daily) and vitamin B12 (1,000 mg subcutaneously given at least 1 week before starting therapy, and then repeated every three cycles). To date, there is no evidence to suggest that vitamin supplementation adversely affects the clinical efficacy of pemetrexed or pralatrexate.
5-FLUOROPYRIMIDINES
The fluoropyrimidine, 5-fluorouracil (5-FU) was synthesized by Charles Heidelberger in the mid 1950s. Uracil is a normal component of RNA; as such, the rationale leading to the development of the drug was that cancer cells might be more sensitive to decoy molecules that mimic the natural compound than normal cells. 5-FU and its derivatives are an integral part of treatment for a broad range of solid tumors (see Table 19.1), including GI malignancies (esophageal, gastric, pancreatic, colorectal, anal, and hepatocellular cancers), breast, head and neck, and skin cancers.11 It continues to serve as the main backbone for combination regimens used to treat metastatic colorectal cancer (mCRC) and as adjuvant therapy of early-stage colon cancer.
Mechanism of Action
5-FU enters cells via the facilitated uracil base transport mechanism and is then anabolized to various cytotoxic nucleotide forms by several biochemical pathways. It is thought that 5-FU exerts its cytotoxic effects through various mechanisms, including (1) the inhibition of TS, (2) incorporation into RNA, and (3) incorporation into DNA (Fig. 19.1). In addition to these mechanisms, the genotoxic stress resulting from TS inhibition may also activate programmed cell-death pathways in susceptible cells, which leads to the induction of parental DNA fragmentation.
Mechanisms of Resistance
Several resistance mechanisms to 5-FU have been identified in experimental and clinical settings. Alterations in the target enzyme TS represent the most commonly described mechanism of resistance. In vitro, in vivo, and clinical studies have documented a strong correlation between the levels of TS enzyme activity/TS protein and chemosensitivity to 5-FU. In this regard, cell lines and tumors with higher levels of TS are relatively more resistant to 5-FU. Mutations in the TS protein have been identified that lead to reduced binding affinity of the 5-FU metabolite fluorodeoxyuridine monophosphate (FdUMP) to the TS protein. Reduced expression and/or diminished activity of key activating enzymes may interfere with the formation of cytotoxic 5-FU metabolites. Decreased expression of mismatch repair enzymes, such as human mutL homolog 1 (hMLH1) and human mutS homolog 2 (hMSH2), and increased expression of the catabolic enzyme dihydropyrimidine dehydrogenase (DPD) are associated with fluoropyrimidine resistance. At this time, the relative contribution of each of these mechanisms in the development of cellular resistance to 5-FU in the actual clinical setting remains unclear.
Clinical Pharmacology
5-FU is not orally administered, given its erratic bioavailability resulting from high levels of the catabolic enzyme DPD present in the gut mucosa. After intravenous bolus doses, metabolic elimination is rapid, with a half-life of 8 to 14 minutes. More than 85% of an administered dose of 5-FU is enzymatically inactivated by DPD, the rate-limiting enzyme in the catabolism of 5-FU.
A pharmacogenetic syndrome has been identified in which partial or compete deficiency in the DPD enzyme is present in 3% to 5% and 0.1% of the general population, respectively. As DPD catalyzes the rate-limiting step in the catabolic pathway of 5-FU, a deficiency of DPD can result in a clinically dangerous increase in the anabolic products of 5-FU. Unfortunately, patients with DPD deficiency do not manifest a phenotype only until they are treated with 5-FU, and in that setting, they can develop severe GI toxicity in the form of mucositis and/or diarrhea, myelosuppression, neurologic toxicity, and in rare cases, death. In patients being treated with 5-FU or any other fluoropyrimidine, it is important to consider DPD deficiency in patients who present with excessive, severe toxicity.12 It is now increasingly appreciated that DPD mutations are unable to account for all of the observed cases of excessive 5-FU toxicity, because up to 50% of patients who experience 5-FU toxicity will have no documented alterations in the DPD gene. Moreover, individuals with normal DPD enzyme activity may be diagnosed with high plasma levels of 5-FU, resulting in increased toxicity. Although DPD enzyme activity can be assayed from peripheral blood mononuclear cells in a specialized laboratory, routine phenotypic and genotypic screenings for DPD deficiency prior to 5-FU therapy are not yet available.
Biomodulation of 5-FU
Significant efforts have focused on enhancing the antitumor activity of 5-FU through biochemical modulation in which 5-FU is combined with various agents, including leucovorin, MTX, N-phosphonacetyl-L-aspartic acid, interferon-α, interferon-γ, and a whole host of other agents.13 For the past 20 to 25 years, the reduced folate LV has been the main biochemical modulator of 5-FU. An alternative approach has been to alter the schedule of 5-FU administration. Given the S-phase specificity of this agent, prolonged exposure of tumor cells to 5-FU would increase the fraction of cells being exposed to the drug. Overall response rates are significantly higher in patients treated with infusional schedules of 5-FU than in those treated with bolus 5-FU, and this improvement in response rate has translated into an improved progression-free survival. Moreover, the overall safety profile is improved with infusional regimens. A hybrid schedule of bolus and infusional 5-FU was originally developed in France, and this regimen has shown superior clinical activity compared with bolus 5-FU schedules. This hybrid schedule has now been simplified by using only the 46-hour infusion of 5-FU and completely eliminating the 5-FU bolus doses.
Toxicity
The spectrum of 5-FU toxicity is dose- and schedule-dependent (Table 19.2). The main side effects are diarrhea, mucositis, and myelosuppression. The dermatologic HFS is more commonly observed with infusional 5-FU therapy. Acute neurologic symptoms have also been reported, and they include somnolence, cerebellar ataxia, and upper motor signs. Treatment with 5-FU can, on rare occasions, cause coronary vasospasm, resulting in a syndrome of chest pain, cardiac enzyme elevations, and electrocardiographic changes. Cardiac toxicity seems to be related more to infusional 5-FU than bolus administration.14
CAPECITABINE
Capecitabine is an oral fluoropyrimidine carbamate that was rationally designed to allow for selective 5-FU activation in tumor tissue.15 This oral agent was initially approved in anthracycline-and taxane-resistant breast cancer and subsequently approved for use in combination with docetaxel as second-line therapy in metastatic breast cancer and in combination with lapatinib, a tyrosine-kinase inhibitor of human epidermal growth factor receptor type 2 (HER2) and epidermal growth factor receptor (EGFR) in women with HER2-positive metastatic breast cancer following progression on trastuzumab-based therapy.16 This agent is also approved by the U.S. Food and Drug Administration (FDA) for the first-line treatment of mCRC and as adjuvant therapy for stage III colon cancer when fluoropyrimidine therapy alone is preferred.17 In Europe and throughout much of the world, the combination of capecitabine plus oxaliplatin (XELOX) is approved for the treatment of mCRC as well as for the adjuvant therapy of stage III colon cancer.18 In addition, recent studies have documented the noninferiority of capecitabine to 5-FU when combined with cisplatin in the treatment of metastatic gastric cancer.
Clinical Pharmacology
Capecitabine is rapidly and extensively absorbed by the gut mucosa, with nearly 80% oral bioavailability. It is inactive in its parent form and undergoes enzymatic conversion via three successive steps. Of note, the third and final step occurs in tumor tissue and involves the conversion of 5′-deoxy-5-fluorouridine to 5-FU by the enzyme thymidine phosphorylase (TP), which is expressed at much higher levels in tumors when compared with corresponding normal tissue. Capecitabine and capecitabine metabolites are primarily excreted by the kidneys, and in contrast to 5-FU, caution must be taken in the presence of renal dysfunction, with appropriate dose modification. The use of capecitabine is absolutely contraindicated in patients whose creatinine clearance is less than 30 mL per minute. The FDA and Roche have added a black box warning and strengthened the precautions section on the capecitabine label about the drug–drug interaction between warfarin and capecitabine-based chemotherapy. It is generally recommended to do weekly monitoring of the coagulation parameters (prothrombin time/international normalized ratio [PT/INR]) for all patients receiving concomitant warfarin and capecitabine, with an appropriate adjustment of warfarin dose.
Toxicity
Similar to what is observed with infusional 5-FU, the main side effects of capecitabine include diarrhea and HFS. Of note, the incidence of myelosuppression, neutropenic fever, mucositis, alopecia, and nausea/vomiting is lower with capecitabine when compared with 5-FU. Elevations in indirect serum bilirubin can be observed, but are usually transient and clinically asymptomatic. Patients in the United States appear to be unable to tolerate as high doses of capecitabine as European patients, either as monotherapy or in combination with other cytotoxic chemotherapy.19 Although the underlying reasons for this discrepancy are not known, it may in part be related to the increased fortification of the US diet with folate and the increased focus on vitamin and folic acid supplementation.
S-1
S-1 is an oral fluoropyrimidine that consists of tegafur (FT), a prodrug of 5-FU, combined with two 5-FU biochemical modulators: 5-chloro-2,4-dihydroxypyridine (gimeracil or CDHP), a competitive inhibitor of DPD, and oteracil potassium, which inhibits phosphorylation of 5-flurouracil in the GI tract, thereby decreasing serious GI toxicities such as nausea/vomiting, mucositis, and diarrhea.20 As with other oral agents, S-1 offers several advantages over 5-FU, including ease of administration, no risks associated with use of central venous access such as infection, thrombosis, etc., and reduced toxicities, especially neurotoxicity. Although S-1 has yet to be approved by the FDA, it has been approved for the treatment of gastric cancer, head and neck, colorectal cancer (CRC), non–small-cell lung, breast, pancreatic, and biliary tract cancers in several countries in Asia and for the treatment of advanced gastric cancer in combination with cisplatin in a large number of European countries.
Clinical Pharmacology
S-1 was designed to provide continuous 5-FU plasma exposure comparable to the intravenous (IV) infusion. FT, the 5-FU prodrug, is absorbed in the small intestine and converted to 5-FU through the liver microsomal P-450 metabolizing enzyme system (CYP2A6). Most of the 5-FU is degraded (85%) by DPD, leading to the formation of fluoro-beta-alanine (FBAL).21 CDHP inhibits DPD, thus allowing higher concentrations of 5-FU to enter the anabolic pathway and enhance its therapeutic effect. Additionally, the inhibition of DPD leads to a decreased amount of FBAL formation, which presumably leads to reduced neurotoxicity. Oteracil is the final component of the S-1 formulation, and it inhibits orotate phosphoribosyltransferase in the GI mucosa, which prevents the formation of fluorouridine monophosphate (FUMP), thereby decreasing GI toxicity.
The maximum tolerated dose was established at 80 mg/m2 in two divided doses for a Japanese population and 25 mg/m2 twice a day for a Caucasian population. This interethnic variability of S-1 pharmacokinetics and pharmacodynamics has been attributed to differences in the CYP2A6 genotypes.22 Studies have demonstrated a high frequency of allelic variants CYP2A6*4, *7, and *9 in East Asians than in Caucasians, which might be associated with reduced enzymatic activity and decreased activation of FT. On the other hand, higher FT metabolism is seen in Caucasian patients due to higher CYP2A6 activity. However, investigators have established similar 5-FU exposure between these two ethnic groups. These findings were explained by higher CDHP exposure in Asians, resulting in increased DPD inhibition and slower catabolism of 5-FU, despite having low CYP2A6 activity, whereas Caucasians had higher CYP2A6 activity but faster 5-FU clearance.
Clinical Toxicity
Clinical studies have shown that the GI toxicities associated with S-1, such as diarrhea, nausea, vomiting, and hyperbilirubinemia, are more prominent in Western patients, whereas hematologic toxicities are more prevalent in Japanese patients. The difference in safety profile cannot be explained by differences in 5-FU exposure, because pharmacokinetic studies have shown that overall drug exposures are similar. A potential explanation might involve interethnic variations in TS promoter enhancer region polymorphisms, which are more frequently seen in Asians or in Caucasians on a higher folate diet.
CYTARABINE
Cytarabine (ara-C) is a deoxycytidine nucleoside analog isolated from the sponge Cryptotethya crypta, and it differs from its physiologic counterpart by virtue of a stereotypic inversion of the 2′-hydroxyl group of the sugar moiety.23A regimen of ara-C, combined with an anthracycline and given as a 5- or 7-day continuous infusion, is considered the standard induction treatment for acute myeloid leukemia (AML). Ara-C is active against other hematologic malignancies, such as non-Hodgkin’s lymphoma, chronic myelogenous leukemia, and acute lymphocytic leukemia (see Table 19.1). However, this agent has absolutely no activity against solid tumors.
Mechanism of Action
Ara-C enters cells via nucleoside transport proteins, the most important one being the equilibrative inhibitor-sensitive (ES) receptor. Once inside the cell, ara-C requires activation for its cytotoxic effects.23,24The first metabolic step is the conversion of ara-C to the monophosphate form ara-cytidine monophosphate (ara-CMP) by the enzyme deoxycytidine kinase (dCK) with subsequent phosphorylation to the di- and triphosphate metabolites, respectively. Ara-cytidine triphosphate (ara-CTP) is a potent inhibitor of DNA polymerases α, β, and γ, which in turn interferes with DNA chain elongation, DNA synthesis, and DNA repair. Ara-CTP is also incorporated directly into DNA and functions as a DNA chain terminator, interfering with chain elongation. Catabolism of ara-C involves two key enzymes, cytidine deaminase and deoxycytidylate deaminase. These breakdown enzymes convert ara-C and ara-CMP into the inactive metabolites, ara-uridine (ara-U) and ara-uridine monophosphate (ara-UMP), respectively. The balance between intracellular activation and degradation is critical in determining the amount of drug that is ultimately converted to ara-CTP and, thus, its subsequent cytotoxic and antitumor activity.
Mechanisms of Resistance
Several resistance mechanisms to ara-C have been described. An impaired transmembrane transport, a decreased rate of anabolism, and an increased rate of catabolism may result in the development of ara-C resistance.23,25,26 The level of cytidine deaminase enzyme activity has been shown to correlate with clinical response in patients with AML undergoing induction chemotherapy with ara-C–containing regimens.
Clinical Pharmacology
Ara-C has poor oral bioavailability given its extensive deamination within the GI tract. Thus, ara-C is administered intravenously via continuous infusion. After administration, ara-C undergoes extensive metabolism in the liver, plasma, and peripheral tissues. Within 24 hours, up to 80% of drug is recovered in the urine as the ara-U metabolite. Ara-C crosses the blood–brain barrier when used at high doses, with cerebrospinal fluid levels between 7% and 14% of plasma levels and reaching peak levels of up to 10 μM.
Toxicity
The toxicity profile of ara-C is highly dependent on the dose and schedule of administration. Myelosuppression is dose-limiting with a standard 7-day regimen. Leukopenia and thrombocytopenia are observed most frequently, with nadirs occurring between days 7 and 14 after drug administration. GI toxicity commonly manifests as a mild-to-moderate degree of anorexia, nausea, and vomiting along with mucositis, diarrhea, and abdominal pain. In rare cases, acute pancreatitis has been observed. The ara-C syndrome has been described in pediatric patients with hematologic malignancies, usually begins within 12 hours after the start of drug infusion, and is characterized by fever, myalgia, bone pain, maculopapular rash, conjunctivitis, malaise, and occasional chest pain.
The administration of ara-C at high doses (2 to 3 g/m2 with each dose) is associated with profound myelosuppression.27 Severe GI toxicity in the form of mucositis and/or diarrhea is also observed. Neurologic toxicity is significantly more common with high-dose ara-C than with standard doses, and presents with seizures, cerebral and cerebellar dysfunction, and peripheral neuropathy. Clinical signs of cerebellar dysfunction occur in up to 15% of patients and include dysarthria, dysmetria, and ataxia. Change in alertness and cognitive ability, memory loss, and frontal lobe release signs reflect cerebral toxicity. Despite discontinuation of therapy, clinical recovery is incomplete in up to 30% of affected patients. Pulmonary complications may include noncardiogenic pulmonary edema, acute respiratory distress, and pneumonia, resulting from Streptococcus viridans infection. Other side effects associated with high-dose ara-C include conjunctivitis (often responsive to topical corticosteroids), a painful HFS, and rarely, anaphylactic reactions.
GEMCITABINE
Gemcitabine (2′,2′-difluorodeoxycytidine) is a difluorinated deoxycytidine analog. Despite its similarity in structure, metabolism, and mechanism of action to ara-C, the spectrum of antitumor activity of gemcitabine is much broader.23,28 This compound has significant clinical activity against several human solid tumors, including pancreatic, bile duct, gall bladder, small cell and non–small-cell lung, bladder, ovary, and breast cancers as well as hematologic malignancies, namely Hodgkin’s and non-Hodgkin’s lymphoma (see Table 19.1).
Mechanism of Action
The transport of gemcitabine into cells requires the nucleoside transporter system. Gemcitabine is inactive in its parent form and requires intracellular activation for its cytotoxic effects. The steps involved in the metabolic activation of gemcitabine are similar to those observed with ara-C, with both drugs being activated by the same enzymatic machinery to the active triphosphate metabolite (see Fig. 19.2). Gemcitabine triphosphate is then incorporated into DNA, resulting in chain termination and the inhibition of DNA synthesis and function, or the triphosphate form can directly inhibit DNA polymerases α, β, and γ, which in turn, interferes with DNA chain elongation, DNA synthesis, and DNA repair. The triphosphate metabolite is also a potent inhibitor of ribonucleotide reductase, which further mediates inhibition of DNA biosynthesis by reducing the levels of key deoxynucleotide pools.29
Mechanisms of Resistance
Several mechanisms of resistance to gemcitabine have been described in various preclinical experimental models.30 Gemcitabine is a polar nucleoside analog that requires the activity of human equilibrative nucleoside transporter 1 (hENT1) to enter cells and exert its cytotoxic effects. Preclinical data in human pancreatic cancer cell lines showed that gemcitabine resistance is negatively correlated with hENT1 expression and can be induced by specific inhibitors of hENT1.31 Clinical data also support the concept that a lack of hENT1 may be predictive of resistance to gemcitabine. CO-101, a lipid-drug conjugate of gemcitabine, was rationally designed to enter cells independently of hENT1. Unfortunately, two studies in pancreatic cancer failed to show any benefit of CO-101.
Additionally, several enzymes involved in the intracellular metabolism of gemcitabine have been implicated in the development of cellular drug resistance, including reduced expression and/or deficiency in dCK enzyme activity as well as increased expression and/or activity of the catabolic enzymes cytidine deaminase and dCMP deaminase. Recent studies have also identified a subset of CD44-positive cancer stem cells within pancreatic tumors that sustain tumor formation and growth, and are resistant to gemcitabine therapy.33
Clinical Pharmacology
Gemcitabine is administered via the intravenous route, typically over a 30-minute intravenous infusion, and it undergoes extensive metabolism by deamination to the catabolic metabolite, difluorodeoxyuridine (dFdU), with more than 90% of the metabolized drug being recovered in urine. Plasma clearance is about 30% lower in women and in elderly patients, and this pharmacokinetic difference may result in an increased risk of toxicity in these respective patient populations. The initial findings from pilot pharmacokinetic studies suggested that gemcitabine, when given at a fixed dose rate (FDR) intravenous infusion of 10 mg/m2 per minute, produced the highest accumulation of active dFdCTP metabolites in peripheral blood mononuclear cells, which led to a randomized phase II trial that compared gemcitabine 1,500 mg/m2 by FDR or 2,200 mg/m2 of gemcitabine over 30 minutes. Although this phase II study suggested an improved overall survival with FDR, a subsequent phase III trial failed to confirm the survival advantage of gemcitabine by FDR over its conventional administration schedule.34
Toxicity
Gemcitabine is a relatively well-tolerated drug when used as a single agent. The main dose-limiting toxicity is myelosuppression, with neutropenia more commonly experienced than thrombocytopenia. Toxicity is schedule dependent, with longer infusions producing greater hematologic toxicity. Transient flulike symptoms, including fever, headache, arthralgias, and myalgias, occur in 45% of patients. Asthenia and transient transaminasemia may occur. Renal microangiopathy syndromes, including hemolytic-uremic syndrome and thrombotic thrombocytopenic purpura, have been reported rarely.
6-THIOPURINES
The development of the purine analogs in cancer chemotherapy began in the early 1950s with the synthesis of the thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG). 6-MP has an important role in maintenance therapy for acute lymphoblastic leukemia, whereas 6-TG is active in remission induction and in maintenance therapy for AML (see Table 19.1).
Mechanism of Action
The thiopurines, 6-MP and 6-TG, act similarly with respect to their cellular biochemistry.34 In their respective monophosphate nucleotide forms, they inhibit enzymes involved in de novo purine synthesis and purine interconversion reactions. The triphosphate nucleotide forms can get directly incorporated into either cellular RNA or DNA, leading to the inhibition of RNA and DNA synthesis and function, respectively.
Mechanisms of Resistance
The development of cellular resistance to 6-thiopurines results from a decreased level of key cytotoxic nucleotide metabolites, either through decreased formation or increased breakdown. Resistant cells have been identified that express either complete or partial deficiency of the activating enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT). In clinical samples derived from patients with AML, drug resistance has been associated with increased concentrations of a membrane-bound alkaline phosphatase or a conjugating enzyme, 6-thiopurine methyltransferase (TPMT), the end-result being reduced formation of cytotoxic thiopurine nucleotides. Finally, the decreased expression of mismatch repair enzymes, including hMLH1 and hMSH2, has been associated with cellular drug resistance.
Clinical Pharmacology
Oral absorption of 6-MP is highly erratic, and the relatively poor oral bioavailability is mainly related to rapid first-pass metabolism in the liver. The major route of drug elimination is via metabolism by several enzymatic pathways. 6-MP is oxidized to the inactive metabolite 6-thiouric acid by xanthine oxidase. Enhanced 6-MP toxicity may result from the concomitant administration of 6-MP and the xanthine oxidase inhibitor allopurinol. In patients receiving both 6-MP and allopurinol, the 6-MP dose must be reduced by at least 50% to 75%. 6-MP also undergoes S-methylation by the enzyme TPMT to yield 6-methylmercaptopurine.35
6-TG is administered orally in the treatment of AML. Its oral bioavailability is erratic, with peak plasma levels occurring 2 to 4 hours after ingestion. The catabolism of 6-TG differs from 6-MP in that it is not a direct substrate for xanthine oxidase.
TPMT enzyme activity may vary considerably among patients as a result of point mutations or loss of alleles of TPMT.36 Approximately 0.3% of the Caucasian population expresses either a homozygous deletion or a mutation of both alleles of the TPMT gene. In these patients, grossly elevated thiopurine nucleotides concentrations, profound myelosuppression with pancytopenia, and extensive GI symptoms are observed after only a brief course of thiopurine treatment. An estimated 10% of patients may be at increased risk for toxicity because of heterozygous loss of the gene or a mutant allele coding for a less enzymatically active TPMT.
Toxicity
The major dose-related toxicities of the thiopurines are myelosuppression and GI toxicity in the form of nausea/vomiting, anorexia, diarrhea, and stomatitis.37 In TPMT-deficient patients, dosage reduction to 5% to 25% of the standard dosage is necessary to prevent severe excessive toxicity. Thiopurine hepatotoxicity occurs in up to 30% of adult patients and presents mainly as cholestatic jaundice, although elevations of hepatic transaminases may also be seen. Combinations of thiopurines with other known hepatotoxic agents should be avoided, and liver function should be closely monitored. The thiopurines are also potent suppressors of cell-mediated immunity, and prolonged therapy results in an increased predisposition to bacterial and parasitic infections.
FLUDARABINE
Fludarabine (9-β-D-arabinosyl-2-fluoroadenine monophosphate, F-ara-AMP) is an active agent in the treatment of chronic lymphocytic leukemia (CLL) (see Table 19.1).38,39 It is also active against indolent non-Hodgkin’s lymphoma, prolymphocytic leukemia, cutaneous T-cell lymphoma, and Waldenström macroglobulinemia. This agent has also shown promising activity in mantle cell lymphoma. In contrast to its activity in hematologic malignancies, this compound has virtually no activity against solid tumors.
Mechanism of Action
The active cytotoxic metabolite is the triphosphate metabolite F-ara-ATP, which competes with deoxyadenosine triphosphate (dATP) for incorporation into DNA and serves as a highly effective chain terminator. In addition, F-ara-ATP directly inhibits enzymes involved in DNA replication, including DNA polymerases, DNA primase, DNA ligase I, and ribonucleotide reductase.37 F-ara-ATP is also incorporated into RNA, causing the inhibition of RNA function, processing, and mRNA translation. In contrast to other antimetabolites, fludarabine is active against nondividing cells. In fact, the primary effect of fludarabine may result from activation of apoptosis, through an as yet ill-defined mechanisms.39 This finding may explain the activity of fludarabine in indolent lymphoproliferative diseases with relatively low growth fractions.
Mechanisms of Resistance
The decreased expression of the activating enzyme dCK resulting in diminished intracellular formation of F-ara-AMP is one of the main resistance mechanisms identified in preclinical models.38 A high degree of cross-resistance develops to multiple nucleoside analogs, requiring activation by dCK, including cytarabine, gemcitabine, cladribine, and clofarabine. Reduced cellular transport of drug has also been identified as a resistance mechanism.
Clinical Pharmacology
Peak concentrations of F-ara-A are reached 3 to 4 hours after intravenous administration.40 The main route of elimination is via the kidneys, with about 25% of a given dose of drug being excreted unchanged in the urine.
Toxicity
Myelosuppression and immunosuppression are the major side effects of fludarabine as highlighted by dose-limiting and possibly cumulative lymphopenia and thrombocytopenia. Suppression of the immune system affects T-cell function more than B-cell function. Fevers, often in the setting of neutropenia, occur in 20% to 30% of patients. Lymphocyte counts, specifically CD4-positive cells, decrease rapidly after the initiation of therapy, and recovery of CD4-positive cells to normal levels may take longer than 1 year. Common opportunistic pathogens include the varicella-zoster virus, Candida, and Pneumocystis carinii. In general, patients are empirically placed on sulfamethoxazole trimethoprim prophylaxis to prevent the development of P. carinii infection.
CLADRIBINE
Cladribine (2-CdA) is a purine deoxyadenosine analog, and it is the drug of choice for hairy cell leukemia with activity in low-grade lymphoproliferative disorders (see Table 19.1).41,42 Salvage treatment of patients previously treated with interferon-α or splenectomy is as effective as first-line treatment. Retreatment with cladribine results in a complete response in up to 60% of relapsing patients. In addition, this agent has promising activity in patients with CLL and non-Hodgkin’s lymphoma.
Mechanism of Action
Upon entry into the cell, 2-CdA undergoes an initial conversion to cladribine-monophosphate (Cd-AMP) via the reaction catalyzed by dCK, and Cd-AMP is subsequently metabolized to the active metabolite, cladribine-triphosphate. The triphosphate metabolite competitively inhibits incorporation of the normal dATP nucleotide into DNA, a process that results in the termination of chain elongation.43 Progressive accumulation of the triphosphate metabolite leads to an imbalance in deoxyribonucleotide pools, thereby inhibiting further DNA synthesis and repair. Finally, the triphosphate metabolite is a potent inhibitor of ribonucleotide reductase, which further facilitates the inhibition of DNA biosynthesis.
Mechanisms of Resistance
Resistance to 2-CdA has been attributed to altered intracellular drug metabolism. A reduction in the activity of dCK, the enzyme responsible for generating cytotoxic nucleotide metabolites, is a major determinant of acquired resistance. The monophosphate and triphosphate metabolites are dephosphorylated by the cytoplasmic enzyme 5′-nucleotidase. Interestingly, resistant cells derived from a patient with CLL exhibited both low levels of dCK expression and high levels of 5′-nucleotidase.
Clinical Pharmacology
2-CdA is orally bioavailable, with 50% of an administered dose orally absorbed. Approximately 50% of an administered dose of drug is cleared by the kidneys, and 20% to 35% of the drug is excreted unchanged in the urine. Of note, this nucleoside can cross the blood–brain barrier with penetration into the cerebrospinal fluid.
Toxicity
At conventional doses, myelosuppression is dose limiting. After a single course of drug, recovery from thrombocytopenia usually occurs within 2 to 4 weeks, whereas recovery from neutropenia takes place in 3 to 5 weeks. GI toxicities are generally mild, with nausea/vomiting and diarrhea. Mild-to-moderate neurotoxicity occurs in 15% of patients and is at least partly reversible with discontinuation of the drug. Immunosuppression accounts for the late morbidity observed in 2-CdA–treated patients. Lymphocyte counts, particularly CD4-positive cells, decrease within 1 to 4 weeks of drug administration and may remain depressed for several years.44 After discontinuation of 2-CdA, a median time of up to 40 months may be required for complete recovery of normal CD4-positive counts. Although opportunistic infections occur, they do so less frequently than with fludarabine therapy. Infectious complications correlate with decreases in the CD4-positive count, and they include herpes zoster, Candida, Pneumocystis, Pseudomonas aeruginosa, Listeria monocytogenes, Cryptococcus neoformans, Aspergillus, P. carinii, and cytomegalovirus.
CLOFARABINE
Clofarabine is a purine deoxyadenosine nucleoside analog, and it is approved for the treatment of pediatric patients with relapsed or refractory acute lymphoblastic leukemia (see Table 19.1).45 Ongoing studies are exploring the benefit of clofarabine alone and in combination with other agents in less heavily pretreated patients and in the use of different dose schedules for other hematologic malignancies.46
Mechanism of Action
Clofarabine is inactive in its parent form and, like other purine analogs, it requires intracellular activation by dCK to form the monophosphate nucleotide, which undergoes further metabolism to the cytotoxic triphosphate metabolite. Clofarabine triphosphate is then incorporated into DNA, resulting in chain termination, and inhibition of DNA synthesis and function or the triphosphate form can directly inhibit DNA polymerases α, β, and γ, which in turn, interferes with DNA chain elongation, DNA synthesis, and DNA repair. The triphosphate metabolite is also a potent inhibitor of ribonucleotide reductase, further mediating the inhibition of DNA biosynthesis by reducing the levels of key deoxyribonucleotide pools.
Mechanisms of Resistance
Several resistance mechanisms have been identified in various preclinical systems, and they include decreased activation of the drug through the reduced expression of the anabolic enzyme deoxycytidine kinase, the decreased transport of drug into cells via the nucleoside transporter protein, and the increased expression of CTP synthetase activity resulting in increased concentrations of competing physiologic nucleotide substrate dCTP. To date, the precise resistance mechanism(s) that are relevant in the clinical setting remain to be determined.
Clinical Pharmacology
Approximately 50% to 60% of an administered dose of drug is excreted unchanged in the urine, and the terminal half-life is on the order of 5 hours. To date, the pathways for nonrenal elimination have not been well defined. Caution should be exercised in patients with abnormal renal function, and concomitant use of medications known to cause renal toxicity should be avoided during drug treatment.
Toxicity
Myelosuppression is dose limiting with neutropenia, anemia, and thrombocytopenia. The capillary leak syndrome (systemic inflammatory response syndrome) presents with tachypnea, tachycardia, pulmonary edema, and hypotension.47 In essence, this adverse event is part of the tumor lysis syndrome and results from rapid cytoreduction of peripheral leukemic cells following treatment.47 Other side effects may include nausea/vomiting, reversible liver dysfunction (hyperbilirubinemia and elevated serum transaminases), renal dysfunction (approximately 10%), and cardiac toxicity in the form of tachycardia and acute pump dysfunction.
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