New insights to multi-drug resistance

Researchers discover possible reasons why anti-cancer drugs fail

By Jerry Call
Life Raft Science Coordinator

Proteins that move drugs out of cells have been suspected to be a cause of resistance to cancer therapy for many years. “Multi-drug resistance” or MDR is the term used to describe the process where cancer cells are able to “pump” drugs out of the cell before the drug is concentrated enough to kill the cell. The ability of some cancer cells to increase the number/activity of these drug pumps can lead to resistance across a spectrum of drugs, hence the term “multi-drug resistance.”

Understanding of multi-drug resistance proteins has largely been limited to their role in pumping drugs out of tumor cells (drug efflux). Recently, several groups have demonstrated that drug transport into the cell (drug influx) and effects on drug bioavailability might also be important factors in multi-drug resistance in patients treated with Gleevec.

One of the largest, and perhaps best studied, families of drug transporters is the ABC transporter family. This family includes seven subfamilies, ABCA through ABCG. Perhaps the most well known member of this family is ABCB1, also known as MDR1 or Pglycoprotein.

Most of the MDR research to date has focused on the ability of drug transporters to pump drugs out of the cell, especially P-glycoprotein. In a recent paper pre-published online in the journal Blood, Julia Thomas et al, of the University of Liverpool, Royal Liverpool University Hospital, U.K., described how another drug transporter, hOCT1, actively transports Gleevec into cells. Further, it is not only drug efflux that determines drug concentration in cells, but that it is the difference between how much Gleevec is transported into a cell and how much is transported out of a cell that determines the concentration.

The past focus on drug efflux, especially P-glycoprotein, has led to studies where P-glycoprotein inhibitors such as verapamil have been used to try to increase intracellular drug levels of chemotherapy. These studies have not been very successful. Munir Pirmohamed, one of the authors of the Liverpool study, speculates that this may be because some of these agents, like verapamil, inhibit not only Pglycoprotein (an efflux pump), but also inhibit hOCT1 (one of the influx pumps). For that matter, verapamil actually decreased Gleevec levels in cells in-vitro studies, apparently because its effects on hOCT1 were more important than its effects on Pglycoprotein.

Using several different cell lines, Thomas et al were able to show that Gleevec is a substrate (a molecule which can be acted on by an enzyme, in this case the transport pumps) for hOCT1, but not the influx pumps hOCT2 or hOCT3. They also confirmed earlier work by Dai, and Mahon that Gleevec was a substrate of P-glycoprotein (ABCB1). There is another study by Ferrao that suggested that P-glycoprotein might not play a significant role in Gleevec resistance. However, this study only used a single cell line, K562, and since this cell line expressed low levels of both Pglycoprotein and hOCT1, it may not have been a good cell line to use for this type study, according to the Liverpool researchers.

After demonstrating that Gleevec was a substrate for hOCT1 and Pglycoprotein, the Liverpool team next showed that several different types of chronic myelogenous leukemia (CML) cells expressed both hOCT1 and Pglycoprotein. They speculated on the clinical implications of their findings. “The net effect of these transport processes may be a decrease in the intracellular concentration of imatinib (Gleevec).” The consequences are that cells might become resistant to Gleevec.

In the laboratory, at least two CML cell lines have been made resistant to Gleevec by culturing the cells in Gleevec at lower-than-optimal concentrations. In one case it led to an increase in BCR-ABL protein expression, causing one cell line to become resistant to Gleevec. The second cell line developed a resistant clone that had a secondary point mutation in BCR-ABL.

Lucy Crossman, Brian Druker, and Michael Deininger extended the laboratory work of the Liverpool team to CML patients. In a letter to the editor of the journal Blood, they gave a short report on their experience with a small group of CML patients. They divided the patients into responders (achieved a complete cytogenetic response to Gleevec within the first year) and nonresponders (remained at least 65 percent Philadelphia-chromosome positive during the first 10 months of Gleevec). In general, responders (n=15) and non-responders (n=15) were closely matched except that the median time from diagnosis to starting Gleevec was 20 months for responders and 41.7 months for non-responders.

The Crossman team found that baseline expression of hOCT1 (the influx pump) was variable and not significantly different from healthy bone marrow donors. Interestingly however, the pre-Gleevec expression level of hOCT1 in non-responders was one eighth that seen in responders (P=.005). Once on Gleevec, six of the non-responders had a further twofold decrease in the expression of hOCT1 compared to baseline. The implications were that in the non-responders, not as much Gleevec was being pumped into the tumor cells.

In contrast to hOCT1, the pre-Gleevec expression levels of the efflux pumps ABCA2, ABCG2 and ABCB1 were similar for responders, nonresponders and normal bone marrow patients. After starting Gleevec, six, five and three non-responders had at least a doubling of ACBA2, ABCG2 and ABCB1 expression respectively, but when the whole group of nonresponders was considered, the results for the efflux pumps were not statistically significant.

Crossman noted in her correspondence to the editor, “Since hOCT1 actively transports imatinib (Gleevec) into cells, patients with low baseline expression of hOCT1 may be unable to achieve adequate intracellular concentrations of imatinib, and hence fail to achieve a cytogenetic response. Although our study is small, our observations add weight to Thomas et al’s proposal that differential expression of hOCT1 may affect patients’ responses to imatinib. We believe that further work is warranted to explore the interaction of hOCT1 and other drug transporters as a cause of primary cytogenetic resistance to imatinib.”

In the same issue of Blood, the Liverpool group also reported their own experience with a larger group of 67 CML patients in whom expression of hOCT1 varied between responders and non-responders.

The research team of Herman Burger, Kees Nooter, et al is investigating the effects of multi-drug resistance proteins on the bioavailability of Gleevec. Their work was recently reported in a paper published in the July edition of Cancer Biology & Therapy. MDR proteins are not limited to tumor cells. They also exist on other cells in the body and have a variety of effects. For instance, MDR proteins may play a part in preventing many drugs from reaching the brain -- i.e., crossing the “blood-brain barrier” -- or in reducing drug absorption from the intestines. The Burger team of primarily European researchers has demonstrated that chronic exposure to Gleevec can “induce” the ABCG2 and ABCB1 drug efflux pumps, and that this has the potential to affect the oral bioavailability of Gleevec. The Burger team had previously demonstrated that Gleevec was a substrate for ABCG2.

In an earlier study for the European Organisation for Research and Treatment of Cancer, Ian Judson et al, found that Gleevec levels in the body tended to decrease over time. This retrospective pharmacokinetic study was small, so not statistically conclusive, but the results were interesting. The Judson team found that apparent Gleevec clearance increased by 33 percent from day one to 12 months and exposure to Gleevec decreased by 42 percent. An article about this study can be found in the January issue of the LRG newsletter. The Burger study provides a possible explanation for the decreased levels of Gleevec over time.

Oral bioavailability refers to the percentage of an oral drug that reaches the systemic circulation. It is a comparison of the blood level of a drug given orally versus the same amount of drug given via an intravenous injection. An oral drug with 50 percent oral bioavailability would reach half of the concentration in the blood compared to the same amount of drug given intravenously.

The oral bioavailability of Gleevec is about 98 percent, which is excellent. But the studies that determined Gleevec bioavailability were given in healthy patients who were only exposed to the drug for a short time. Long-term Gleevec bioavailability is, to our knowledge, unknown (or at least unpublished). Oral bioavailability is highly dependent on gastrointestinal absorption and first-pass drug clearance.

Burger and his team used a Caco-2 cell line model to examine the longterm effects of Gleevec on the expression of intestinal drug pumps. Caco-2 cell lines have been widely used as an in vitro model for studying drug transport across the intestinal epithelial barrier. They found that continuous exposure with Gleevec (up to 100 days) specifically up-regulates the expression of both of the multi-drug resistant proteins, ABCG2 and ABCB1. Although the degree of up-regulation varied over time, in both cases it tended to eventually stabilize at fivefold higher levels of expression compared to starting levels. The hypothesis of the Burger team is that “… chronic use of imatinib may induce enhanced expression of intestinal drug transport pumps and drugmetabolizing enzymes, which may then limit the bioavailability and efficacy of imatinib. Moreover, this may eventually lead to decreased drug uptake and lower drug plasma levels, development of cellular resistance, and subsequent treatment failure.”

The conclusions of the Burger team were “… our in-vitro data suggest that, at the systemic level, these drug pumps may play a role in acquired [pharmacokinetic] resistance to imatinib by lowering the bioavailability after oral dosing. Furthermore, these in vitro data and the clinical data discussed above (the EORTC pharmacokinetic study which showed that Gleevec levels decrease over time) suggest that it is of great importance to address the question whether in vivo exposure of imatinib will indeed result in enhanced expression of ABC transporters in the epithelial cells of the gastrointestinal tract of cancer patients. In that case, it might have far reaching consequences for the clinical use of imatinib and a different dosing approach may be the ultimate outcome.”

As Berger acknowledges, there is still more work to do to verify whether oral bioavailability actually changes over time in patients. Patients currently taking Gleevec are still left with questions about the optimal dose of Gleevec. This study adds a little more weight to the side that suggests higher doses of Gleevec may work better in the long run. It also seems to support the concept of starting Gleevec at a lower dose and, in the absence of unacceptable toxicity, escalating the dose as Gleevec blood levels drop and side effects diminish over time. Previous GIST studies that have supported the theory that higher Gleevec doses may produce better results include the Phase III EORTC randomized clinical trial, the EORTC pharmacokinetic study on Gleevec blood levels, and the Life Raft Group relapse survey that found that Life Raft Group patients actually taking higher doses of Gleevec reported few relapses than patients actually taking lower doses of Gleevec.

The work of the researchers mentioned in this article is an example that demonstrates how CML/GIST and Gleevec are models for molecularly targeted therapies. The tremendous focus that results from having one target and one drug being so important to controlling CML and GIST results in a more thorough inquiry into every aspect of what can go wrong. In the case of multi-drug resistance, this has resulted in looking at an old problem in new ways. Additional work is needed to translate these initial findings into clinical benefit.