Growth Factors Bind to their Rcccptors


Scheme 10.31 • Schematic of the activation of growth factor receptors.

Figure 10.25 • Schematic of the Ras pathways leading to uncontrolled cell proliferation.

cascade effect, whereby one kinase phosphorylates and activates more of the next kinase in the pathway amplifying the signal in the process. Stimulation of this pathway results in the activation of several transcription factors involved in the expression of growth factors, cyclin D1 (involved in controlling cell division, see Fig. 10.1), and transcription factors Fos and Jun, which were first discovered as viral oncogenes and are known to be elevated in several human cancers. In addition, overactivation of this pathway may result in loss of contact inhibition, loss of anchorage dependence, and changes in cell shape, all of which are characteristic of cancer cells.

The second major pathway activated by Ras is PI3K-PIP3-Akt/PKB whose activation increases cell proliferation but perhaps most importantly, inhibits apoptosis. When Ras is activated, phosphatidylinositol(4,5)-diphosphate (PIP2) is further phosphorylated to give phosphatidylinosi-tol(3,4,5)-triphosphate PIP3, which remains embedded in the plasma membrane but attracts and activates other ki-nases namely protein kinase B (PKB), a serine-threonine kinase. Overactivity of this pathway results in decreased apoptosis, increased proliferation and increased cell size. Specific effects include inactivation of the proapoptotic protein Bad (Fig. 10.2) and activation of antiapoptotic NF-kB.

The third pathway involves Ras-mediated activation of Ral-GEF (Guanine nucleotide exchange factor), which acts on Ral-A and Ral-B to stimulate their exchange of GDP for GTP and hence their activation. The activation of this pathway, although less well understood, seems to allow cancer cells to metastasize and invade.

Overactivity of these pathways is present in several different cancers, and consequently, several agents have been developed to correct this. One such approach is the inhibition of kinases whose normal activity has been amplified.

Working in Philadelphia in the 1960s, scientists discovered a genetic abnormality present in 95% of patients with CML.145 The abnormality was a translocation between chromosomes 22 and 9 in which the ABL gene (analogous to the Abelson proto-oncogene found in mice) originally on chromosome 9 was translocated to chromosome 22, such that it was fused with BCR gene (breakpoint cluster region). The new chromosome was designated as the Philadelphia translocation or the Philadelphia chromosome (Ph1).146 The fusion of these two genes resulted in a protein that encoded the entire ABL gene and as many as three additional proteins from BCR. The product of this fusion was found to be a TK that was always activated. This altered TK utilizes ATP to phosphorylate and activate numerous pathways including MAPK and Akt/PKB along with several others. This leads to proliferation of white cells that normally lasts about a year, when the disease becomes more difficult to treat and eventually to the terminal blast phase lasting several months.

Imatinib (Fig. 10.26) was developed to specifically inhibit this unique TK and does so rather selectively by binding to the ATP-binding pocket and stabilizing an inactive form of the enzyme. Protein kinases have similar structures and consist of C- and N-terminal lobes with the active site formed by a cleft between the two lobes. The activity of the enzyme is regulated by an activation loop that is extended in the active form of the enzyme and provides for substrate binding. X-ray crystal studies of imatinib binding to Abl show that the drug binds in the cleft formed by the two lobes of the enzyme through the formation of several hydrogen bonds and hydrophobic interactions with the pyridine ring assuming the postion occupied by ATP in the active enzyme.147 Imatinib binding locks the enzyme in a conformation in which the activation loop is oriented so as to block substrate binding. Specificity for the BCR-ABL kinase is not complete because of structural similarity of TKs, and those associated with the PDGF receptor and the receptor for SCF known as KIT are also inhibited (Table 10.3). The agent has been found to be effective in 90% of patients in the chronic stage (the chronic stage last 4-6 years and precedes the proliferation stage mentioned previously). Blast-phase patients respond at a rate of 60% but usually relapse after several months.148

The tremendous success of imatinib led to other agents that could be used in the case of resistance. Specifically, dasatinib can bind to either an active or inactive form of the enzyme and is 100 times more potent compared with ima-tinib. It has the ability to overcome resistance associated with amino acid alterations that leads to decreased affinity for imatinib. As the structure is altered, binding to other TKs (BCR-ABL, PDGF, SRC, KIT) is manifested, which may eventually lead to other indications but at this time, it is used primarily in those cases when patients fail to respond to imatinib.149

This general idea of targeting TKs was extended to the EGF receptor. It was hoped that by targeting EGF TKs the inhibitors would be applicable to a wide range of cancers. Recall that after the receptors dimerize, they transphospho-rylate each other and act as TKs. Currently, there are two structurally similar agents in use, gefitinib and erlotinib, which bind to the ATP-binding site and thereby inhibit the resulting cascade that would normally occur from activation of the EGF receptor.150,151 The benefit of the agents was hoped to be caused by inhibition to the Ras pathway so that ultimately, all three arms could be affected (MAPK, Akt/PKB, and Ral-GEF). The agents are modestly selective for this kinase. Currently both compounds have indications for NSCLC generally as alternatives after the failure of more traditional therapy, and gefitinib has an additional indication against pancreatic cancer. Initial results have not been as dramatic as that seen with imatinib. There are also several known mutations and deletions in the EGF receptor of cancer patients and this may affect the activity of these compounds. Specifically, point mutations in the KRAS gene (a member of the Ras family) have been associated with resistance to these agents and tests have become available to detect this prior to initiating therapy.

The activation of numerous tyrosine and serine-threonine kinases offers many theoretical targets for drug intervention when these pathways become overactive as is often the case in neoplastic cells. These kinase enzymes are all thought to have evolved from a common ancestor and as such possess a great deal of structural similarity that complicates the development of agents, which are selective for a single kinase. This has led to the strategy of simultaneously inhibiting numerous kinase enzymes, and consequently there are several multikinase inhibitors now available. Sorafenib was originally developed using high-throughput testing and parallel synthesis as an inhibitor of the serine-threonine kinase Raf.152 It proved to be a potent inhibitor of this enzyme with an IC50 = 6 nM and acted by stabilizing an inactive form of

Lapatinib p

Figure 10.26 • Protein kinase inhibitors.

the enzyme similar to imatinib. Subsequent x-ray analysis of the drug receptor complex showed that the pyridine ring occupied the ATP-binding site of Raf, whereas the trifluo-romethyl-chloro-substituted phenyl ring occupied a hy-drophobic pocket in the cleft formed by the two lobes of the protein.153 The urea moiety forms two hydrogen bonds, one through interaction of the amide proton with the carboxylate of Glu500 and the second through interaction of the car-bonyl oxygen with the peptide amide N-H of Asp593. The compound also has the capacity to inhibit several other TKs associated with growth factor receptors including PDGF-R, VEGF-R, RET-kinase, and c-Kit with IC50s in the range of 20 to 90 nM. In some cancer models, the compound has been shown to be effective even though the MAPK pathway was not inhibited and this has been explained by the com pounds inhibition of angiogenesis This has been attributed to the effects on inhibition of PDGF-R and VEGF-R phos-phorylation and the role these growth factor receptors play in the creation of new blood vessels. The agent was investigated and subsequently approved for use in renal cell carcinoma (RCC) because of the fact that this particular cancer is often associated with increased activity of the targets inhibited by sorafenib and the high level of neoangiogenesis that is seen in this cancer.

As a group of agents, the protein kinase inhibitors have the advantage of oral administration and better patient tol-erability. The major adverse effects include a skin rash that normally appears early in therapy on the upper torso and is generally mild but may become more serious in some cases. Mild diarrhea and nausea are also commonly seen but are generally controlled with the administration of antiemetics and antidiarrheals. Mild myelosuppression is also seen with several of these agents. There has been concern about car-diotoxicity associated with protein kinase inhibitors because of the fact that multiple kinases may be inhibited. This could potentially have several effects on signaling processes in cardiac tissue. For example if the Akt/PKB pathway was blocked, apoptosis may become activated and cardiac cells would die (Table 10.3). There has been some evidence for cardiotoxicity in this class and the agents have been ranked with imatinib, dasatinib, sorafenib, and suni-tinib having known or likely cardiotoxicity and gefitinib, erlotinib, and lapatinib having low cardiotoxicity. Concern of cardiotoxicity is also a result of the fact that other drugs such as the anthracyclines and several of the monoclonal antibodies directed against the EGF-R are known to have cardiotoxicity associated with their use.154 Therefore, patients who have already been exposed to these agents may experience additional adverse effects to their cardiovascular system if a protein kinase inhibitor were utilized and it also produced significant cardiotoxicity. Studies for the most part have not evaluated the cardiotoxicity of these agents and the given ranking is based on preliminary data, but there is concern about the cardiovascular effects of this class of agents.


Imatinib is available in 100- and 400-mg capsules for oral administration and is indicated for the treatment of CML, gastrointestinal stromal tumors (GIST) that express Kit and acute lymphoplastic leukemia that is positive for the Philadelphia chromosome.

Bioavailability of the agent is nearly 100% by the oral route. The agent is highly protein bound and metabolized to the N-desmethyl derivative by CYP3A4-mediated removal of the piperazinyl methyl group. The resulting metabolite is similar to the parent in activity. Elimination occurs primarily in the feces, and the terminal half-life is 18 hours for the parent and 40 hours of the N-desmethyl metabolite. Resistant forms of the TK are known, which have altered amino acids that prevent binding. In addition, there may be increased levels of the kinase itself. The drug is also a substrate for Pgp and an additional efflux transporter known as breast cancer resistance protein (BCRP), both of which remove the drug from the cell. These transporters are also inhibited by the agent as well. Severe side effects include as-cites, neutropenia, thrombocytopenia, skin rash, and pulmonary edema. Less serious side effects include nausea/vomiting, heartburn, and headache but overall, the agent is better tolerated than most other medications used in treating the disease.


Dasatinib is available in 20-, 50-, and 70-mg tablets for oral administration in the treatment of CML and ALL that are Ph1 positive. Although dasatinib is more potent than imatinib, bioavailability is much lower with values ranging between 14% to 34%. The agent is extensively metabolized with as many as 29 metabolites seen as result of oxidation by primarily CYP3A4 and phase II conjugation. The agent may act as an inhibitor of CYP3A4 and CYP2C8. Metabolism does give an active metabolite, but this accounts for only 5% of the total and is not believed to be important for the overall activity of the agent. Dasatinib is 95% protein bound with a terminal half-life of 3 to 5 hours. The majority of the drug and metabolites are eliminated in the feces. The most common side effects are skin rash, nausea, diarrhea, and fatigue. Serious side effects include myelosuppression appearing as neutropenia and thrombocytopenia, bleeding of the brain and GI tract, and fluid retention.


Geftinib is available as 250-mg tablets for oral administration in the treatment of NSCLC for those patients who have failed to respond to platinum-based therapies and docetaxel and has also been used against squamous cell cancers of the head and neck. The agent is an inhibitor of the TK of EGF-R and possibly other TKs as well. Gefitinib is both a substrate and inhibitor of Pgp and BCRP. The agent is absorbed slowly after being administered orally with 60% bioavailability. Metabolism occurs in the liver and is mediated primarily by CYP3A4 to give eight identified metabolites resulting from defluorination of the phenyl ring, oxidative-O-demethylation, and multiple products arising as a result of oxidation of the morpholine ring. The O-demethylated product represents the predominate metabolite and is 14-fold less active compared with the parent. The parent and metabolites are eliminated in the feces with a terminal elimination half-life of 48 hours. The drug appears to be well tolerated with the most commonly reported side effects being rash and diarrhea. It may also cause elevations in blood pressure especially in those patients with preexisting hypertension, elevation of transaminase levels, and mild nausea and mucositits.


Erlotinib is available as 25-, 100-, and 150-mg tablets for oral administration and is used after failure of first-line therapy in metastatic NSCLC and as first-line therapy in combination with gemcitabine in the treatment of metasta-tic pancreatic cancer, and in treating malignant gliomas. The structural similarity to gefitnib imparts similar phar-macokinetic behavior with bioavailability of 60% and protein binding of 93%. The agent is extensively metabolized primarily by CYP3A4. Three major metabolic pathways have been identified, involving oxidative-O-demethylation of the side chains followed by further oxidation to give the carboxlic acids, oxidation of the acetylene functionality to give a carboxylic acid, and aromatic hydroxylation of the phenyl ring para to the electron-donating nitrogen. The metabolites are primarily eliminated in the feces, and the terminal half-life is 36 hours.155 The major toxicities seen with the agent are dose-limiting skin rash and diarrhea. Other common adverse effects include shortness of breath, fatigue, and nausea.


Sorafenib is available in 200-mg tablets for oral administration and is used in the treatment of RCC and colon cancer. The agent is classified as a multikinase inhibitor because of its action on numerous kinase enzymes including the PDGF-R, VEGF-R, Kit, and Raf. Sorafenib is 39% to 48%

bioavailable and CYP3A4-mediated metabolism gives eight identified metabolites including the N-oxide, which is equally active with the parent. However, the majority of the drug in plasma is present as the parent compound. Sorafenib is highly protein bound (99.5%). The drug is eliminated primarily in the feces (77%) with 19% appearing in the urine as glucuronides (UGT1A1) and has a elimination half-life of 24 to 48 hours. The most commonly seen toxicity is skin rash that normally occurs within the first 6 weeks of therapy. Other adverse effects include hypertension, fatigue, increased wound healing time, and increased risk of bleeding.


Lapatinib is available in 250-mg tablets for oral administration and is used in combination with cabecitabine in the treatment of breast cancer for those patients that over express the type 2 EGF-R and who have previously received taxane, anthracycline, and trastuzumab therapy. The type 2 EGF-R is one subtype of this receptor and is also known as HER2 or ErbB-2. The agent is a receptor TK inhibitor targeting the ErbB-1 and ErbB-2 subtypes. Binding occurs at the ATP-binding site and thereby prevents phosphoryla-tion and the subsequent activation of other kinase enzymes. ErbB-1 overexpression occurs in approximately 27% to 30% of breast cancers, while ErbB-2 is over expressed in 20% to 25% of cases.156 The agent has demonstrated IC50 values of <0.2 ^M against ErbB-1 and 2 from several different cancer cell lines and dissociates slowly (i1/2 = 300 min) from these receptor TKs.157 The drug is both a substrate and an inhibitor of the efflux transporters Pgp and BCRP. It is also an inhibitor of the hepatic uptake transporter OATP1B1, which is an organic anion transporter.158 The absorption of lapatinib is incomplete and variable after oral administration. The agent is extensively metabolized by CYP3A4 and CYP3A5, with minor contributions from CYP2C19 and CYP2C8. Lapatinib inhibits CYP3A and CYP2C8 at clinically relevant concentrations. The agent is highly (99%) protein bound and eliminated primarily in the feces. The half-life of the agent increase upon repeated dosing, taking 6 days to reach steady state that gives an effective half-life of 24 hours. The most commonly seen adverse effects of lapatinib therapy are skin rash and diarrhea. Skin rash is commonly seen with many of the other TK inhibitors and agents that target ErbB-1. Lapatinib-induced diarrhea is usually mild to moderate. There have been reports of decreases in left ventricular ejection fraction associated with the agent, although this appears to occur only rarely and is reversible upon discontinuation of therapy.


Sunitinib is available in 12.5-, 25-, and 50-mg capsules for oral administration for the treatment of advanced RCC and GIST upon the failure of imatinib. The agent is a multiki-nase inhibitor and has been shown to inhibit PDGF-R, VEGF-R, Kit, RET, and the colony-stimulating factor receptor (CSR-1R). The result of this spectrum of activity is a slowing of tumor progression and inhibition of angiogen-esis both of which are useful in the highly vascularized cancers, RCC and GIST. The median TTP (time to tumor progression) was 27.3 weeks for sunitinib and 6.4 weeks for placebo. Progression-free survival was also significant with a median time of 24.1 weeks for sunitinib and 6 weeks for placebo. The agent is well absorbed upon oral administration, and both the parent and major metabolite are highly (90%-95%) protein bound. Metabolism is mediated primarily by CYP3A4 to give the N-desethyl derivative, which is the major metabolite (23%-37%), equally active with the parent and undergoes further metabolism by CYP3A4. The terminal elimination half-life for the parent and N-desethyl derivative are 40 to 60 hours and 80 to 110 hours, respectively. Elimination occurs primarily via the feces. Common adverse effects of sunitinib include fatigue, diarrhea, yellow skin discoloration, anorexia, nausea, and mucositis. Mild myelosuppression has been reported in patients with GIST including neutropenia, lymphopenia, thrombocy-topenia, and anemia. There have been reports of cardiotox-icity including decreases in left ventricular ejection fraction, which occurred in 11% of patients during a GIST study.

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