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Antiangiogenic activity & VEGF pathway inhibition of Tarceva


gpawelski

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The AngioRx Assay has identified potential responders to Avastin, Nexavar, Sutent and other anti-angiogenic drugs and assessed previously unanticipated direct and potentiating anti-angiogenic effects of targeted therapy drugs such as Tarceva and Iressa.

Tarceva is a tyrosine kinase inhibitor. However, it also has an anti-angiogenic effect on cancer cells. There are a number of classes of drugs that target angiogenesis (VEGF). At the protein level is Avastin. At the tyrosine kinase level is Iressa, Nexavar, Sutent and Tarceva. At the intracellular metabolic pathway mTOR level is Afinitor and Torisel.

When chemotherapy drugs work, they often cause tumors to shrink a lot, sometimes even making them disappear. But anti-angiogenesis drugs don't seem to work in the same way. In some cases they shrink tumors, but in others they just seem to stop them from growing any larger.

Newer approaches to treatment that combine anti-angiogenesis drugs with chemotherapy, other targeted drugs, or radiation may work better than using them alone. For instance, early studies that tested the drug Avastin by itself did not find that it helped people with cancer to live longer. But later studies found that when it was combined with chemotherapy to treat certain cancers, it helped people (some subsets of patients) live longer than if they got the chemotherapy alone.

Doctors aren't sure why this is the case. One theory is based on the fact that chemotherapy drugs may have a hard time getting to cells in the middle of tumors. Tumor blood vessels grow in a short amount of time and in an abnormal environment, so they are not as well-made and stable as normal blood vessels.

Because of this, they tend to be leaky. This affects how well drugs can reach the inside of the tumor. The theory is that anti-angiogenesis drugs may somehow stabilize these tumor blood vessels for a short period of time, allowing the chemotherapy to reach more tumor cells and be more effective.

J Intern Med. 2008 Sep;264(3):275-87.

Cell culture detection of microvascular cell death in clinical specimens of human neoplasms and peripheral blood.

Weisenthal LM, Patel N, Rueff-Weisenthal C.

Source

Weisenthal Cancer Group, Huntington Beach, CA 92647, USA. [email protected]

Abstract

BACKGROUND: Angiogenesis studies are limited by the clinical relevance of laboratory model systems. We developed a new method for measuring dead microvascular (MV) cells in clinical tissue, fluid and blood specimens, and applied this system to make several potentially novel observations relating to cancer pharmacology.

METHODS: Dead MV cells tend to have a hyperchromatic, refractile quality, further enhanced during the process of staining with Fast Green and counterstaining with either haematoxylin-eosin or Wright-Giemsa. We used this system to quantify the relative degree of direct antitumour versus anti-MV effects of cisplatin, erlotinib, imatinib, sorafenib, sunitinib, gefitinib and bevacizumab.

RESULTS: Bevacizumab had striking anti-MV effects and minimal antitumour effects; cisplatin had striking antitumour effects and minimal anti-MV effects. The 'nib' drugs had mixed antitumour and anti-MV effects. Anti-MV effects of erlotinib and gefitinib were equal to those of sunitinib and sorafenib. There was no detectable VEGF in culture medium without cells; tumour cells secreted copious VEGF, reduced to undetectable levels by bevacizumab, greatly reduced by cytotoxic levels of cisplatin + anguidine, and variably reduced by DMSO and/or ethanol. We observed anti-MV additivity between bevacizumab and other drugs on an individual patient basis. Peripheral blood specimens had numerous MV cells which were strikingly visualized for quantification with public domain image analysis software using bevacizumab essentially as an imaging reagent.

CONCLUSIONS: This system could be adapted for simple, inexpensive and sensitive/specific detection of tissue and circulating MV cells in a variety of neoplastic and non-neoplastic conditions, and for drug development and individualized cancer treatment.

http://onlinelibrary.wiley.com/doi/10.1 ... 955.x/full

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Cancer treatment has focused primarily on killing rapidly dividing cells because one feature of cancer cells is that divide rapidly. Unfortunately, some of our normal cells divide rapidly too, causing multiple side effects.

Targeted therapy is about identifying other features of cancer cells, like looking for specific differences in the cancer cells and the normal cells. This information is used to create targeted therapy to attack the cancer cells without damaging the normal cells, thus leading to fewer side effects.

Each type of targeted therapy works a little bit differently but all interfere with the ability of the cancer cell to grow, divide, repair and/or communicate with other cells.

There are different types of targeted therapies, defined in three broad categories.

First. Some targeted therapies focus on the internal components and function of the cancer cell. These use small molecules that can get into the cell and disrupt the function of the cells, causing them to die.

Second. There are a variety of target receptors that are on the outside or surface of the cell. This form of targeted treatment includes the monoclonal antibodies.

Third. Antiangiogenesis inhibitors target the blood vessels that supply oxygen to the cancer cells, ultimately causing the cells to starve and die.

Tarceva is designed to block tumor cell growth by targeting a protein EGFR (epidermal growth factor) that is present on the surface of some cancer cells and some normal cells.

Tarceva inhibits an enzyme within the cell (tyrosine kinase) that is associated with EGFR, however, the specifics of how this inhibition functions is not fully understood.

Mol Ther. 2007 Feb;15(2):279-86.

Oncolytic HSV and erlotinib inhibit tumor growth and angiogenesis in a novel malignant peripheral nerve sheath tumor xenograft model.

Mahller YY, Vaikunth SS, Currier MA, Miller SJ, Ripberger MC, Hsu YH, Mehrian-Shai R, Collins MH, Crombleholme TM, Ratner N, Cripe TP.

Source

Division of Hematology/Oncology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA.

Abstract

Malignant peripheral nerve sheath tumors (MPNSTs), driven in part by hyperactive Ras and epidermal growth factor receptor (EGFR) signaling, are often incurable. Testing of therapeutics for MPNST has been hampered by lack of adequate xenograft models. We previously documented that human MPNST cells are permissive for lytic infection by oncolytic herpes simplex viruses (oHSV). Herein we developed and characterized a xenograft model of human MPNST and evaluated the antitumor effects of oHSV mutants (G207 and hrR3) and the EGFR inhibitor, erlotinib. Additive cytotoxicity of these agents was found in human MPNST cell lines, suggesting that EGFR signaling is not critical for virus replication. Mice bearing human MPNST tumors treated with G207 or hrR3 by intraperitoneal or intratumoral injection showed tumor-selective virus biodistribution, virus replication, and reduced tumor burden. oHSV injection demonstrated more dramatic antitumor activity than erlotinib. Combination therapies showed a trend toward an increased antiproliferative effect. Both oHSV and erlotinib were antiangiogenic as measured by proangiogenic gene expression, effect on endothelial cells and xenograft vessel density. Overall, oHSVs showed highly potent antitumor effects against MPNST xenografts, an effect not diminished by EGFR inhibition. Our data suggest that inclusion of MPNSTs in clinical trials of oHSV is warranted.

PMID: 17235305 [PubMed - indexed for MEDLINE]

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Effect of ABC transporters on Tarceva disposition in pharmacokinetic studies

Mol Cancer Ther. 2008 Aug;7(8):2280-7.

Effect of the ATP-binding cassette drug transporters ABCB1, ABCG2, and ABCC2 on erlotinib hydrochloride (Tarceva) disposition in in vitro and in vivo pharmacokinetic studies employing Bcrp1-/-/Mdr1a/1b-/- (triple-knockout) and wild-type mice.

Marchetti S, de Vries NA, Buckle T, Bolijn MJ, van Eijndhoven MA, Beijnen JH, Mazzanti R, van Tellingen O, Schellens JH.

Department of Experimental Therapy and Medical Oncology, The Netherlands Cancer Institute, Plesmanlaan 121, Amsterdam, The Netherlands.

Abstract

We tested whether erlotinib hydrochloride (Tarceva, OSI-774), an orally active epidermal growth factor receptor tyrosine kinase inhibitor, is a substrate for the ATP-binding cassette drug transporters P-glycoprotein (P-gp; MDR1, ABCB1), breast cancer resistance protein (BCRP; ABCG2), and multidrug resistance protein 2 (MRP2; ABCC2) in vitro and whether P-gp and BCRP affect the oral pharmacokinetics of erlotinib hydrochloride in vivo. In vitro cell survival, drug transport, accumulation, and efflux of erlotinib were done using Madin-Darby canine kidney II [MDCKII; wild-type (WT), MDR1, Bcrp1, and MRP2] and LLCPK (WT and MDR1) cells and monolayers as well as the IGROV1 and the derived human BCRP-overexpressing T8 cell lines. In vivo, the pharmacokinetics of erlotinib after p.o. and i.p. administration was studied in Bcrp1/Mdr1a/1b(-/-) (triple-knockout) and WT mice. In vitro, erlotinib was actively transported by P-gp and BCRP/Bcrp1. No active transport of erlotinib by MRP2 was observed. In vivo, systemic exposure (P = 0.01) as well as bioavailability of erlotinib after oral administration (5 mg/kg) were statistically significantly increased in Bcrp1/Mdr1a/1b(-/-) knockout mice (60.4%) compared with WT mice (40.0%; P = 0.02).

CONCLUSION: Erlotinib is transported efficiently by P-gp and BCRP/Bcrp1 in vitro. In vivo, absence of P-gp and Bcrp1 significantly affected the oral bioavailability of erlotinib. Possible clinical consequences for drug-drug and drug-herb interactions in patients in the gut between P-gp/BCRP-inhibiting substrates and oral erlotinib need to be addressed.

PMID: 18723475 [PubMed - indexed for MEDLINE]

Tarceva antagonizes ABC transporters like P-glycoprotein

Cancer Res. 2007 Nov 15;67(22):11012-20.

Erlotinib (Tarceva) antagonizes ATP-binding cassette subfamily B member 1 and ATP-binding cassette subfamily G member 2-mediated drug resistance.

Shi Z, Peng XX, Kim IW, Shukla S, Si QS, Robey RW, Bates SE, Shen T, Ashby CR Jr, Fu LW, Ambudkar SV, Chen ZS.

Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John's University, Jamaica, New York 11439, USA.

Abstract

It has been reported that gefitinib (Iressa), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), has the ability to modulate the function of certain ATP-binding cassette (ABC) transporters and to reverse ABC subfamily B member 1 (ABCB1; P-glycoprotein)- and ABC subfamily G member 2 (ABCG2; breast cancer resistance protein/mitoxantrone resistance protein)-mediated multidrug resistance (MDR) in cancer cells. However, it is unknown whether other EGFR TKIs have effects similar to that of gefitinib (Iressa). In the present study, we have investigated the interaction of another EGFR TKI, erlotinib (Tarceva), with selected ABC drug transporters. Our findings show that erlotinib (Tarceva) significantly potentiated the sensitivity of established ABCB1 or ABCG2 substrates and increased the accumulation of paclitaxel or mitoxantrone in ABCB1- or ABCG2-overexpressing cells. Furthermore, erlotinib (Tarceva) did not significantly alter the sensitivity of non-ABCB1 or non-ABCG2 substrates in all cells and was unable to reverse MRP1-mediated MDR and had no effect on the parental cells. However, erlotinib (Tarceva) remarkably inhibited the transport of E(2)17 beta G and methotrexate by ABCG2. In addition, the results of ATPase assays show that erlotinib (Tarceva) stimulated the ATPase activity of both ABCB1 and ABCG2. Interestingly, erlotinib (Tarceva) slightly inhibited the photolabeling of ABCB1 with [(125)I]iodoarylazidoprazosin (IAAP) at high concentration, but it did not inhibit the photolabeling of ABCG2 with IAAP. Overall, we conclude that erlotinib (Tarceva) reverses ABCB1- and ABCG2-mediated MDR in cancer cells through direct inhibition of the drug efflux function of ABCB1 and ABCG2. These findings may be useful for cancer combinational therapy with erlotinib (Tarceva) in the clinic.

PMID: 18006847 [PubMed - indexed for MEDLINE]

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P-glycoprotein (PGP) is a plasma membrane protein which acts as a localized drug transport mechanism, actively exporting drugs out of the cell. The effects of PGP on the distribution, metabolism and excretion of drugs -- including protease inhibitors -- in the body is great. PGP activity decreases the intracellular concentration of cancer drugs, enabling resistance to develop to them.

The normal physiological function of PGP in the absence of therapeutics or toxins is unclear. Studies of MDR-1 knock-out mice (mice bred in the lab specifically for the absence of the MDR-1 gene and, therefore, no PGP activity) show that they have normal viability, fertility and a range of biochemical and immunological parameters.

Predictably, they do have delayed kinetics and clearance of vinblastine, and they accumulate high levels of certain drugs (vinblastine, ivermectin, cyclosporin A, dexamethasone and digoxin) in their brains. The mice also demonstrated marked increases in the levels of these drugs in the tests, ovaries and adrenal gland compared with wild-type mice. It has been reported that some MDR-1a knock-out mice develop a severe, spontaneous intestinal inflammation similar to human inflammatory bowel disease.

The majority of published data suggest that PGP acts as a transmembrane pump which removes drugs from the cell membrane and cytoplasm. It has further been proposed that PGP acts like a hydrophobic vacuum cleaner or "flippase," transporting drugs from the inner leaflet of the plasma membrane lipid bilayer to the outer leaflet or to the external medium.

There have been various attempts to classify compounds based on their effect on or interaction with PGP. A number of chemicals, including anticancer drugs, have been categorized based on their effect on ATPase activity of human PGP.

Class I compounds in low concentrations stimulate ATPase activity and in high concentrations inhibit it. Kinetic analyses show they have high affinity for the active site and low affinity for the inhibitory site. They include vinblastine, verapamil and taxol.

Class II compounds stimulate ATPase activity in a dose-dependent manner without any inhibition and interact only with the active site. They include bisantrene, valinomycin and diltiazem.

Class III compounds, which bind to the inhibitory site with high affinity, inhibit both basal and verapamil-stimulated ATPase activity. They include cyclosporin A, rapamycin and gramicidin D.

Some studies support a model of PGP in which there is a region or multiple regions of interaction rather than one or two simple binding sites. Molecules interacting with PGP may be classified as "substrate" or "antagonist."

It has also been demonstrated that one possible mechanism of action for PGP-mediated resistance to chemotherapeutic agents is through gene rearrangement.

Source: Treatment Action Group

Tamoxifen and the P-glycoprotein Transmembrane Efflux Pump

The discovery of ABC transporters (superfamily of carrier proteins) was recognized as the basis for the human P-glycoprotein drug resistance mechanisms. The over expression of P-glycoprotein involved in cellular transport is a frequent cause of multiple drug resistance. A genetic predisposition causes multiple proteins to dot the surface of tumor cells. These proteins are known as P-glycoprotein (PGP). PGP is a transmembrane efflux pump. It pumps harmful things from the inside of the cell to the outside of the cell. As soon as drugs enter the cancer cells, the PGP pumps start pumping the drugs out.

The presence of P-glycoprotein signals that the patients would not respond well to chemotherapy. PGP is primarily responsible for inducing multi-drug resistance, in which the tumors become resistant to many chemotherapy drugs. PGP effectively pumps the drug out of tumor cells before it has time to kill the cells. Harpole et al, found that patients with PGP survived 20.9 months on average, while patients without PGP had an average survival of more than 5 years after diagnosis.

High-dose tamoxifen significantly inhibits the P-glycoprotein (gatekeeper in the blood-brain barrier) multidrug resistant membrane pump, as well as inhibiting protein kinase C (preventing the increase in vascular resistance).

Although a cytostatic agent like Tamoxifen does not induced programmed cell death (apoptosis) and the functional profiling platform usually does not give strong cell-death signals for Tamoxifen exposure in most tumors, high-dose Tamoxifen can be a potentiator (make more potent) for a cytotoxic drug and also act as an anti-angiogenic effect (limiting formation of new blood vessels).

In cell functional analysis that Rational Therapeutics and Weisenthal Cancer Group provide, the P-glycoprotein is not measured, per se. What is measured is a drug alone, a drug with high-dose Tamoxifen, and high-dose Tamoxifen alone. Sometimes a drug alone doesn't work and high-dose Tamoxifen alone doesn't work, but a drug PLUS high-dose Tamoxifen works brilliantly. This can be tested in any of the cell-death endpoints they use: DISC, MTT, ATP, resazurin, or potentially others.

High-dose Tamoxifen can significantly inhibit the P-glycoprotein (gatekeeper in the blood-brain barrier) multidrug resistant membrane pump, as well as inhibit protein kinase C (preventing the increase in vascular resistance).

Source: Rational Therapeutics, Inc. and Weisenthal Cancer Group

Research by Giovanna Ames, Piet Borst, Peter Wielinga, Michael Dean, et al and others, has recognized ABC (ATP-Binding Cassette) genes that represent the largest family of transmembrane proteins (transporters) as the basis for P-glycoprotein drug resistance mechanisms. The over expression of P-glycoprotein (PGP) involved in cellular transport is a frequent cause of multiple drug resistance. In many cases, PGP is responsible for a patient's decreased sensitivity to anti-cancer drugs. As soon as the drug enters the cancer cells, the PGP pumps start pumping the drug out. PGP effectively pumps the drug out of tumor cells before it has time to kill the cells. Tarceva is not exported from the PGP and other ABC transporters placed at the luminal membrane of brain capillaries. Thus the higher concentration of Tarceva in the central nervous system (CNS).

The Human ATP-Binding Cassette (ABC) Transporter Superfamily

http://www.ncbi.nlm.nih.gov/books/NBK31/

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The blood–brain barrier (BBB), an anatomic structure consisting of endothelial vessel cells, astrocytes and pericytes with tight junctions (TJs) and a number of carrier proteins, controls and limits the passage of molecules to the brain. In the presence of an intact BBB, only small lipophilic molecules (molecular weight [MW] <400 Da) can cross the BBB by diffusion, while the passage of other molecules is regulated by the carrier proteins.

Apart from a few drugs, such as temozolomide, melphalan, carmustine and irinotecan, chemotherapeutic agents, which are large hydrophilic molecules, are unable to cross the BBB, which is furthermore characterized by a high concentration of multidrug resistance efflux pumps, which may be another cause of the low concentration of drugs reaching the site of action.

Recently, Lin et al. underlined the role of astrocytes in preventing chemotherapeutic drugs from reaching metastatic sites in the brain, and observed in murine models that in pathological conditions, astrocytes are activated and come into direct contact with tumor cells, thus confirming the role of the microenvironment in brain metastases (1). This may lead to calcium sequestration, with a *consequent marked reduction in chemotherapy-induced apoptosis.

The role of the BBB in drug resistance has been called into question, since macroscopic intracranial lesions cause BBB disruption due to neoangiogenesis, which produces vessels that lack tight junction molecules and cannot therefore create an effective barrier (2,3).

Nonetheless, BBB disruption may be a characteristic of larger lesions, while the barrier could remain effective in small metastases, as demonstrated by contrast-enhanced images showing enhancement in large intracranial lesions but not in small infiltrative tumors.

Furthermore, the loss of tight junction molecules in the tumor vascular system does not necessarily incur the loss of other biological BBB components, such as detoxification and drug resistance mechanisms, which may remain effective and compromise drug *concentrations in BM (4).

1. Lin Q, Balasubramanian K, Fan D et al. Reactive astrocytes protect melanoma cells from chemotherapy by sequestering intracellular calcium through gap junction communication channels. Neoplasia 12(9), 748–754(2010).

2. Muldoon LL, Soussain C, Jahnke K et al. Chemotherapy delivery issues in central nervous system malignancy: a reality check. J. Clin. Oncol. 25(16), 2295–2305(2007).

3. Papadopoulos MC, Saadoun S, Binder DK, Manley GT, Krishna S, Verkman AS. Molecular mechanisms of brain tumor edema. Neuroscience 129(4), 1011–1020(2004).

4. Lee G, Dallas S, Hong M, Bendayan R. Drug transporters in the central nervous system: brain barriers and brain parenchyma considerations. Pharmacol. Rev. 53(4), 569–596(2001).

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Relationship between skin rash and outcome in non-small-cell lung cancer patients treated with anti-EGFR tyrosine kinase inhibitors: A literature-based meta-analysis of 24 trials

Fausto Petrelli, Karen Borgonovo, Mary Cabiddu, Veronica Lonati, Sandro Barni

Azienda Ospedaliera di Treviglio, Piazzale Ospedale 1, 24047, Treviglio (BG), Italy

Lung Cancer; Volume 78, Issue 1 , Pages 8-15, October 2012

Abstract

Background:

Dermatological toxicity, usually in the form of acneiform rash, is frequently observed in non-small-cell lung cancer (NSCLC) patients treated with anti-EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs). The objective of this review was to assess the predictive value of skin rash for outcome in patients with NSCLC treated with erlotinib and gefitinib.

Methods:

We searched PubMed for articles reporting a correlation of skin rash with survival, progression and response rate. In total, 349 prospective or retrospective studies presenting data regarding patient outcome and skin toxicity were screened. Hazard ratios (HRs) with 95% confidence intervals for progression and survival and risk ratios (RRs) for response rate were obtained from these publications and pooled in a meta-analysis.

Results:

This meta-analysis included 24 publications (17 prospective trials and 7 retrospective case series). Skin rash was found to be an independent predictive factor for survival (HR: 0.30; p<0.00001) and progression (HR: 0.50; p<0.00001). In addition, patients who developed grade 2–4 rash were more likely to respond to treatment respect to patients with no rash (42% vs. 7%). The result for survival meta-analysis appears to be similar for gefitinib and erlotinib.

Conclusion:

These results are noteworthy, because patients with severe skin rash may be reassured over treatment outcome Skin rash during treatment with anti-EGFR TKIs for NSCLC represents a significantly strong predictor of the efficacy in particular for patients with unknown EGFR mutation status.

http://www.lungcancerjournal.info/article/S0169-5002(12)00390-X

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