HER1/EGFR-Tyrosine Kinase Inhibitors: Experimental Evidence
by Manuel Hidalgo, MD
Properties of Erlotinib
My job will be to briefly discuss some of the key experimental data with Tarceva (erlotinib) and some of the evidence that this is a drug that inhibits the growth of human tumors.
These are the areas that I would like to cover in my presentation: provide some background information on erlotinib; discuss the implications of the extent of the inhibition of the epidermal growth factor receptor (EGFR) on downstream signaling pathways; review some of the core experimental in vitro and in vivo data; and provide some evidence that the drug inhibits tumors that are transformed as a consequence of mutations in the EGFR.
This is the chemical structure of erlotinib; it is a small molecule. It is a quinazoline and is orally available.
The drug is able to inhibit the kinase activity of the purified EGFR very specifically. As shown in this graphic, it is quite selective for human epidermal growth factor receptor 1 (HER1) vs HER2, another membrane, and intercytoplasmic receptor. This is the effect of increasing the concentration of erlotinib as a function of phosphorylation of the HER1 receptors, so it is a drug very specific for EGFR. Erlotinib and Kinase Activation
As a consequence of inhibition of EGFR, we expect that erlotinib would inhibit tyrosine signaling pathways that are activated by this receptor. There are 2 principle pathways: the mitogen-activated protein (MAP) kinase pathway and the P13K signaling pathway. In order to have an antitumor effort, we need to inhibit one or both of these pathways. Inhibiting the receptor alone, if these pathways remain activated, probably will not be translated into a significant antitumor effect.
It is important to notice that increasing concentration of the drug is able to inhibit not only the EGFR receptor in these 2 breast cancer cell lines, but it is also able to inhibit one of the downstream signaling pathways, in this case, the MAP kinase pathway. So, here is increasing concentration of the drug and proportional response for relative inhibition of HER1 and MAP kinase. What these data suggest is that we may need higher concentrations of the drug to inhibit the downstream signaling pathways vs the concentrations that are needed to inhibit the receptor itself. This appears to be true in situations in which the proportion of receptor in the membrane is different, as you can see in these cells with different concentrations of HER1 and HER2 receptors in the membranes.
The same appears to be correct for inhibition of Akt; the drug is able to inhibit Akt phosphorylation. It inhibits Akt phosphorylation as a consequence of the inhibition of EPO receptor binder 1 (ERB1), but the concentrations that are needed to achieve that effort appear to be higher, at least in vitro, as compared with what is needed to inhibit the receptor itself.
What these data probably are telling us is that in order to achieve inhibition of downstream signaling pathways and, therefore, in order to have a chance to have antitumor effect, we will need to be able to inhibit the majority of receptors in the membrane. That is probably an effect related to the concentration of the drug that can be achieved in vitro system, in vivo system, or in plasma Experimental Data on Erlotinib
Where are we now in terms of experimental data with erlotinib?
This is perhaps a better-known figure representing the effects of oral administration of erlotinib. In a xenograft model of head and neck carcinoma (HN5), increasing concentration of the drug to a level of probably 12-15 mg/kg/day orally is very effective in inhibiting the growth of this particular tumor. You can take these data as an indication that concentrations above 12-15 mg/kg/day are not more efficacious than concentrations of that magnitude; however, this is probably an artifact of this model being very sensitive to EGFR inhibition.
When you look at the same data -- in this case using perhaps a more realistic model, which is a non-small cell lung cancer model -- the effect, indeed, is related to the concentration of the drug, of the dose administered to these mice; 100 mg is more effective than 25 mg. When you work with this drug in the laboratory, in the majority of models, you need to give doses above 100 mg to have a meaningful tumor effect.
In these preclinical in vivo studies, the drug demonstrated antitumor activity. There was a clear demonstration that the antitumor effect, the tumor-blocking inhibition -- in this case in the head and neck carcinoma in vivo model -- was related to the degree of inhibition of the pathway. I think we can take these data to make the comment that we need to be inhibiting the target in order to have an antitumor effect; therefore, these have significant implications in terms of how we develop this drug. We may start thinking of dosages and the schedules in which this effort is optimized. The problem is how do we apply this to a clinical trial, but that is a different question.
This is more evidence of erlotinib activity in a preclinical model -- in this case the HN5 head and neck carcinoma model -- in which increasing concentration of the drug, to the effective dose of 1 mcM, is able to induce a very brisk apoptosis response in these cells. This is overcome when you add a growth factor, indicating that the apoptosis response is growth factor inhibition dependent Use of Erlotinib in Combination
I am going to present some data illustrating combinations of erlotinib with conventional chemotherapy, radiation therapy, and some novel agents.
As we are aware of some of the studies with Iressa (gefitinib) in combination with chemotherapy, we were a little scared of talking about combinations of EGFR inhibitors with chemotherapy, but when you look at the rationale -- at least from the basic science from the preclinical data -- to do these combinations is very important. Combining agents with different mechanisms of action may improve efficacy by targeting different pathways, and it also may be a way to overcome the development of resistance. So, there are ongoing preclinical, as well as clinical, studies combining erlotinib with chemotherapy agents and radiation therapy. What is of major interest is the combination of these drugs with other targeted agents, if we consider that we probably will need inhibition of different levels of the signal transduction pathway in order to have a meaningful antitumor effect.
There have been a significant number of preclinical studies combining erlotinib with conventional chemotherapy agents; for the most part, there was a positive interaction in terms of additivity or synergy. When combined with cisplatin, doxorubicin, paclitaxel, gemcitabine, and capecitabine, there were no interactions with other drugs; in none of these studies was there evidence of increased toxicity in the animals. The question then becomes how do you translate this into the clinic? I am not quite sure that the experimental design that is commonly utilized in these preclinical studies is really the same thing that we do in the clinic; that may be the reason why, sometimes, we have seen discrepancies in terms of the preclinical data.
This is a representative example of an interaction between erlotinib and cisplatin, in a head and neck carcinoma model, in terms of tumor-growth inhibition, demonstrating that the combination of the drugs is more effective than either of the agents alone. This motivated the design of clinical trials. We do not know the results of these trials, so we need to wait to see what happens.
The same thing occurs preclinically when you combine erlotinib with radiation therapy. In this case, the combination of erlotinib with radiation therapy is able to stimulate caspase activity and, therefore, to stimulate the entry of cells in apoptosis. The combination of the drug with radiation therapy decreases the survival fraction, which is the classic parameter of radiation efficacy in preclinical tumor models.
In addition to combinations of erlotinib with conventional drugs and radiation therapy, there is mounting evidence that the combination of the drug with other targeted compounds has increased efficacy. A combination that is very attractive preclinically, and one that is currently well advanced in terms of clinical development, is the combination of erlotinib with an angiogenesis inhibitor. These are data in colorectal carcinoma indicating that erlotinib by itself, in general, does not have very pronounced activity in this particular colon cancer model, something that we then learn is also true in the clinic. Bevacizumab (Avastin) is an effective drug, at least in some models, but what is of major interest is that in the majority of the models, when you combine the 2 drugs, you have a significant increment in tumor growth inhibition. Erlotinib and Epidermal Growth Factor Receptor Variant III
In the final part of the presentation, I would like to present some data presented at the American Society of Clinical Oncology (ASCO) recently, demonstrating that erlotinib is able to inhibit this mutant EGFR. This has significant implications in many cancers, including non-small cell lung cancer.
This truncated EGFR variant III (EGFRvIII) has a deletion of exons 2 to 7. It is detected in a high percentage of primary human glial tumors, and we ask why the drug appears to be so effective in this particular tumor model. EGFR has also been found to be mutated in other common solid tumors, including non-small cell lung cancer, prostate cancer, and breast cancer. What is important is that this mutation results in a constitutive ligand-independent activation of the receptor.
The studies that have been conducted indicate that erlotinib is able to inhibit the phosphorylation or activation of EGFRvIII. It appears also to be able to decrease the amount of receptor, particularly when exposure is prolonged. On the left, you have Western blots of total receptor at control, exposed to erlotinib, and exposed to AG1478, which is an inhibitor of EGFRvIII. Two hours of exposure does not change the amount of the receptor; however, when you prolong the exposure, there appears to be a clear decrement in the amount of this mutated receptor. What is also of major importance is that the drug is able to inhibit the phosphorylation (the activation) of this particular receptor, both in 2 hours and in 24 hours. So, we are leading to higher concentrations, higher dosages to inhibit downstream signaling pathways, but perhaps, for some models, prolonged exposure is also of interest because there can be this decrement in the membrane receptor.
When you compare the activity of erlotinib in tumors with mutated receptor vs tumors with wild-type receptors, such as MB468 and the HN5, erlotinib is able to inhibit the phosphorylation of the receptor. Erlotinib is also able to achieve an inhibitory concentration of 50% (IC50) in that regard, but the concentrations that you need for this particular effect are higher.
This is translated to the same effect when you look at, in this case, tumor growth or cell proliferation, targeting not only inhibition, but now proliferation as well. The data show that the drug is able to inhibit the activation of the receptor, probably to decrease the amount of the receptor in the membrane. This is clearly translated into inhibition of cell proliferation in this assay.
What is probably more important is that the same effect is noticed when you look at clonogenic survival; concentrations above 1 mcM are able to inhibit the capacity, or the capability, of these cells to form colonies in soft agar experiments.
This is the summary of the data that I have presented. Erlotinib is an orally available, ATP-specific, competitive, reversible, and selective inhibitor of EGFR. It produces stasis and regression of tumor xenografts with a 10-15 mg/kg dose. Some models suggest that higher dosages are more effective. High exposure and prolonged exposure may be necessary to inhibit the relevant downstream signaling pathways, the ones that are associated with inhibition of proliferation and induction of apoptosis. There is an additive effect when combined with the majority of conventional chemotherapeutic agents, radiation therapy, and other targeted agents. There is convincing evidence that the drug is effective in tumors that have this constitutively active mutation of the EGFRvIII. *** POSTED JULY 27, 2004 *** |
