Jeffrey W. Clark
From the first identification of mutations unique to neoplastic cells, the dream has been that targeting those mutations would provide a means of specifically killing malignant cells while sparing normal tissues. Discoveries in biological mechanisms of cancer cells at the DNA, RNA, and protein level are continuing to lead to rapid improvements in understanding the specific processes important for the survival, proliferation, and metastasis of different types of neoplastic cells. By combining these with technologic advances that allow sophisticated manipulation and analysis of nucleic acids and proteins, agents that target proteins or genes critical to the neoplastic process have been identified and further developed. A variety of compounds, including designed small molecules, monoclonal antibodies (mAbs), peptidomimetics, siRNAs, antisense oligonucleotides, and expressed genes, and other molecularly targeted therapies are being evaluated for potential treatment of cancer (Table 30.1). An increasing number of these have sufficient clinical activity to be important components of current therapy for a number of different malignancies (Table 30.2). Approved mAbs include trastuzumab (Herceptin) for breast cancer; rituximab, ibritumomab tiuxetan (Zevalin with Yttrium-90), and tositumomab + I131 (Bexxar) for follicular B-cell non-Hodgkin's lymphomas (NHLs); alemtuzamab for B-cell chronic lymphocytic leukemia (B-CLL); gemtuzumab ozogamicin for acute myelogenous leukemia (AML); and bevacizumab and cetuximab for colorectal cancer. Targeted small molecules include imatinib (Gleevec) for chronic myeloid leukemia (CML) and gastrointestinal stromal tumors (GISTs) and gefitinib (Iressa) and erlotinib (Tarceva) for non–small cell lung cancer (NSCLC). The immunotoxin denileukin diftitox (diphtheria toxin-interleukin 2 [IL-2] fusion protein, DAB486IL-2) is approved for the treatment of cutaneous T-cell lymphomas. A schematic representation of signaling through growth factor receptors and, where currently approved, targeted anticancer agents that inhibit this process is shown in Figure 30.1. A large number of other agents are in clinical trials, and this list is certain to grow.
Conceptually, targeting a specific gene or protein appears straightforward. Theoretically, antitumor agents can be designed based on known sequence data, obviating the need to empirically screen a large number of compounds. However, many issues in the successful development of these agents remain to be addressed. It is important that the targeted protein in the neoplastic cell either be sufficiently different (if mutant) to provide a specific target or else not be critical for the survival of normal cells in order to decrease the risk of serious toxicity. Targeting one gene or protein may have limited effects on growth of neoplastic cells unless that gene or protein is vital for the proliferation or survival of those cells. In many cases, which gene (or genes) should be targeted is unclear. Genes important for the process by which a cell becomes a neoplastic cell may not be important for the continued proliferation or survival of the cell and therefore may be irrelevant targets for treating established malignancies. Inhibition of many genes, even if they are important for neoplastic cell growth, may only be cytostatic. It would be more useful to target genes whose inhibition (or stimulation) induces cell death (i.e., by apoptosis) or terminal differentiation.1 Ultimately, these approaches must be capable of eliminating (or at least leading to prolonged growth suppression of) all tumor cells, either by themselves or in combination with other agents, if they are to be effective in curing patients. Agents with cytostatic effects might need to be used in combination with other therapy.
TABLE 30.1 TYPES OF MOLECULARLY TARGETED COMPOUNDS
These are all important issues that need to be addressed for successful development of targeted therapies. Despite the difficulties, strategies using each of the just-mentioned approaches have been shown to be effective in animal models, and, as outlined above, a number of targeted molecules have significant clinical activity against several human tumors. Clearly, a number of genes could be targeted simultaneously or sequentially, and combinations of approaches inhibiting certain genes (e.g., oncogenes) or their protein products and enhancing expression of others (e.g., tumor suppressor genes) may ultimately be used. Continued analysis of the human genome and improved understanding of complex interactions among different genes and proteins will continue to provide a large range of potential targets in the coming decades. With numerous potential targets, determining at an early stage which of these are most fruitful to pursue in treating cancer remains an important challenge.
RATIONALE FOR TARGETING GROWTH FACTOR RECEPTORS AND DOWNSTREAM SIGNALING PATHWAYS
Until the past decade, cancer therapy primarily used drugs that lack selectivity for tumor cells. These agents are predominantly cytotoxins, lethal for both neoplastic and normal cells, with a narrow therapeutic index. They were discovered through cytotoxic screens rather than efforts to exploit targets that are specific for malignant cells. They have improved treatment of many solid tumors and have cured some hematologic malignancies and selected solid tumors, but with significant toxicity to normal tissues. They have also improved cure rates for several malignancies when used in the adjuvant setting or in combination with radiation therapy. However, they have not been curative for the majority of patients with metastatic solid tumors. Therefore, new approaches for the treatment of the majority of cancers are needed.
The rapid growth in knowledge of cancer biology has led to a more rational approach to therapeutic discovery through the targeting of pathways and proteins that are essential for the survival of cancer cells and, either quantitatively or qualitatively, unique to cancer. 1, 2, 3, 4, 5, 6, 7 Studies in cancer biology have revealed a number of such pathways and proteins. Many of these are either overexpressed or in some way altered in cancer cells. Possible targets include the overexpression of growth factors or their receptors, such as the epidermal growth factor receptor (EGFR) family, including the HER-2-neu tyrosine kinase; angiogenic pathways (e.g., vascular endothelial growth factor [VEGF] or platelet-derived growth factor [PDGF]–mediated angiogenesis) that provide a blood supply for the expanding tumor; antiapoptotic mechanisms that antagonize cell death, such as overexpression of Bcl-2 or decreased BAX expression; and enhanced activity of intracellular signaling pathways that promote growth, impede apoptosis, or both. 1, 2, 3, 4, 5, 6, 7 The most unique targets on cancer cells are mutant genes. Examples of mutations of growth factor receptors themselves include c-KIT in GIST and EGFR in certain cases of NSCLC, both of which have proven clinically useful targets for small molecular inhibitors of their tyrosine kinase activity. Mutations of genes in signaling pathways downstream of growth factor receptors, such as activation of one of the ras family of proteins, can lead to unrestrained growth. In addition, or alternatively, important brakes on proliferation induced by stimulation of constitutively active mutant growth factor receptors can be lost, such as by mutations or other processes that inhibit the function of p53, retinoblastoma (RB), or the phosphate and tensin homolog (PTEN), which regulates the PI3-kinase pathway. 5, 6, 7, 8
TABLE 30.2 APPROVED MOLECULARLY TARGETED AGENTS
Figure 30.1 Schematic of growth factor ligand binding to cell surface receptors and the subsequent downstream signaling cascade that leads to biological effects such as survival, proliferation, metastasis, and angiogenesis. Double asterisks indicate steps at which approved monoclonal antibodies inhibit the process. Single asterisks indicate steps at which small tyrosine kinase molecules inhibit the process.
Recognition of a potential target for drug discovery is only the first step. The target requires “validation” if it is to warrant the extensive efforts required to develop a clinically effective antitumor agent. A number of theoretical and practical questions must be answered before the investment is justified. Among the most relevant questions are the following:
1. Are the subject gene and its protein found in human tumors, and is there selective expression in tumors versus normal tissues?
2. Is function of the overexpressed or mutated target essential to the transformed behavior of the malignant cells? Does inhibition of the gene product change the phenotype of these cells? Does inhibition lead to the desired result (such as a decrease in metastasis) in an animal model? Experiments evaluating the biological effects when the subject gene is mutated, deleted, or neutralized with short RNAs with specific sequence that have been shown to selectively inhibit expression of genes within cells (small interfering or siRNAs) are important for addressing these questions. The discovery of the ability of siRNAs to inhibit specific genes within cells has provided a powerful tool for analyzing the role those genes play in the biology of the cell. Not only has this revolutionized the study of the effects of silencing specific genes because of its relative ease of use technically compared to the other techniques, but siRNAs also have potential as therapeutic agents. This is an area that is being actively investigated.
3. In the case of an overexpressed (as opposed to mutant) protein in a tumor, is the protein also expressed in key proliferating normal tissues, such as intestinal epithelium and bone marrow progenitors, or even nonproliferating tissues, such as heart, kidney, or brain, and does the protein therefore carry the risk for significant toxicity if targeted? Patterns of drug toxicity are often difficult to predict, but the profile of gene expression in normal tissues may provide helpful clues about potential toxicity of an agent directed against that target gene. Does a knockout of the gene have fatal consequences for the host (in animal models), indicating that inhibition of that gene or its protein product might lead to significant toxicity?
4. Are there closely related proteins that are essential for normal tissue function and survival of the host and that might be cross-targeted by the agent, making it nonselective as a molecularly targeted inhibitor?
These considerations are paramount in determining the choice of a target and the probability of success. Obviously, even the most validated target may not be amenable to a drug discovery strategy for a number of reasons. Unanticipated toxicities, interactions with previously inapparent receptors or proteins, pharmacologic problems in drug distribution, and unfavorable pharmacokinetic (PK) properties may defeat the most rational strategy.
A number of excellent reviews of high-priority molecular targets for cancer therapy are available. 1, 2, 3, 4, 5, 6, 7 Angiogenesis inhibitors and mAbs are covered elsewhere in this book. The following is a brief review of several of the growth factor receptor and downstream signaling pathways that have yielded substantial new leads for cancer treatment.
POTENTIAL MOLECULAR TARGETS
The current choice of a molecular target for anticancer therapy is often dictated by an important practical consideration. It is vastly easier to design an inhibitor of the function of a protein that is either constitutively activated by mutation or has increased functional activity by another mechanism, such as by being overexpressed, than it is to replace an inactive or deleted function. Thus, although mutations in tumor suppressor genes, such as those affecting the p53 or RB pathways, play a prominent role in tumorigenesis, it is difficult to restore the function of these proteins without restoring a fully functional gene. In those cases where protein function is suppressed as opposed to mutated, such as by the binding of MDM2 to p53, inhibitors of that binding might be able to restore function, but in this case the target is the inhibitor of the normal function of these genes. Thus, at the present time, the primary choices for targeting remain proteins that are either activated by a mutation or have increased activity by being overexpressed.1
APPROACHES TO IDENTIFYING AND SYNTHESIZING MODULATORS FOR MOLECULAR TARGETS
The diversity of approaches to developing inhibitors or modulators for identified targets in cancers is large, although small molecules, antibodies, and modified peptides are the agents with clearly established clinical value at this time.
One approach to developing agents targeting specific genes or proteins is to empirically screen a large number of compounds for activity and subsequently design better ones based on the structure of the active compound (this is the approach by which most anticancer agents have been developed). High-throughput screens allowing rapid evaluation of a large number of compounds have enhanced the utility of this approach. Alternatively, compounds can be designed based on the structure of the specific region being targeted, using known sequence and other information available about the gene or protein (e.g., x-ray crystallography, nuclear magnetic resonance [NMR] imaging, computer molecular modeling, and analysis). Clearly, some combination of these two approaches might be most useful—for example, lead identification by random high-throughput screening followed by lead optimization through structural studies of the inhibitor and target. This allows more rational design of antineoplastic molecules and, at the same time, efficient screening of potentially therapeutic agents with a wide range of structures.
The complexity of molecular structures and the frequent interaction between proteins and other proteins or molecules makes designing compounds based on actual structure (as opposed to DNA sequence) a significant undertaking that requires sophisticated technology (e.g., x-ray crystallography, extensive computer analysis, and molecular modeling). Certain proteins, such as growth factor receptors (GFRs), which are among the most attractive targets for anticancer therapy because of their accessibility and potential importance in proliferation and survival pathways, are large and therefore still difficult to analyze even with current analytic approaches. It is not yet possible to predict which properties of an agent might influence subsequent development of resistance (which remains a major obstacle to the ultimate effectiveness of any treatment), because mechanisms of resistance to the compound cannot be fully predicted beforehand. Nonetheless, knowledge of the critical surface for ligand interaction with the target and how this might be modified by changes in the protein or interacting proteins may allow the design of a series of compounds that could overcome resistance due to mutations in the targeted protein or in the proteins with which it interacts. These could be given either simultaneously, to prevent the selection of resistant cells, or possibly sequentially, to maintain the response for a longer time. The complexity of the body's handling of compounds makes predicting toxicology and PKs difficult. At the present time, pharmacologic features can only be determined from careful preclinical and clinical studies.
As with all forms of systemic cancer therapy, the ultimate usefulness of these treatment approaches depends on the ability to effectively deliver the agent to tumor cells; adequate binding to neoplastic cells (in the case of mAbs or other compounds used to activate the immune system or deliver radioactive compounds) or uptake by neoplastic cells; the presence or expression of the agent on or within cells for a sufficient time to lead to death or differentiation of those cells; a high gradient of concentration for the compound in malignant versus normal cells; the rate of elimination of the agent from normal and malignant cells; and the toxicity of the agent for normal tissues, acutely and chronically. Heterogeneity of neoplastic cells within tumors limits single-drug therapy and is also a problem for single molecularly targeted therapy. Therefore, approaches that target heterogeneous cell populations, including mechanisms for overcoming resistance, need to be part of the strategic plan in designing therapeutic use of these compounds.
There are a number potential negative pharmacologic factors for certain classes of targeted compounds, such as mAbs, antisense oligonucleotides, and modified peptides. These include relatively large size, which makes delivery more difficult; complex structure, which contributes to decreased absorption, enhanced hepatic clearance, and decreased permeation from blood into tissues; natural nucleases and proteases that rapidly break down unmodified compounds; specific receptors for these agents that circulate in the blood or are expressed on normal cells and that alter their distribution; and potential immunogenicity, because these compounds often have features that make them directly immunogenic or inducers of host cytokine release, a property that can limit their long-term use.
Growth Factor Receptors
Growth factor receptors or their ligands represent some of the most attractive molecular targets for cancer therapy. They are overexpressed on a number of malignancies, mutated in several, and in both cases have increased activity, so that they are targets for inhibition. The importance of growth factors and downstream signaling pathways in a number of cellular processes essential for proliferation and survival of cells has been demonstrated. Studies have established the importance of growth factors (such as VEGF in the angiogneic process for a number of malignances) or their receptors (such as HER-2 in breast cancer) in the neoplastic process. There is significant evidence for the role of growth factors and their receptors (such as VEGF, VEGFR, EGF ligands, and EGFR) in the metastatic process.9, 10 Their presence on the cell surface makes them more readily accessable than many proteins. Thus, the rationale for targeting growth factors and their receptors for anticancer therapy is well established.
The members of the EGFR family of receptors, including HER-2, are especially attractive targets. The majority of cancers arise from epithelial cells that express EGFR. As outlined, EGFR family members have been shown to have a role in tumor cell survival and proliferation as well as the metastatic process. Inhibition of EGFR in certain preclinical models leads to death of malignant cells and significant antitumor response. As discussed above, mutant proteins provide the most specific target in cancer. EGFR are mutated in a subset of NSCLC tumors with adenocarcinoma or adenocarcinoma with bronchoalveolar cancer histology.11, 12 Mutations in c-KIT are found frequently in GIST tumors, and their targeting by imatinib has been very successful clinically.13, 14 Growth factor receptors have been shown to be overexpressed in certain malignances. These include overexpression of VEGF/VEGFR in a number of malignancies, platelet-derived growth factor receptors (PDGFRs) in brain tumors, and EGFRs in head and neck cancer as well as a number of other epithelial malignancies. 15, 16, 17
Downstream Signaling Pathways of Growth Factor Receptors
A number of signaling pathways downstream of growth factor receptors have been identified and shown to play important roles in cancers. The potential for targeting these pathways directly either alone or in combination with anti–growth factor receptor agents as a means of treating cancer continues to be actively investigated.
One of the first oncogenes to be recognized in human tumors was the mutation and constitutive activation of ras.18 Ras proteins play central roles in transducing signals important for a variety of critical processes in cells, including proliferation and differentiation.18 One of the key roles played by Ras proteins is in transmitting signals from GFRs to downstream signaling molecules. A scheme for ras function is shown in Figure 30.1. The ras protein family (K-ras, N-ras, and H-ras) are activated by upstream signaling from tyrosine kinase receptors, such as the EGFRs. Ras proteins are activated by binding guanosine triphosphate (GTP), and subsequently they activate downstream targets in the signaling cascade, including the raf kinase. In the process, GTP is hydrolyzed to guanosine diphosphate, and ras is inactivated. Mutations at codons 12, 13, or 61 of the ras genes constitutively activate ras by locking it in the GTP-bound state. This leads to activation of raf and the signal transduction pathway in the absence of growth factor stimulation. Ras must be bound to the plasma membrane to activate raf. In concert with other mutations, ras is transforming in normal cells. Ras mutations occur relatively frequently in a number of malignancies. For example, K-ras mutations are found in approximately 40 to 50% of colon cancers, 70 to 90% of pancreatic cancers, and 30% of adenocarcinomas of the lung. 18, 19, 20 N-ras is mutated in approximately 20 to 30% of acute nonlymphocytic leukemias.13 H-ras is mutated in a minority of bladder and head and neck cancers. Thus, as a molecular target, ras has attractive features.
There are several potential methods of inhibiting ras. The unprocessed native protein is inactive and requires sequential posttranslational modification to allow insertion in the plasma membrane, which is required for its active signaling function.18 It must first be farnesylated (attachment of a 15-carbon, lipophilic group) by soluble prenylation enzymes. The carbon terminal (C-terminal) CAAX motif of ras then directs the prenylated protein to the endoplasmic reticulum and Golgi, in which the C- terminal AAX residues are cleaved by a specific protease. The terminal prenylcysteine is then methylated by a prenyl cysteine methyl transferase found in the endomembrane system. The final product is exported to its active site in the plasma membrane. In the case of N- and H-ras, this occurs after further lipid attachment (palmitic acid) to another cysteine or cysteines. K-ras, which possesses a polybasic region upstream from the C-terminal peptide, does not require palmitoylation to localize in the plasma membrane. 18, 19, 20, 21
Initial attempts to develop compounds blocking ras function have been devoted to the discovery of inhibitors of the farnesylation reaction,22 although there has also been interest in exploring inhibition of prenyl cysteine methyl transferase and the ras proteases. In addition, targeting of downstream effectors of ras, such as the raf kinase, are also being pursued. 23 Potent and selective farnesyl transferase inhibitors (FTIs) with preference for H-ras inhibition have been isolated by selecting lead compounds in high-throughput screening. With subsequent structural refinement, a number of compounds entered clinical trial. Although some limited antitumor activity has been seen with several of these compounds, the ultimate clinical use of any of these compounds remains uncertain.
Experience with the FTIs taught valuable lessons about targeted drug discovery. A number of initial assumptions have proven to be invalid: (a) Contrary to what was initially believed, it is now clear that an ever growing number of proteins other than ras proteins undergo farnesylation. One or more other farnesylated proteins, such as RhoB, may be the critical targets in the inhibition of tumor cell growth by FTIs.24 (b) K-ras can be inserted into the cell membrane and thus activated through geranyl-geranylation, bypassing FTI inhibition. (c) Ras plays a role in signaling via a complex set of pathways in cells, and it is not always clear what effects might be produced by inhibition of its function in different cellular circumstances. 21, 22, 23, 24, 25 Not surprisingly, given these facts, antitumor activity of FTIs in cell culture does not necessarily correlate with the presence of ras mutation. In addition, the FTIs as a class are not selective for tumors but demonstrate a spectrum of toxicities in humans, including diarrhea, hepatotoxicity, neurotoxicity, cardiac conduction abnormalities, and myelosuppression.
Other targets within the ras pathway are also being attacked. As mentioned above, the raf kinase is an immediate downstream target of ras. Raf inhibition has been shown to have significant antitumor effects in preclinical models and remains a potentially valid target. 23 A molecule designed to target the raf kinase, BAY 43-9006, has had sufficient antitumor activity to be tested in clinical trials for a number of malignancies.23, 26 This compound is not entirely specific for the raf kinase. It has significant inhibiting activity for another potentially important target (VEGFR-2), making it difficult to determine what role the inhibition of raf is playing in its antitumor activity. Other inhibitors of raf as well as inhibitors of further downstream targets (such as ERK) in the ras pathway are also undergoing study.
A second prominent target that modifies signaling from growth factor receptors is the RB pathway.6 This pathway is named for the critical role played by the product of the RB gene, a protein that in its underphosphorylated state inhibits E2F, a transcription factor that promotes synthesis of messenger RNAs (mRNAs) for a number of proteins involved in DNA synthesis. The function of RB is, in turn, tightly controlled by a complex sequence of protein interactions that regulate its phosphorylation state. Two of the responsible kinases, cdk4 and cdk6, are activated by cyclin D and inhibited by p16 and p21. Multiple sites of mutation or alteration in this pathway can be involved in the neoplastic process; essentially any mutation or modification that eliminates or inactivates RB function (including by phosphorylation) will activate E2F and allow cell cycle progression. These alterations include loss of RB itself in patients with retinoblastoma; activation of cdk4 in melanoma; overexpression of cyclin D in many human tumors; and loss of p16 function (such as by mutation), which can occur in a number of malignancies. Most human tumors display an alteration of at least one component of this pathway, most frequently p16 deletion or cyclin D overexpression. Experimental models of RB loss or inactivation have confirmed the tumorigenic effect of mutations in this pathway. Thus, antitumor therapy targeted at inhibiting RB phosphorylation is rational.
An inhibitor of cdk4, flavopiridol, has entered clinical trials. Thus far it has had modest antitumor activity in phase I and early phase II studies, although it has had moderate activity against B-CLL.7 It fulfills many of the hypothesized advantages of molecularly targeted therapies. It has limited toxicity for normal proliferating tissues, induces apoptosis in tumor cells, and enhances cytotoxicity of traditional drugs. However, its mechanism of action, competitive inhibition of the adenosine triphosphate–binding site of the kinase, is characteristic of compounds that are not highly selective for only one kinase. This is true for a number of kinase inhibitors, including imatinib.
In fact, although flavoperidol inhibits cdk4, it lacks specificity for cdk4 in that it inhibits cdk1, cdk2, cdk7, cdk9, and, at higher (micromolar) concentrations, a number of other kinases. In addition, it suppresses expression of cyclin D1, the important activator of cdk4. Thus, whether its antitumor effects are attributable to inhibition of RB phosphorylation is unclear. This question can only be answered by detailed studies of the correlation of changes in RB phosphorylation status in tumors with the response of those malignancies to flavoperidol. The fact that flavoperidol has some antitumor activity (especially against B-CLL), that it potently induces apoptosis in various human tumor cells in a p53-independent manner, and that it has synergy with cytotoxins is reason enough to pursue its clinical development and that of related compounds.7
Mutant Proteins That Lead to Unrestrained Growth of Malignant Cells
The 9:22 translocation in CML has proven to be a particularly attractive target.27 The translocation places the Abl tyrosine kinase activity on chromosome 9 in juxtaposition to the breakpoint cluster region of chromosome 22. The resulting protein has a complex variety of functions, including a constitutively active tyrosine kinase that affects a number of signaling pathways within the cell. It is capable of cell transformation in mice. Antisense to the bcr-ablgene reverses the malignant phenotype and induces apoptosis in CML cells in vitro. Thus, a large body of preclinical data argues that bcr-ablinhibition should produce significant anti-CML effect. In fact, imatinib, a potent inhibitor of the tyrosine kinase activity of Bcr-Abl, is highly effective against CML, as discussed elsewhere in this chapter.
Few mutations or biological processes associated with human cancer provide such a clear target for drug development as the bcr-abl protein. Most epithelial cancers represent the evolution of multiple mutations, and targeting different genes may be necessary to kill individual clones of cells within a given cancer. Mutations or cellular processes that might be good candidates for targeting neoplastic cells more effectively include apoptosis, telomerase, critical growth factor signaling pathways [e.g., EGFR and HER-2-neu receptor], PTEN gene mutations and deletion in the PI3-kinase pathway, and various angiogenic targets, such as the VEGF and its receptor (see Chapter 35). 16, 17, 28, 29, 30 Clinical responses in patients treated with mAbs to HER-2-neu (using trastuzumab) or the EGFR (using cetuximab) and with EGFR small molecule inhibitors (gefitinib and erlotinib) have shown that interruption of growth signals can be a useful strategy for drug design. 11, 12, 29, 30, 31 The success of bevacizumab in combination with chemotherapy in treating colorectal cancer has established the benefit of inhibiting VEGF, a critical molecule in tumor angiogenesis. Approaches targeting each of the other proteins or pathways discussed earlier are under active investigation.
SPECIFIC TARGETING AGENTS OF GROWTH FACTOR RECEPTORS, CELL SURFACE PROTEINS, AND DOWNSTREAM SIGNALING PATHWAYS
The ability to generate highly specific antibodies against the antigen of choice makes such antibodies excellent targeting agents. They can be used alone or to deliver radionuclides, toxins, or chemotherapy to malignant cells or specific tissues. MAbs directed against growth factor receptors (GFRs) and other cell surface antigens have undergone extensive testing in treatment of a number of cancers.32 Those that have been approved for treating patients include trastuzumab against the HER-2 member of the EGFR family (for breast cancer); bevicizumab against VEGF and cetuximab against EGFR (for colorectal cancer); three antibodies directed against the CD20 antigen: rituximab, zevalin (coupled to yttrium 90 to deliver radiation therapy), and tositumomab (coupled to I131 to deliver radiation therapy) (Bexxar) (for B-cell NHL); alemtuzamab, which binds to CD52 (for B-CLL); and gemtuzamab ozogomicin (Mylotarg), which binds to CD33 and contains a cytotoxic agent (for AML). A number of others are currently undergoing clinical evaluation. Continued improvements utilizing antibodies to target agents, such as radioactive compounds or chemotherapeutic agents, in an attempt to selectively kill tumor cells continue to be explored. Combinations of antibodies and radiation therapy or chemotherapeutic agents are also being investigated. Radiolabeled antibodies are also useful as imaging agents for cancer. This is an area of continuing study directed toward enhancing specificity and sensitivity in imaging tumors. See Chapter 31 for a more complete discussion of monoclonal antibodies.
The ability to rapidly screen and iteratively redesign a large number of molecules, in combination with favorable pharmacokinetic (PK) properties compared to other compounds, make small molecules among the most attractive agents for targeted therapy in cancer. The approach used by Drucker and colleagues in identifying the activity of imatinib in the 1990s is a prototypic example of the potential development of small molecules for molecular targeting.27 Using high-throughput screening against recombinant bcr-abl protein, they identified a peptidomimetic molecule (imatinib) that has a high affinity for the adenosine triphosphate–binding site of the Abl tyrosine kinase and is capable of suppressing proliferation and inducing apoptosis of bcr-abl transfected tumor cells in vitro and in vivo.27 The subsequent clinical development of this compound to treat CML and GIST has paved the way for further development of small molecular inhibitors targeted at aberrant tyrosine kinase activity in tumors.
Mechanism of Action
Imatinib's structure is given in Figure 30.2. It is a member of the two-phenylaminopyrimidine class of tyrosine kinase inhibitors. Imatinib mesylate is a potent inhibitor of certain protein tyrosine kinases, including bcr-abl, which is constitutively active in CML; c-KIT, which is frequently mutated in GIST; and PDGFR-alpha, which is over expressed in a number of malignant cells. 32, 33, 34, 35
Cellular Pharmacology and Metabolism
Imatinib is orally active and freely soluble in water. It is rapidly taken up by cells. It undergoes extensive hepatic metabolism. The major enzyme that metabolizes it is the Cyp3A4 member of P450 family of enzymes in the liver involved in the metabolism of many drugs. Minor metabolism occurs by other P450 enzymes. The elimination half-life is approximately 14–20 hours for the parent compound and 40 hours for the major active metabolite, N-demethylated piperazine derivative (N-desmethyl-imatinib). This metabolite has approximately the same potency as the parent compound. Elimination is primarily in the feces, approximately 85 to 90%, with approximately 10 to 15% in the urine. The majority of the drug is secreted within 7 days, with unchanged imatinib accounting for approximately 25% of the dose. Clearance varies by about 40% between patients, which means that it is important to monitor for toxicity in individual patients.
Figure 30.2 Structure of imatinib mesylate, a tyrosine kinase inhibitor.
In vitro, imatinib has been shown to be a potent inhibitor of a number of P450 enzymes and, especially, cyp3A4. There is significant increase in imatinib exposure (with increases in both maximum concentration [Cmax] and the area under the curve [AUC]) when imatinib is given with cyp3A4 inhibitors such as ketoconazole. Similarly, imatinib significantly increases the concentration of simvastatin, another cyp3A4 substrate, when they are given together. Inducers of cyp3A4, such as phenytoin, significantly decrease imatinib exposure.
After oral administration, the drug is relatively quickly absorbed (within 2 to 4 hours) and exhibits high bioavailability (98%). It is 95% bound to plasma proteins, primarily albumin and alpha 1-acid glycoprotein. The AUC increases proportionally with increasing dose. There is no significant change in PK with repeated dosing. There is a 1.5- to 2.5-fold accumulation at the steady state when the dose is given daily. It has poor penetration into the CSF.
The major toxicities seen with imatinib include
· hematological toxicity with neutropenia, thrombocytopenia, and anemia
· hepatotoxicity, usually manifested by elevated liver enzymes (toxicity can be decreased by holding or adjusting the dose, but occasionally it can be severe, especially if the agent is given with acetaminophen; thus, acetaminophen should be used with caution in patients who are taking imatinib)
· fluid retention or edema (often periorbital edema, which is usually manageable though occasionally serious, with a low incidence of ascites, pleural effusions, and brain edema)
· musculoskeletal pains and cramps
· occasional diarrhea
· GI irritation (imatinib should be taken with food and a large glass of water)
· GI bleeding or intratumoral bleeding (especially in patients with GIST), which can be significant Holding of doses or appropriate dose modification should be done for toxicities as necessary. Dose modifications are dependent on the specific disease being treated (PDR). For example, for chronic phase CML or GIST, dose should be held for ANC < 1 × 109 or platelets < 50 × 109 and then resumed at the same dose (the CML starting dose is 400 mg/day; the GIST starting dose is 400 or 600 mg/day) once ANC has recovered to 1.5 × 109 and platelets to ≥ 75,000. If hematological toxicity recurs, then the dose should be reduced to 300 mg if the starting dose was 400 mg or reduced to 400 mg if starting dose was 600 mg once counts have returned to the parameters outlined above. For patients with CML in accelerated phase, when the ANC < .5 × 109 or platelets < 10,000, then bone marrow should be checked. If the decreased ANC is not disease related, then imatinib should be reduced to 400 mg/day (from the starting dose of 600 mg/day). If cytopenias persist for 2 weeks, then dose should be further reduced to 300 mg/day. If cytopenias persist at 4 weeks, imatinib should be held until ANC ≥ 1 × 109 and platelets ≥ 20,000, then the dose should be resumed at 300 mg/day. For hepatotoxicity, imatinib should be held for T. Bili more than three times the upper limit of normal or transaminases more than five times the upper limit of normal. The dose should then be resumed after reduction (100-mg reduction for 400-mg dose or 200-mg reduction for 600-mg dose) once the bilirubin has decreased to less than 1.5 times the normal and the transaminases have decreased to less than 2.5 times the normal. Of course the PDR package insert should be followed for appropriate standard dose modifications of any drug.
As discussed above, because imatinib is metabolized by cyp3A4, it is important to modify the dose when given with cyp3A4 inhibitors (e.g., itraconazole, erythromycin, clarithromycin). Similarly, exposure to imatinib may be decreased by inducers of cyp3A4 (e.g., dexamethasone, phenytoin, carbamazepine, rifampin, or phenobarbital). Drugs that might have increased exposure when given with imatinib include simvastatin, cyclosporine, pimozide, warfarin, and certain HMG coA reductase inhibitors. Whenever possible, alternate drugs that do not interact with cyp3A4 should be utilized, such as low molecular weight heparin instead of warfarin. Grapefruit and grapefruit juice inhibit cyp3A4 and should be avoided. In addition, a number of compounds used as alternative therapies might influence cyp3A4 function and should be avoided.
Imatinib is approved for treatment of patients with CML. It produces clinical hematologic responses in the majority of patients (approximately 90 to 95%) with CML in chronic phase either previously untreated or refractory to interferon, at doses that have acceptable toxicity (400 mg/m2 per day). It produces cytogenetic complete remission in approximately 54% of previously untreated patients and 32% of those with previous interferon therapy. The median time to cytogenetic response is approximately 1 month. At higher dose (600 mg/m2 per day), it also has activity against CML in accelerated phase, with approximately 28 to 37% complete hematological response and 20% major cytogenetic response. Although less active in patients with blast crisis, when used at the 600 mg/m2 per day dose level, it still produces a small percentage of complete hematological responses (4 to 7%) and major cytogenetic responses (14 to 15%). It has had less clinical activity in patients with acute lymphoblastic leukemia who have the 9:22 translocation, possibly because of a greater role for src family kinases, although the actual mechanism remains to be fully elucidated. The drug's key features are given in Table 30.3.
TABLE 30.3 KEY FEATURES OF IMATINIB MESYLATE
Gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors arising from the GI tract.36, 37 They originate from the interstitial cell of Cajal, an intestinal pacemaker cell. Although they can arise from anywhere in the gastrointestinal tract or even the omentum, mesentery, or retroperitoneum, they most commonly occur in the stomach, followed by the intestine. Many of these tumors were formerly called “leiomyosarcomas.” The primary therapy for GIST is surgical resection. However, a fairly high rate of recurrence or metastatic disease is noted. Historically, these tumors have been largely unresponsive to standard chemotherapeutic agents, including those active against sarcomas.
Imatinib has significant inhibitory effects on the tyrosine kinase activity of the c-KIT receptor and the PDGF receptor (PDGFR).38 Immunohistochemical staining for c-KIT is usually positive in GIST tumors. Furthermore, the c-KIT receptor is mutated in a high proportion of patients with GIST (approximately 85%). A number of different mutations in the c-KIT receptor in GIST tumors have been identified (Table 30.4). The clinical antitumor activity of imatinib against GIST correlates with specific mutations in c-KIT, as shown in Table 30.4. The most common mutation is in exon 11, which occurs in approximately two thirds of patients. The highest response rates are seen in patients with exon 11 mutations. The second most common mutation is in exon 9, which occurs in approximately 17% of cases. These mutations so far have only been found in tumors that originate in either the small bowel or colon. Mutations also occur occasionally in exons 13 or 17. In those GIST tumors with wild-type c-KIT, the PDGFR-α is mutated in approximately 40% of cases.36, 37
TABLE 30.4 C-KIT MUTATIONS AND RESPONSE TO IMATINIB
The approved dose of imatinib for GIST is 400 mg/day or 600 mg/day. The potential benefit of dose escalation for those who stop responding to imatinib is uncertain at the present time, although no responses were seen in a small group of patients who were escalated to the higher dose after they stopped responding to 400 mg/day. Ongoing clinical trials are evaluating imatinib against PDGFR-expressing tumors, such as gliomas and prostate cancer.
Mechanism of Action
Gefitinib's structure is shown in Figure 30.3. It is a member of the anilinoquinazoline class of tyrosine kinase inhibitors. Gefitinib (Iressa) is a specific inhibitor of the EGFR tyrosine kinase (Table 30.5). 11, 12, 39, 40, 41, 42, 43, 44, 45 Similar to imatinib, which inhibits the bcr-abl tyrosine kinase, it is targeted at the ATP-binding site of the EGFR tyrosine kinase. Since EGFR signaling is involved in the survival and proliferation of certain neoplastic cells, inhibition of the tyrosine kinase activity leads to inhibition of cell proliferation and apoptosis of these cells in vitro.
Cellular Pharmacology and Metabolism
Gefitinib is rapidly taken up by cells and leads to inhibition of the intracellular tyrosine kinase domain of the EGFR. Pharmacodynamic data using skin biopsies at 1 month into treatment have shown that significant inhibition of EGFR activation occurred at all doses greater than 150 mg/day, and inhibition of downstream signaling (such as through map kinase) was also noted with increased cyclin-dependant kinase expression and apoptosis.
Figure 30.3 Structure of gefitinib EGFR tyrosine kinase inhibitor.
Gefitinib is orally bioavailable. Its uptake is slow and not significantly affected by food. It is distributed extensively, with maximal plasma concentration between 3 and 7 hours after dose. It is approximately 60% bioavailable. The terminal half-life is approximately 40–50 hours with multiple dosing, and the steady-state level is reached by 7 to 10 days. It is dose proportional with increasing dose. There is interpatient variability of up to 56% and intrapatient variability of up to 30%. Ninety-one percent of the drug is plasma protein bound. Gefitinib does not alter the PKs of the chemotherapeutic agents with which it has been used to date, but gefitinib exposure is increased when it is given with carboplatin and paclitaxel.43 However, this has not significantly altered toxicity.
Gefitinib undergoes extensive hepatic metabolism. The metabolism of gefitinib is complex, with at least five identified metabolites and three sites of biotransformation. None of the metabolites are thought to be bioactive. It is primarily cleared through the bile, with approximately 86% cleared in feces and less than 4% cleared in urine.
Similar to imatinib, it is processed by cyp3A4, with all the same potential drug interactions mentioned in the discussion of imatinib. For example, activation of cyp3A4 (e.g., by barbiturates) increases clearance, whereas inhibitors of cyp3A4 (e.g., ketoconazole) increase exposure to gefitinib. It is also an inhibitor of cyp2D6 and therefore increases levels of coadministered cyp2D6 substrates such as amitriptyline and codeine.
TABLE 30.5 KEY FEATURES OF GEFITINIB
Diarrhea (occurring in 37 to 50 % of patients) and skin rash, often acneiform (occurring in approximately 40 to 50% of patients), are the most common toxicities. Nausea, pruritus, elevated liver function tests, and asthenia all can be seen. Uncommon but potentially serious toxicities include eye changes (corneal erosion or ulcer) and interstitial pneumonitis, which can be life-threatening or fatal.
Gefitinib has activity against approximately 10 to 12% of NSCLCs that have progressed after two lines of chemotherapy. Gefitinib's antitumor activity does not correlate with intensity of immunohistochemical staining for EGFR in the tumor, suggesting that the level of EGFR expression does not predict response. However, similar to the situation for imatinib mesylate, which has increased activity against GIST tumors with specific c-KIT mutations, the activity of gefitinib appears to correlate with somatic mutations that are clustered around the ATP-binding pocket of the intracellular catalytic tyrosine kinase domain of the EGFR. 11, 12, 39, 40, 41, 42, 43, 44, 45 These mutations increase both EGF-induced activation as well as gefitinib inhibition of the receptor. The mutations are heterozygous, and thus the mutant protein appears to have a dominant effect. In two studies, 13 of 14 of the responding patients had a mutation in EGFR, versus no mutations in 11 nonresponders.11, 12 To date, all of the mutations have been found in tumors that are adenocarcinomas with or without bronchoalveolar features. Mutations are found more frequently in patients who are nonsmokers, women, and Japanese. The reason for the association of EGFR mutations with these features is currently unknown. In addition, mutational status does not appear to account for all of the clinical activity. Studies are ongoing to deliniate this further.
Gefitinib did not increase the clinical activity of chemotherapy when used in combination in large trials.42, 43 This was also true for erlotinib, an EGFR inhibitor similar to gefitinib, when used with chemotherapy in treatment of NSCLC (see next section). Both of these agents are currently being evaluated for potential treatment of other malignancies alone and in combination with chemotherapy or other agents. The absence of survival advantage for patients who received gefitinib has led to restriction of its use to patients already benefiting from it.
The small molecules approved for tyrosine kinase targeted therapy to date have so far shown significant clinical activity primarily against mutant proteins (bcr-abl and c-KIT in the case of imatinib, EGFR in the case of gefitinib and erlotinib). This provides greater antitumor selectivity but also potentially decreases the population that might benefit. Whether this is going to be generally true for clinically active small molecular inhibitors of tyrosine kinases is not known. However, as noted above, mutational status of EGFR is only part of the mechanism defining effectiveness of erlotinib. This remains an active area of investigation.
Mechanism of Action
Erlotinib (Tarceva) is a potent specific inhibitor of the EGFR tyrosine kinase.46, 47 Similar to gefitinib, it is targeted at the ATP-binding site of the molecule.
Cellular Pharmacology and Metabolism
Erlotinib is orally active. Similar to gefitinib, it is metabolized by cyp3A4, with all the potential drug interactions and other caveats discussed in the section on gefitinib.
PK studies have shown it to be dose proportional with increasing dose; to have a half-life of approximately 36 hours; to have increased Cmax and AUC when taken with food, along with a delayed Tmax; and to have relatively low interpatient variability (range less than twofold). The maximum tolerated dose is 150 mg/day.
The toxicities are similar to those seen with gefitinib. At the recommended dose of 150 mg/day, these include skin changes, including rash, dermatitis, and pruritus, totaling 83% overall, with 13% grade 3 or 4; diarrhea (38% overall, 6% grade 3), nausea (33%), fatigue (18%), and rare cases of interstitial pneumonitis, which can be life-threatening (PDR).
Erlotinib has a level of activity similar to that of gefitinib against NSCLC (approximately 12% response rate). 46, 47, 48 This was associated with prolongation of survival in patients who had previously failed two previous chemotherapy regimens for metastatic lung cancer.48 As with gefitinib, the response rate is higher in patients who have adenocarcinoma with or without bronchoalveolar features and whose tumors contain the mutation in the ATP-binding pocket of the catalytic domain of the EGFR.49 However, survival did not correlate with EGFR mutational status. The FDA has approved Tarceva for potential treatment of NSCLC.
Two other targeted small molecular inhibitors that are in clinical trial have sufficient activity against renal cell cancer that they are currently in pivotal trials. These are Bayer 43-4009 and SU-011248.23, 26, 50, 51 Bayer 43-4009 is an orally available compound that was initially developed as an inhibitor of the raf kinase. As discussed earlier, the raf protein is one of the immediate downstream targets of ras in signal transduction, and thus inhibiting raf could inhibit proliferative signaling from growth factor receptors. However, subsequent evaluation has shown that it has activity against a number of other kinases, including the VEGFR2 tyrosine kinase. Thus, it has potential as an antiangiogenic agent in addition to its potential activity directly against malignant cells by blocking the ras-raf pathway. It is not known at present exactly which inhibitory effect or effects are responsible for Bayer 43-4009′s antitumor activity. In any case, early clinical trials showed it to have antitumor activity against a number of malignancies, especially renal cell cancer, with acceptable toxicity. Preliminary data from a phase III trial evaluating the potential clinical efficacy of Bayer 43-9006 against renal cell cancer have shown that it prolongs progression free survival compared with placebo. 51a Participants in the study are currently being followed for overall survival. A number of other studies are evaluating its efficacy against other malignancies.
SU-011248 is an orally available small molecular inhibitor of the tyrosine kinase activity of the VEGFR-2, PDGFR, and KIT receptors. As with BAY43-4009, it exhibited some antitumor activity against a number of malignancies in phase I studies, including against renal cell cancer, also with acceptable toxicity. It is also currently in clinical trials against a number of malignancies, including renal cell cancer but also GIST tumors that have progressed during imatinib treatment.
A large number of other small molecules targeting a variety of tyrosine kinases are currently undergoing clinical investigation. Some of these are chosen to have greater specificity for specific kinases, whereas others have activity against a number of kinases. At present, it is not known whether having specific activity (and potentially combining different agents each with specific activity) or having broader activity will be more clinically effective. Both strategies are being pursued in the development of kinase inhibitors. Strategies for combining different classes of targeted agents (e.g., mAbs and small molecules) are also being pursued.
A number of issues remain that need to be addressed through further investigation of small molecular inhibitors of kinases as anticancer agents, including these:
· The three malignancies that have been responsive to small molecular tyrosine kinase inhibitors to date (CML, GIST, and lung adenocarcinoma with or without bronchoalveolar features) have all had specific mutations in the protein being targeted (bcr-abl in CML, KIT in GIST, and EGFR in lung adenocarcinoma). Efforts are being made to identify other potentially mutated tyrosine kinases in other tumors that might be targets for therapy. A corollary question is whether small molecular inhibitors of tyrosine kinase activity might be clinically active in tumors that have overexpressed but not mutated tyrosine kinases (such as the EGFR in head and neck cancer).
· Might combinations of monoclonal antibodies and small molecular inhibitors directed against the same target have greater activity in combination than when used alone? Although conceptually these combinations might appear to be redundant and therefore no more active, because they attach to different sites on the molecule, they could have enhanced activity.
· What is the best way to target multiple steps in GFR pathways, and will there be increased antitumor activity or clinical benefit from targeting multiple steps in the best way possible?
· How can multiple receptors or pathways be most effectively targeted?
· How can resistance to the targeted agent (which remains a significant problem) best be prevented or overcome?
Research is addressing each of these issues.
Other Modified Peptides and Peptidomimetics
As discussed in the sections on imatinib mesylate and gefitinib, modified peptides or peptidomimetic compounds designed to bind to and inhibit the active sites of proteins are natural candidates for effective inhibitors of protein function. The ubiquitous presence of proteases, the extremely short half-lives of most naturally occurring peptides, and their rapid hepatic and renal clearance make unmodified molecules impractical for clinical use. Therefore, modified compounds or, more commonly, small organic molecules that mimic peptides functionally, so-called peptidomimetics, have been the major focus of study. Modifications include incorporation of altered amino acids (e.g., phosphonates) that are less prone to degradative attack, incorporation of compounds (e.g., benzodiazepine analogs) that structurally mimic amino acids but are not targets of proteases, and use of totally synthetic polymers that structurally mimic peptides but are not targets for enzymatic degradation.
For these agents to be effective, a target that is therapeutically relevant must be chosen (Table 30.6). This is an obvious but critical point that can be forgotten in the excitement of knowing the sequence or structure of a gene that may not be an ideal candidate from the standpoint of tumor biology. Defining the best genes to target is especially important given the large number of potential targets and the high cost of evaluating each of them. Continued studies are helping to define the properties of optimal targets. Structures of the appropriate active sites of the target protein must be known. Ideally, structures of the specific regions being targeted are known from x-ray crystallography, allowing initial evaluation of potential binding compounds by computer analysis. Short peptidomimetics can be synthesized based on known sequences of active binding sites critical for the function of target proteins. Those predicted to have the best binding properties can be synthesized. Thus, the process requires the production, purification, and crystallization of the known protein, followed by x-ray crystallographic and computer analysis.
TABLE 30.6 MODIFIED PEPTIDE TARGETS
These can be used for initial screening and provide the basis for subsequent modifications to improve efficacy. Clinically, the most extensively studied of the modified peptide compounds to date have been somatostatin analogs.52, 53 Thus, to illustrate pharmacological features of these compounds, somatostatin analogs are discussed as prototypical examples of synthetic peptide or peptide-like structures. Where appropriate, other peptidomimetics or general features of these types of compounds are discussed. Peptide analogs of luteinizing hormone–releasing hormone (LHRH) are also extensively used clinically, but because the antitumor effect is mediated via hormonal manipulation, these are discussed under hormonal therapies.
Mechanisms of Action
Uncoupled Modified Peptides
Somatostatins are, in general, growth inhibitory for a wide variety of cells, including neoplasms. 52, 53, 54, 55, 56 Receptors for somatostatin have been found on a number of tumors, including lung, colon, pancreas, breast, and neuroendocrine tumors. Most animal models suggest that the biologic effects of these compounds (or analogs) are mediated via binding to receptors, although a few studies have not shown a direct correlation between receptor numbers and the biologic effects of the compound.
Somatostatin itself has too short a half-life (approximately 3 minutes) to be clinically useful. In addition, withdrawal of somatostatin can produce rapid rebound effects. Therefore, a number of analogs have been developed that have greater stability, have more selectivity, and possibly induce less of a rebound effect than somatostatin itself. 52, 53, 54, 55, 56 Somatostatin analogs that are significantly more active than the parent compound have been studied extensively. They vary in potency for different somatostatin effects, suggesting some specificity of different analogs. For example, sandostatin is 45 times more potent than somatostatin in inhibiting GH release, 11 times more potent in inhibiting glucagon, and 1.3 times more potent in inhibiting insulin. 52, 53, 54,55, 56 Binding of various somatostatin analogs varies between different tissues, suggesting that there are possibly different subtypes of receptors. 52, 53, 54, 55,56 Antiproliferative effects of somatostatin analogs appear to be mediated by a number of mechanisms. One mechanism is inhibition of centrosomal separation and cell proliferation induced by epidermal growth factor (EGF).57 This may be due, at least in part, to stimulating a tyrosine phosphatase that inhibits signaling via the EGFR pathway, although additional studies are necessary to further define the mechanism.58 A large number of potential indirect mechanisms of tumor inhibition by somatostatin analogs exist, including suppression of growth hormone (GH) and prolactin, which can be growth stimulatory for breast cancer; inhibition of GH, with subsequent suppression of a number of growth factors (e.g., insulin-like growth factor I) that are important for tumor cell growth; and suppression of plasma-EGF levels. 52, 53, 54, 55, 56 Somatostatin analogs have been shown to inhibit tumor growth in vitro and in a number of animal model systems. 52, 53, 54, 55, 56 The effect appears to be primarily cytostatic, with return of tumor growth once analogs are removed.
Peptide analogs that recognize the binding site of other receptors also have been synthesized to bind to and inhibit receptor function directly. These have been shown to be effective in vitro, and if these or modified peptides that would be more stable in vivo can be effectively delivered to tumors, then they might be clinically useful. 57, 58, 59
In addition to development of peptidomimetics targeted to cell surface receptors, much of the current interest in these molecules is focused on their potential as inhibitors of specific steps in signal transduction pathways from cell surface to nucleus. The identification of the mammalian target for rapamycin (mTOR, which is a downstream effector in the phosphatidylinositol 3-kinase/Akt pathway that is involved in controlling cell cycle progression and proliferation) has led to the development of a number of analogs that are currently in clinical evaluation.60 Clearly, a large number of other potential targets for peptidomimetics are present within cells, including proteins involved in other signaling pathways, enzymes, or sequences important for protein-protein interactions.61 Many of these are being actively pursued as potential therapeutic targets.
Targeting Cytotoxic Agents via Modified Peptides
Similar to mAbs directed against GFRs, peptide growth factors can be used to target toxic compounds to cells. Given the rapid degradation of growth factors once they are internalized, peptide growth factors are most useful for targeting toxic compounds.62, 63 This approach has been shown to be effective in inhibiting the growth of neoplastic cell lines in culture and tumors in animal models, with tolerable toxicity. Human trials also have shown efficacy for this approach using an IL-2–diphtheria hybrid toxin (denileukin diftitox) to treat cutaneous T-cell lymphomas (CTCLs) and other hematological malignancies that express the CD25 component of the IL-2 receptor. It is approved for the treatment of persistent or recurrent CTCL that expresses the CD25 antigen.64
Given the immunogenicity of toxins, the short plasma half-life of most small peptides, and the tendency of many peptides (e.g., growth factors) administered in vivo to concentrate in organs, such as the liver and kidney, these compounds must be modified to make them consistently useful therapeutic agents for systemic therapy. A potential means of decreasing systemic side effects and increasing the amount delivered to tumor cells is to deliver therapy locally. Approaches to doing this, such as intravesicular therapy or hepatic arterial infusion, are being pursued.
Peptides can be modified in a number of ways to enhance their potential as effective therapeutic agents. One of these is coupling them to compounds, such as dextran, that prevent the extremely rapid degradation of peptides that normally occurs in cells and therefore allow longer retention in these cells.65 If these constructs are coupled with radioactive or cytotoxic compounds, they can provide a potentially effective system for delivering the toxic compound to malignant cells.
Cellular Pharmacology and Metabolism
The factors important for the cellular pharmacologic properties of modified peptides targeted to GFRs (or other cell surface antigens) are similar to those for mAbs. The activity, half-life, and metabolism of these peptides are dependent on whether the ligand-receptor complex is internalized. Internalization is not necessarily essential and might be pharmacologically detrimental for compounds that bind to and inhibit the receptor directly or are ligated to radionuclides. On the other hand, for toxin or chemotherapy conjugates, internalization is essential. Furthermore, some agonistic ligands that promote the growth of normal cells paradoxically produce growth arrest and apoptosis in tumor cells (CD40, CD25). Modified peptides targeted at intracellular proteins clearly need to be delivered into cells. As discussed earlier for ras, compounds with these properties can be constructed. It is impossible to generalize the pharmacologic properties of different modified peptides because they are affected by the presence, concentration, and properties (e.g., internalization) of the proteins to which they bind in plasma as well as on the cell surface.
Somatostatin analogs are rapidly absorbed after subcutaneous injection. The peak plasma concentrations are seen in approximately 25 to 30 minutes. The t½α values are approximately 10 to 15 minutes, and t½β values are approximately 100 to 115 minutes when the dose is given by subcutaneous injection and approximately half as long when given by intravenous bolus. Approximately one third of the dose is excreted unchanged in the urine. A number of analogs have different biologic and pharmacologic effects. Somatuline, which has been studied in the treatment of pancreatic cancer, has a plasma half-life of approximately 90 minutes.66 The short half-life of initially developed somatostatin analogs meant that, in order to have sufficient concentrations for therapeutic effect for any duration, they had to be given by subcutaneous injection three to four times per day or continuously either intravenously or subcutaneously. Therefore, a long-acting somatostatin analog, an LAR depot form for intramuscular use given once monthly, was developed and has been approved for clinical use. Steady-state octreotide serum concentrations are achieved after the third dose. Although effective in treating the carcinoid syndrome and for controlling symptoms related to GH and prolactin-secreting pituitary tumors, it has been only minimally evaluated in other tumor settings.
Denileukin diftitox is usually administered as intravenous therapy over at least 15 minutes once daily for 5 days in a row, with treatment repeated every 21 days. It is rapidly cleared from the serum, with a short distribution phase of 2 to 5 minutes and a terminal half-life of 70 to 80 minutes.67 There is not a significant difference in PKs between bolus and 90-minute infusion schedules. As with antibodies, there is significant interpatient variability in PKs. The majority of patients developed antibodies against the toxin, against the IL-2, or against both by the third cycle of therapy, and the specific types of antibodies that develop may enhance clearance rates, which tend to be two to three times more rapid by the third course.
Given the short half-lives of peptides, a number of approaches to improving their in vivo pharmacologic properties have been investigated. For example, polyethylene glycol conjugated to peptides might improve their PKs by a number of mechanisms, including prolonged plasma half-life, decreased immunogenicity (although occasionally it may actually enhance immunogenicity), increased solubility, resistance to proteolysis, and better coupling to liposomes. Innovative approaches, such as controlled-release polymers, may enhance peptide delivery for a prolonged period.61 Additionally, mathematical models have been developed to define parameters required for in vivo use of targeted therapies, and these might be useful in attempting to optimize the design of compounds for clinical use.68 Ultimately, clinical trials are necessary to define PK properties and toxicity for any given compound.
Somatostatin analogs are quite nontoxic, with median lethal dose values not being reached in rats. There is no evidence of chronic toxicity in studies conducted up to 2 years in dogs.66 In addition, they have been given to patients for many years with no discernible long-term side effects. Side effects include pain at the injection site, abdominal pain, cramps, and diarrhea. Chololithiasis can occur.
Denileukin diftitox is moderately well tolerated.64, 67 Constitutional (flu-like) and gastrointestinal symptoms (low-grade fevers and chills, asthenia, anorexia, nausea/ vomiting, headache, diarrhea, arthralgias/myalgias) develop in approximately 90% of patients but usually can be treated symptomatically. Hypersensitivity reactions (dyspnea, chest tightness, hypotension, back pain, pruritus and flushing) occur in approximately 60% of patients, but, again, most of these are controllable or preventable by slowing the rate or temporarily interrupting the infusion and/or symptomatic therapy with antihistamines and acetaminophen. Occasionally, glucocorticoids have been necessary. Vascular leak syndrome occurs in about one quarter of patients. It usually is self-limited and can be treated symptomatically. Rashes occur in approximately one third of patients. A significant number of patients have some elevations of hepatic enzymes, but this is not accompanied by other liver abnormalities. Renal insufficiency defined the maximum tolerated dose. Anemia, thrombocytopenia, hemolysis, and proteinuria are also seen.
The toxicity profiles of new modified peptides will be defined by the results of trials using those agents.
Clearly, somatostatin analogs have activity in controlling symptoms related to GH and prolactin-secreting pituitary tumors, GI neuroendocrine tumors (especially carcinoid tumors), and diarrhea related to certain chemotherapeutic agents.66, 68 Clinical trials have demonstrated minor activity against prostate cancer, minimal activity in breast and pancreatic cancer, and no activity against small cell lung cancer. 66, 69, 70, 71 High-dose therapy using somatuline (in the range of 12 mg/day by continuous infusion subcutaneously) has biologic activity as measured by suppression of GH and IGF-I, but the evaluation of clinical effectiveness against different tumors is still in progress. As new, potentially more potent somatostatin analogs are developed, they continue to be evaluated for treating malignancies.66, 71
Given the relatively low level of antitumor activity of these agents used alone, combinations with other agents have been evaluated in several trials. RC-160 (somatostatin analog) and SB-75 (LH-RH antagonist) inhibit the growth of human pancreatic xenograft in a mouse model.72 However, no beneficial effects were seen in a large trial using LH-RH (not the antagonist) and somatostatin, alone or in combination, to treat patients with pancreatic cancer, and the LH-RH agonist goserelin plus hydrocortisone produced no responses.73 Somatostatin analogs appear to enhance the antitumor effects of tamoxifen in preclinical studies.74 A randomized study suggests that the combination of a somatostatin analog, an antiprolactin agent, and tamoxifen produced a higher response rate and time to disease progression than tamoxifen alone for patients with metastatic breast cancer.75 However, there was no difference in overall survival. The evaluation of combinations of somatostatin analogs and other agents or approaches, including chemotherapy and radiation therapy, is ongoing.76
Conjugated Modified Peptides
In addition to combination therapy, a potentially promising approach is to use modified peptides to deliver cytotoxic agents to tumors expressing high receptor levels. This approach has been studied utilizing radionuclide-coupled somatostatin analogs to treat neuroendocrine tumors. 52, 56, 76, 77, 78, 79 The only significant toxicities to date have been reversible myelosuppression, especially anemia; decreases in lymphocyte counts; mild thrombocytopenia; and mild increases in creatinine. Although this approach has produced some antitumor activity, this activity has not yet been sufficient to establish the approach as clinically useful. Efforts to improve these agents are ongoing. Other analogs coupled to radionuclides, chemotherapeutic agents, and toxins are also being developed and evaluated as potential therapeutic as well as imaging agents. 80, 81, 82
Denileukin diftitox has shown effectiveness against a number of hematopoietic malignancies, including Hodgkin's disease and lymphomas (including cutaneous T-cell lymphomas [CTCLs]).64 It is approved for use in the treatment of patients with advanced or refractory CTCL.
To date, clinically useful targeted compounds have come from one of the three classes of agents already discussed. However, significant research is ongoing to evaluate the following classes of compounds for potential utility as anticancer agents.
Antisense oligonucleotides (oligos) are modified single-stranded DNA molecules usually between 15 and 25 nucleotides in length. They are synthesized to have nucleotide sequences complementary to DNA or mRNA sequences of the specific gene being targeted for inhibition. As drugs, oligos have inherent disadvantages, including large size, significant charge, difficult synthesis, and poor penetration of cells. In part for these reasons, they still have not fulfilled their promise as therapeutic agents. However, development is ongoing, with the hope that they will prove useful after a better understanding is achieved of their clinical pharmacology and how to optimize their modification of gene expression.
Mechanism of Action
When therapy is directed at mRNA, it is based on one of the fundamental features of biology, the specificity of Watson and Crick base pairing. By means of binding an mRNA, the synthesis of the specific protein encoded by that mRNA is ultimately inhibited, and its function blocked. Using mRNA as the target, a number of specific mechanisms can be invoked 83, 84, 85, 86, 87 (Table 30.7), including the activation of the RNA-degrading enzyme, RNaseH.
TABLE 30.7 POTENTIAL MECHANISMS OF MESSENGER RNA (mRNA) FUNCTION OR mRNA MODIFICATION TARGETED BY ANTISENSE OLIGONUCLEOTIDES
Alternatively, specific DNA or protein sequences can be targeted by oligos. The major target in DNA is Hoogsteen and anti-Hoogsteen base pairing in the major groove to produce triple-helical structures, leading to inhibition of transcription. Oligos can be coupled to a number of antineoplastic compounds to specifically target them, especially to DNA.
Hybridization to RNA or DNA is a relatively slow process, and, in general, the association rates of oligos determine their efficacy. 83, 84, 85, 86, 87 The minimum size of oligos required to provide necessary specificity and affinity for these purposes appears to be 15 to 18 bases. A caveat to this specificity is that mismatched oligos can induce degradation of target mRNA, so it is certainly possible that cleavage of nontarget mRNA sequences might occur when specific oligos are used in vivo.
The binding of proteins (e.g., transcription factors) to specific DNA or RNA sequences can be targeted. The sequence length required depends on the precise protein-DNA (or RNA) interaction site. 83, 84, 85, 86, 87 Proteins that do not normally bind to DNA or RNA can also potentially be inhibited by binding to oligos specifically and nonspecifically. An approach to designing specific oligos for this purpose is protein epitope targeting. A large number of partially random oligos are used to screen for binding to a target protein. This allows identification of oligos that bind to specific epitopes. The bound oligos can be amplified by polymerase chain reaction. Identified sequences can then be used to develop improved oligos for targeting the protein therapeutically.
Cellular Pharmacology and Metabolism
Necessary requirements for oligos to be useful therapeutically are outlined in Table 30.8. Oligos appear to bind to the cell surface through receptors, although the exact nature of these receptors remains to be defined. This process is saturable. However, it is not clear whether this saturability of uptake will be important for systemic use of oligos. Once bound to the cell surface, oligo uptake into cells appears to occur primarily by pinocytosis, adsorptive endocytosis, or both. 83, 84, 85, 86, 87 Although certain studies suggest that a significant proportion of oligos remain in endosomes, the consensus is that retention in endosomes is not a major limitation in their effectiveness. Binding by cellular proteins may modify the PKs and efficacy of oligos. 83, 84, 85, 86, 87
Uptake of oligos varies among different cell types (studies suggest greater uptake in carcinoma than leukemic cells) and among cells in the same culture. Various factors (e.g., number of oligo receptors, cell cycle stage, rate of division) can explain differences in oligo uptake. Increased cell density decreases uptake of phosphorothioate oligos.88 As would be expected, differences in the intracellular degradation of oligos by cells can have a profound effect on their efficacy.89 Exocytosis also occurs and is a potentially important determinant of the concentration of oligos within cells over time and therefore of their efficacy. Thus, a number of aspects of cellular pharmacology are critical in determining the activity of these compounds.
Oligos present a special problem as in vivo compounds because of the ubiquitous presence of nucleases that rapidly degrade unmodified constructs.89Fortunately, the potential clinical use of oligos can be enhanced by medicinal chemical approaches. 83, 84, 85, 86, 87 Certain modifications make them potentially better for some purposes but not as good for others. Modifications can be designed to prevent rapid metabolism, increase cellular uptake, increase targeting to tumor cells, stabilize binding of oligos to the target structure, or increase inhibitory efficacy. Oligos can be conjugated with ribozymes (with catalytic RNase activity; see “Ribozymes, Other Nucleases, and Proteases”) or other reagents that cleave specific nucleic acid sequences.90
Peptide nucleic acids can inhibit DNA and RNA function. Attachment of toxic compounds to oligos can target those agents to DNA and enhance cell killing, such as using antisense bcr-abl constructs to target compounds specifically to DNA in CML cells.
TABLE 30.8 REQUIREMENTS FOR OLIGONUCLEOTIDES TO BE THERAPEUTICALLY USEFUL
The most important modifications for in vivo use have been chemical alterations of the bases used to construct oligos. This includes using methylphosphonates or phosphorothioates as the phosphodiester backbone, a change that prevents their destruction by nucleases. 83, 84, 85, 86, 87 Phosphorothioates appear to be the more advantageous of these analogs, because they have higher affinity than dimethyl phosphonates for nucleic acids, and they are more effective in activating RNaseH. Therefore, phosphorothioates have been the most extensively studied modified oligos. Many other modifications to oligos, including incorporation of methylene, carbonate (or carbamate), sulfonamide, amino acid, peptide, phosphorodithioate, C-5 propyne analogs of cytidine and uridine, and pyrimidines modified at the 5 and 6 positions, also have significant activity in vitro. 83, 84, 85, 86, 87, 91 However, these have not yet been extensively developed for clinical use.
A number of methods have been used to increase specificity of delivery of oligos to tumor cells and uptake by those cells. Molecules such as porphyrin, cholesterol (or cholesteryl), poly-L-lysine, and transferrin-polylysine significantly enhance uptake and retention, although they also reduce the time of the interaction with mRNA or DNA. 83, 84, 85, 86, 87 Liposomes (e.g., pH-sensitive liposomes) may provide an effective way of delivering oligos into cells.92Complexes of liposomes with compounds (e.g., the Sendai virus protein coat) can significantly enhance their uptake by cells. Coupling of liposomes (with encapsulated oligos) to targeting peptides (i.e., mAbs directed against antigens expressed on the cell surface) offers another means of specific targeting.93
Ex vivo use of oligonucleotides does not have many of the problems associated with in vivo use. Oligos by themselves, as well as in combination with chemotherapy, are highly effective in inhibiting tumor cell growth ex vivo. These strategies may be useful in purging bone marrow of leukemic or other malignant cells.94 Small molecule alternatives to antisense, with more favorable PK properties, could potentially inhibit site-specific interactions between RNA and proteins or nucleotides.95 This is an area of active study.
In the excitement over the potential use of antisense therapy for the treatment of cancer, the PKs of these agents in vivo have received relatively little attention and study. Until the last decade, the paucity of PK data was at least partially due to the high cost of producing sufficient amounts of the compounds for careful PK studies, even in mice. Fortunately, the cost of producing oligos has decreased dramatically with continued improvements in technology. A second problem has been assay methodology for separation of the parent compound and its stepwise oligo cleavage products. These problems have been sufficiently solved for PK studies of oligos to be performed.
Several animal models have been used for evaluating the targeting, pharmacology, and effectiveness of these compounds. Most studies suggest that oligos are fairly rapidly cleared from the circulation (phosphorothioate analogs, which are resistant to nucleases, water soluble, and readily taken up by cells, have been primarily studied; methylphosphonates have been studied to a lesser extent). 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Most studies in animals have shown that t½α ranges from 5 minutes to several hours after bolus administration; human studies to date have been more variable, with t½α ranging from 30 minutes to as long as 26 hours after bolus administration and approximately 7 hours after subcutaneous administration. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 In any case, overall oligos have relatively short half-lives, arguing for the need for frequent administration if continued inhibition of the target is to be maintained.
Oligonucleotides are distributed to the majority of tissues, including malignancies. In the absence of central nervous system abnormalities, brain takes up little of systemically delivered phosphorothioate or methylphosphonate oligos.96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Oligo length (at least within the 20 to 50 base range), base sequence, and dose (at least in the range up to 150 mg/kg) do not appear to significantly affect the rate of plasma clearance. 99, 100, 101,102, 103, 104, 105 However, the specific base analog used in constructing the oligos does affect their PKs. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105
Given the relatively slow uptake of oligos by cells, rapid clearance from the circulation makes delivery of oligos to tumor cells difficult. Mechanisms for enhancing delivery are important considerations if oligos are to be used successfully as systemic agents. In normal tissues, the highest concentrations accumulate in the kidney and liver, with up to 40% in the liver at 12 hours. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 This can induce some degree of hepatic inflammation, as indicated by elevations in lactate dehydrogenase and transaminases. 96, 97, 98, 99, 100, 101, 102, 103, 104 To a lesser extent, accumulation occurs in other tissues, including intestine, spleen, lung, heart, and muscle. 96, 97, 98, 99, 100, 101, 102, 103, 104 Route of administration may affect organ distribution, with subcutaneous injection possibly favoring accumulation in spleen and muscle. Oligos are stable in most tissues but are more rapidly metabolized in kidney and liver. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Degradation in blood and tissues appears to be primarily due to 3′-exonucleases.99 Rate of degradation does not appear to be dose-dependent, as would be expected given the large amount of nucleases present. The t½β of total-body clearance is approximately 30 to 40 hours. 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Clearance of methylphosphonate and unmodified oligos is more rapid than clearance of phosphorothioates.99 Most of the dose is excreted in urine in 2 to 3 days (with approximately 30% in the first 24 hours) and significantly less via the GI tract. 96, 97, 98, 99, 100, 101, 102,103, 104, 105 Most oligos that are excreted are degraded, so only a small proportion of intact oligos are found in the urine. Modifications to the oligos, including addition of bases, can occur in several tissues, including liver, kidney, and GI tract.96, 97, 98, 99, 100, 101, 102, 103,104, 105 Interactions with other drugs are poorly understood; there continues to be a need for careful evaluation of PKs in the setting of coadministered drugs. However, most studies have not shown significant PK interactions between oligos and chemotherapeutic drugs (see below for further discussion).
Although antisense molecules differ in their sequence and target, they seem to share similar PK and toxicologic properties, independent of effects on the target sequence itself. Although a single dose of 640 mg/kg of anti-HIV phosphorothioate was lethal, single doses of up to 150 mg/kg have been well tolerated in mice.105 Antisense molecules that are cytostatic may require prolonged administration, making potential chronic toxicity an important consideration. Doses up to 100 mg/kg of phosphorothioate oligos for 14 days do not appear to produce serious toxic effects.96, 97, 98, 99, 100, 101, 102, 103, 104, 105 In rhesus monkeys, continuous infusions of oligos at doses up to 1,500 mg for periods as long as 15 days did not produce significant toxicities.106 Mild elevations in liver functions were seen in the majority of animals, and mild neutropenia was seen in one third. Many oligos seem to produce complement activation and thrombocytopenia as their dose-limiting toxicities in animals. Oligos can also accumulate in kidneys, with the potential for nephrotoxicity, although significant nephrotoxicity is primarily seen at higher doses.44, 45, 46, 47, 48, 49, 50
Most of the clinical trials in humans have indicated that oligonucleotides are generally safe. The most common toxicities have been thrombocytopenia, asthenia, fevers, hypotension, hyperglycemia, and minor changes in renal function (Table 30.9).100, 101, 102, 103, 104 All of these are usually mild and rapidly reverse when the oligonucleotide is discontinued. Local skin reactions are also seen when oligos are delivered subcutaneously. Less common toxicities have included activation of complement and clotting abnormalities.
The in vivo effectiveness of antisense oligos has been demonstrated in a number of animal model systems.96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Longer exposure of animals to oligos appears to produce a greater antitumor effect.96 These results indicate that sufficient quantities of oligos can be delivered over time to a variety of tumors in mice to achieve an antitumor effect at doses that do not produce significant toxicity.
TABLE 30.9 TOXICITIES OF SOME OF THE OLIGONUCLEOTIDES IN CLINICAL TRIALS FOR CANCER PATIENTS
Clinical trials have been completed using antisense constructs alone and in combination with chemotherapy.96, 97, 98, 99, 100, 101, 102, 103, 104, 105 Antisense constructs to other mutated cancer genes, including p53 and bcr-abl, as well as to HIV (in patients with HIV-associated malignancies), have been performed. These have shown the following: (a) Target genes can be inhibited in peripheral blood cells and malignant cells in lymph nodes at achievable concentrations of oligonucleotides. (b) Occasional patients have had responses (including a complete response seen in a patient with B-cell NHL treated with Bcl-2 antisense).101, 102, 103, 104 A proportion of patients have had stable disease. However, most patients have not responded. (c) Combinations of oligonucleotides with chemotherapy can be delivered with acceptable toxicity. In addition, these have not indicated significant PK interactions between oligos and chemotherapeutic agents. Although this remains a potential concern, it does not occur commonly. Additional clinical trials are ongoing, and the results will indicate whether sufficient clinical efficacy can be achieved in humans (either efficacy of oligonucleotides themselves or in combination with chemotherapy) for this approach to be of value in treating cancer patients. Other routes of administration (ex vivo, intraperitoneal, transcutaneous, intrathecal) are also being explored. A number of antisense constructs against growth factor receptors, especially the EGFR, have been evaluated in a number of preclinical models and are being considered for clinical trials.
Posttranscriptional Gene Silencing via RNA Interference Other Than by Use of Antisense DNA Compounds
The discovery of the mechanism of RNA interference in which double-stranded RNAs are processed to small interfering RNAs (siRNAs) that ultimately bind to complementary RNA molecules, leading to their cleavage and the posttranscriptional gene silencing (PTSG) of the gene, has provided a very powerful tool for studying the effects of silencing specific genes.107, 108, 109 Although double-stranded RNAs trigger the antiviral response in mammalian cells that interferes with the ability to use them to silence specific genes, siRNAs can bypass this response and can be utilized for this purpose. A large number of studies have utilized siRNAs to evaluate the effects of silencing specific genes on cellular phenotype and function. This is critical in assessing the potential effects of agents targeted to a specific gene. Although still in the early stages of development as therapeutic agents, the potential delivery of siRNAs as therapeutic agents to silence specific genes is actively being evaluated. The identification of the related microRNAs (miRNAs), which are single-stranded RNA molecules processed from stem-looped precursors and are thought to be involved in gene regulation in a variety of organisms (including humans), has provided further insight into the importance of PTSG in regulating gene expression and the potential utility of these agents for targeting the expression of specific genes.110
Ribozymes, Other Nucleases, and Proteases
Ribozymes are RNAs that can catalyze a variety of RNA or DNA cleavage reactions (i.e., splicing or site-specific cleavage) via their tertiary structures, and therefore they function as enzymes or possibly drugs.111, 112, 113, 114, 115, 116 Their advantage over protein enzymes is that one can, through sequence and structure changes, modify ribozymes to carry out specific functions much more readily than can be done in the case of proteins. As with oligos, they need to be modified to make them nuclease resistant to be useful for in vivo purposes. The stability of the RNA being targeted and the turnover rate of ribozyme-substrate interaction are important determinants of the potential usefulness of ribozymes. They can be targeted to cells by coupling with proteins, genetic vectors, or other compounds to be most useful. The pharmacology of these coupled compounds for the most part resembles that of the targeting agent to which they are coupled. The most extensively studied of the ribozymes as anticancer agents to date has been angiozyme.114, 115, 116 Angiozyme is a ribozyme directed against the FLT-1 (VEGFR-1) receptor. It has been shown to down-regulate the FLT-1 receptor in vivo and to have antitumor and antimetastatic activity in an animal tumor model. Phase I studies showed it to be well tolerated when given as a subcutaneous injection, with the major toxicity being mild to moderate local skin reactions. Bioavailability after subcutaneous injection is estimated at greater than 74%. It has an approximately 6-hour half-life after subcutaneous injection. Approximately 10 to 20% of the drug is excreted unchanged in the urine. It has been evaluated in a phase I study in combination with chemotherapy for colorectal cancer. It was well tolerated, and initial results were encouraging, although it is impossible to know whether angiozyme added to the effectiveness of chemotherapy in this setting. Further development of angiozyme is currently being considered. Studies evaluating other ribozymes as possible antiviral agents as well as antitumor agents are ongoing.
Gene therapy uses the insertion of new genetic information into cells and its expression in those cells to alter their biologic behavior for the purpose of achieving therapeutic benefit. The initial promise of gene therapy (and still an important area of investigation) lay in the placement of a normal critical gene into cells that either do not express the gene at all or have a mutated form of the gene. Such placement could also potentially suppress the abnormal function of a mutated gene. This strategy is being pursued for inherited genetic disorders and for introducing tumor suppressor genes into neoplastic cells. However, a number of significant hurdles remain to be overcome if malignancies are to be treated using this approach. These include the need to transfect at least the vast majority of malignant cells for this to be clinically useful, the inefficiency of gene transfer into cells, the difficulties of in vivo delivery to neoplastic cells, and the problem of maintaining continued expression of the transduced gene in those cells over time.
Therefore, much of the emphasis in gene therapy as an approach to treating neoplasms has shifted to making cells more immunogenic in order to activate host-mediated killing of the cells. A number of animal model systems suggest that modification of a subpopulation of tumor cells may lead to an “innocent bystander effect” (by mechanisms that have not been fully elucidated but presumably are, at least in part, immune mediated) whereby nontransfected tumor cells are also eliminated. This approach would not require transfecting every cell to be effective.117, 118, 119, 120, 121, 122, 123, 124, 125 The fact that the innocent bystander effect is seen with a wide variety of transferred genes suggests that the mechanism or mechanisms involved in mediating this effect may not be immunologic (or some may not), but much more study is required to determine why this occurs and how it might be best used. Alternatively, genes that protect normal cells against toxicity of therapeutic agents used to treat the malignancy could be used to protect the host and allow higher-dose delivery of the toxic agents.
Difficulties remain in ensuring that expression of the transfected gene is appropriately controlled in tumor cells, maintaining expression in these cells during a prolonged period (expression of most genes tends to decrease with time) while preventing deleterious function in normal cells. These need to be adequately addressed for gene therapy to be therapeutically useful.
Mechanism of Action
Introduced genetic material can be used to inhibit or augment gene function or produce proteins beneficial to the host by a number of potential mechanisms (Table 30.10).117, 118, 119, 120, 121, 122, 123, 124, 125 For example, such material can be used:
· as RNA decoys (multiple copies of a sequence can be transcribed and compete with other intracellular sequences for protein binding)
· as transdominant altered proteins that can suppress specific protein function in cells (if these happen to be critical proteins for a signal transduction pathway [e.g., ras or raf], one could potentially disrupt the entire pathway)
· in the production of intracellular toxins, leading to cell death
· in the intracellular production of antibodies to block the function of specific proteins
TABLE 30.10 POTENTIAL APPLICATIONS OF GENE THERAPY
· as ribozymes to catalyze the cleavage of specific RNA or DNA sequences
· in the expression of proteins that make tumor cells more immunogenic and therefore prone to elimination by the host's immune system and that generate a systemic immune response against the tumor
· in the expression of genes that make cells susceptible to killing by specific drugs (e.g., the herpesvirus thymidine kinase [TK] gene, which makes cells susceptible to killing by ganciclovir, or the cytosine deaminase gene, which activates 5-fluorocytosine)
· in the expression of a gene (e.g., the dihydrofolate reductase or multiple drug resistance type 1 gene) in normal cells that makes them more resistant to killing by cytotoxic agents or radiation therapy
· to produce proteins beneficial to the host in an ongoing and controllable manner, either in immune-effector or other host cells, such as fibroblasts
Cellular Pharmacology and Metabolism
Two major methods for introducing genetic material into cells are (a) physical approaches, such as via liposomal delivery, DNA-ligand complexes, ballistic techniques (in which multiple pellets coated with DNA are rapidly injected into cells), and direct injection of DNA itself, and (b) viral-mediated transfer. Defective retroviral vectors were the initial ones studied for use as a means of getting genes into and expressed in mammalian cells.117, 118, 119 However, they have a number of limitations for therapeutic use, which has led to the development of other viral vectors for this purpose. Limitations of retroviral vectors include these: it is necessary to have active cell division for retroviral vectors to integrate into chromosomes; cells must have the appropriate receptors to take up the vectors; the integration of viral DNA into random chromosomal sites raises concerns about the potential activation of endogenous genes, which might be transforming, or the inactivation of genes important in controlling cell proliferation; and the vectors may become inactivated, such as by complement, when used in vivo. In fact, the activation of the T-cell oncogene LMO2 after gene therapy for X-linked immunodeficiency, with resultant uncontrolled T-cell proliferation, has confirmed this potential risk.125
For these reasons, construction of other viral vectors that might not carry the same degree of risk for mutagenesis or induction of host genes has been actively pursued. These viral vectors include vaccinia (and other poxviruses), polio, Sindbis (and other RNA viruses), adenovirus, adeno-associated viruses, herpesvirus vectors (e.g., herpes simplex virus 1), and HIV-1, which have all been modified for use in gene therapy.117, 118, 119, 120, 121, 122, 123, 124, 125 As with retroviral vectors, each of these has its potential benefits but also potential problems as delivery systems for gene therapy. There is an ongoing effort to improve each of these vectors in order to achieve better vector systems for therapeutic use. As an example, a possible method for making these vectors safer is by incorporating the TK gene, rendering the cells they infect susceptible to ganciclovir and therefore susceptible to elimination, should this be desired. This approach has been explored to control graft versus host disease mediated by alloreactive donor T cells; however, donor cells have occasionally become leukemic as a consequence of insertional mutagenesis.126
Physical methods of gene transfer not requiring modified viral vectors also have been used, including liposomal delivery, direct injection of genetic material, in vivo lipofection with cationic lipid–DNA complexes, ballistic techniques to “microinject” large amounts of DNA-coated pellets into cells, and targeted DNA-ligand complexes via cell endocytosis. 117, 118, 119 Examples of ligands used for this purpose include liver-specific asialoglycoprotein receptors and transferrin receptors. Special efforts have to be made to inhibit lysosomal degradation of introduced DNA. Chloroquine is one of the compounds used for this purpose. Viral particles (e.g., adenovirus, which can disrupt endosomes) provide another means of preventing DNA degradation. These approaches have not been toxic in animal models. Ultimately, combinations of different delivery systems using the advantages of each may provide the most useful approach to gene therapy.
Gene delivery can either be ex vivo or in vivo. Advantages of ex vivo delivery include high efficiency, ability to enrich infected cells, and ability to assess for presence of the transduced gene before reinfusing the cells.117, 118, 119, 120, 121, 122, 123, 124, 125 Cells can be labeled fluorescently for in vivo tracking, which allows a means of following the fate of ex vivo transfected cells.117, 118, 119, 120, 121, 122, 123, 124, 125 Targeting vectors to cells via specific receptors is also currently being studied.117, 118, 119, 120, 121, 122, 123, 124, 125 In addition, DNA can be injected directly into tissues to produce desired proteins, such as activators of the immune system.117, 118, 119, 120, 121, 122, 123, 124, 125 Direct injection of the genes for enzymes that activate prodrugs into malignant cells, followed by treatment with prodrugs, is another approach. Many of the current trials evaluating the efficacy of gene therapy in treating cancer involve ex vivo introduction of a gene (usually an immunomodulatory molecule, such as granulocyte-macrophage colony-stimulating factor [GM-CSF]) into tumor cells (either autologous or allogeneic), with reintroduction of modified, inactivated cells (inactivated usually by irradiation) into the patient as cancer vaccines.
In vivo, a number of approaches can be used to enhance gene delivery to tumors in specific settings.117, 118, 119, 120, 121, 122, 123, 124, 125 Liposomal delivery can be used to target cells, such as in the liver or lung. Methods of getting compounds across the blood-brain barrier, such as using the high concentration of transferrin receptors in brain capillary endothelial cells, also might be useful for delivery to lesions in the central nervous system. Cationic liposomes may offer an advantage for targeting delivery to specific tissues. Each of these approaches can be combined with tissue-specific promoter-enhancer elements to limit expression of the gene to tissues of interest (see below).117, 118, 119, 120, 121, 122, 123, 124, 125 A number of factors may be important in the expression of transgenes in vivo, including composition of the liposomal lipid, the DNA-liposome ratio, and the promoter-enhancer elements that control the expression of the gene.117, 118, 119, 120, 121, 122, 123, 124, 125 Combinations of viral and physical systems offer yet another approach.
Controlling the expression of genes in vivo remains an important issue. The expression of genes can be limited to target tissues by using tissue-specific transcriptional regulatory sequences upstream of the gene. An example of this is targeting the active form of a drug by virally directed enzyme prodrug therapy; in this method, tissue-specific transcriptional regulatory sequences are placed upstream of the drug-activating gene so that its expression is restricted in a tissue-specific manner.101, 102 The expression of most genes has been relatively low and declines with time due to the death of transfected cells as well as the loss of gene activity by mechanisms that are not yet well delineated but may involve nucleases.
Although there have been a large number of clinical trials evaluating gene therapy for cancer treatment in humans, a significantly smaller number of these have evaluated the PKs of virus, especially delivered systemically. Two recent trials in humans have evaluated the PKs of virus and vector DNA when injected intratumorally.127, 128 Both trials used adenoviral vectors (replication defective in one case and replication competent in the other) and had similar results inasmuch as there was no detectable intact infectious adenovirus in the blood at any time point although vector DNA was detected in the blood. In both trials, an immune response to the vector was induced. In one trial, tumors were resected between 3 and 8 days after the injection and showed the presence of virus in approximately 20% of cells at the dose of 1 × 1011 virus particles.127 Viral particles were seen in tumor cells but also in other cells, including mostly macrophages but also lymphocytes and fibroblasts. In the other trial, biopsies were obtained over time to evaluate the persistence of transgene expression in the target tissue (prostate) in a small number of patients. Expression was seen in 3 of 3 patients at 2 weeks, 1 of 3 patients at 3 weeks, and 0 of 3 patients at 4 weeks.128 As the authors point out, biopsies have the potential for sampling error that might underestimate the persistence of expression elsewhere in the organ. These trials illustrate features that have been commonly found in studies: antibody responses to vectors occur in a significant percentage of patients, infectious particles are not commonly detectable in secreted fluids or blood when the vectors are delivered directly into tumors, viral replication occurs and is detectable for 1 to 2 weeks after delivery, and persistence of expression of the gene of interest over time remains a problem to be solved.
As regards the toxicity of gene therapy, the main concern is the potential toxicity of different viral vectors and the potential for serious toxicity to an organ (such as the liver or lung) from marked immune response or viral replication as well as the possible risk of malignancy from the insertion of retroviral vectors in critical areas of the genome. In fact, the death of a patient from overwhelming toxicity (particularly hepatotoxicity) after receiving a modified adenoviral vector and the development of uncontrolled clonal proliferation of mature T cells in two children with X-SCID treated with the common y chain of the interleukin-2 receptor delivered via a modified retroviral vector that inserted near the proto-oncogene LMO2 promoter showed that these risks are real.127, 129These two events have led to significant reevaluation of how gene therapy can be made safer as well as heightened oversight of gene therapy trials.
It has been shown that the transfection of tumor cells with a wide spectrum of cytokines, antisense constructs, foreign major histocompatibility complex genes, or other genes important in mediating various aspects of the immune response, as well as the reintroduction of these cells into animals, has significant antitumor effects, putatively by immune-mediated mechanisms.117, 118, 119, 120, 121, 122, 123, 124, 125, 128, 130, 131 Most studies suggest that a proportion of tumor cells are killed by an innocent bystander effect (presumably immunologically mediated). However, this effect is seen even with approaches that would not be expected to generate a strong immune response, and the actual mechanisms for the innocent bystander effect remain undetermined.
Gene therapy is undergoing extensive clinical evaluation in humans.117, 118, 119, 120, 121, 122, 123, 124, 125 Approaches demonstrated to be effective in animal studies and currently undergoing clinical trials include (a) inhibition of an oncogene (e.g., K-ras or raf); (b) replacement of a functional tumor suppressor gene (e.g., p53); (c) transfection of activating enzymes for prodrugs (e.g., hTK gene) into tumor cells, followed by treatment with the prodrugs (e.g., ganciclovir); (d) transfer of protecting genes (e.g., MDRI) into normal (especially hematopoietic) cells, followed by high-dose chemotherapy; (e) transduction of immune effector cells with cytokine genes (e.g., TNF or IL-2) to enhance their effectiveness in killing tumor cells; (f) transduction of tumor cells with immunomodulatory molecules (GM-CSF, IL-2, IL-4, HLA-B7) to make them more susceptible to immunologic destruction, as well as to generate a systemic immune response against the tumor; and (g) use of vectors expressing antisense messages to inhibit the function of critical genes for tumor cell survival, proliferation, or metastasis, such as growth factors or their receptors or angiogenic factors (e.g., VEGF) or their receptors.77, 103, 105, 106, 107, 108, 109, 110, 111,112 There are extensive ongoing efforts to optimize dendritic cell activation by those approaches using immunomodulatory molecules, such as GM-CSF and IL-4. These approaches have all been successful in treating tumors in animal models, but clearly their potential use in patients is in the early stages of development. To date, these approaches for treating cancer patients have been shown to be feasible but have had limited clinical efficacy. Efforts to enhance and prolong the expression of transduced genes in vivo, as well as other approaches for enhancing the therapeutic efficacy of these approaches, are being evaluated in ongoing studies.
Sufficient understanding of the biology of cellular processes exists, and tools are now available to manipulate molecular systems for therapeutic benefit, to allow the rational design and delivery of molecules directed at specific proteins or genes important for the survival or growth of malignant cells. Over the past decade, a number of molecularly targeted agents have been developed and are now part of the standard anticancer armamentarium. Targets include mutant proteins, proteins overexpressed on malignant cells, and angiogenic factors. These include growth factor receptors and their ligands as well as proteins involved in the signaling processes downstream from growth factor receptors. Most targeted therapies have been designed to inhibit the target directly, although several deliver cytotoxic compounds to malignant cells. Agents that have been approved include mAbs for treatment of breast cancer, colorectal cancer, AML, B-CLL, and B-cell NHL as well as an immunotoxin for treatment of CTCL; the small molecule bcr-abl and the KIT tyrosine kinase inhibitor imatinib mesylate for CML and GIST, respectively; and the small molecules gefitinib and erlotinib for treatment of patients with NSCLC. A number of other targeted agents are in late clinical development, and the list will continue to grow.
This is an exciting time in the development of rationally targeted therapies. However, there is still a significant amount of study necessary to develop additional clinically useful antitumor approaches using targeted agents. Continued improvement in our understanding of the critical processes in cancer development, growth, and metastasis should provide new targets and new agents for attacking these. Technological advances allowing much more sophisticated evaluation of intracellular and intercellular processes, such as utilization of siRNAs and analysis of protein-protein interactions, will play an increasingly important role. As more information is gathered about the immensely complex interaction of proteins in cells, this effort will require the use of mathematical evaluation by powerful computers. However, carefully performed toxicity and pharmacologic studies to determine how to best deliver these therapies to patients remain critical to the successful clinical development of any of these agents. Because there is no current method for adequately modeling these by in vitro studies, analysis (e.g., by computer models), well-designed and well-performed animal and, most importantly, human clinical studies remain essential to the development of these compounds as therapeutic agents. Knowledge gained from the successful development of targeted agents and also from failed attempts will provide insights into how to best identify and develop new compounds. As studies continue to identify new targets, there will be an ongoing effort to develop new and improved targeted agents against them to improve the treatment of different cancers. Gradually, the dream of targeted therapy is being turned into reality.
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