For the vast stretch of evolutionary time preceding refrigeration, running water, and airtight packaging, life was short and food was dirty. In todays industrialized world, things are different. But the mechanisms that evolved in living cells to protect them from dangerous substances introduced through ingestion, inhalation, and infection remain intact.
Prominent among these mechanisms is a family of cell-membrane-anchored proteins that act as "goalies" for the cell, denying entry to foreign toxins that would otherwise diffuse through the cell membrane. The first such pump to be identified, P-glycoprotein or simply P-gp, is common in the gut, liver, and kidneywhere toxins aboundas well as the testes and blood-brain barrier.
This goalie is ultra-versatile: A variety of structurally diverse chemicals are substrates for P-gp, which makes good evolutionary sense, because there are not enough genes to code for all the pumps that would be required if each were narrowly specific. Unfortunately, many widely used chemotherapy drugs are expelled by P-gp before they can do their job of damaging the cell beyond all hope of replication. In a tumor, clones of cells can spontaneously arise that produce large amounts of P-gp and thereby acquire chemoresistance. Other tumors, derived from tissues expressing P-gp, are resistant to begin with.
Given these observations, it is reasonable to ask: Can blocking P-gp "tripping the goalie"safely and effectively reverse a tumors chemotherapy resistance? After more than two decades of focused attention, there has been no definitive answer to this question, largely because of the absence of ideal P-gp-inhibiting drugs that would provide unambiguous results.
Redundant Mechanisms
"Virtually all of the roughly half-million annual cancer deaths in the United States can be said to have occurred because chemotherapy failed," said Tito Fojo, M.D., Ph.D., a senior investigator at the National Cancer Institutes Center for Cancer Research. Such failures happen because patients tumors either were resistant at the outset or eventually developed resistance after exposure to the drugs.
Is P-gp the major determinant of chemotherapy resistance? Victor Ling, Ph.D., now vice president of research at the British Columbia Cancer Agency in Vancouver, B.C., discovered P-gp in the late 1970s when, after he challenged cultured tumor cells with the cytotoxic drug colchicine, some cells became resistant not only to colchicine but also to chemically unrelated drugs. However, several molecular pumps similar to P-gp (not to mention several unrelated mechanisms) have now been implicated in drug resistance. One such pump is MRP1 (for multidrug resistance protein), discovered in 1992 by Susan Cole, Ph.D., and Roger Deeley, Ph.D. "MRP1 is distributed fairly ubiquitously in almost all normal tissues, usually at low levels," said Cole, professor of pathology, oncology, and toxicology at Queens University in Kingston, Ontario.
There are redundant toxin-expelling systems in the digestive tract. "In colorectal and renal cancer, the large majority of tumors express P-gp," said Branimir Sikic, M.D., an oncologist, professor of medicine, and program director of Stanford University Medical Centers Clinical Research Center. "And we know that, uniformly, P-gp-related cancer drugs arent active in those tumors. Yet, even at doses of compounds that inhibit P-gp substantially in patients, we havent been able to convert inactive drugs into active agents. I think the reason is co-expression of multiple defense mechanisms by the cell."
Still, Sikic and others think that P-gp is the most prevalent and single most important cause of multidrug resistance. In the laboratory, P-gp is the molecular pump whose expression most commonly gets upregulated by cancer cell lines after exposure to chemotherapy drugs. And the pump is overexpressed in acute myelogenous leukemia (AML) and breast, lung, and other tumors whose healthy tissue counterparts typically express only low levels, said Susan Bates, M.D., head of the Molecular Therapeutics Branch at the NCIs Center for Cancer Research.
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With Lings discovery of P-gp, the search was on for P-gp-inhibiting agents that, when co-administered with chemotherapy, could prolong, restore, or enhance the effectiveness of chemotherapeutic drugs. The first batch turned up on pharmacy shelves, already approved for other uses: both the calcium-channel blocker verapamil and the immunosuppressant cyclosporine, for example, inhibited P-gp. But their strong pharmacological effects in their intended indications made them undesirable as P-gp inhibitors. They blocked P-gp in the laboratory, but in humans the doses needed to get pump-blocking quantities into tumors proved toxic to healthy tissues.
These first-generation P-gp blockers were succeeded by a second generation of compounds that were tweaked to reduce side effects and increase potency. Two prominent examples were valspodar (Novartis Pharmaceuticals, East Hanover, N.J.) and biricodar (Vertex Pharmaceuticals, Cambridge, Mass.). Valspodar, in combination with standard chemotherapy, advanced all the way to phase III trials in AML and ovarian cancer, but neither trial yielded statistically significant results.
Neither drug was potent or long-acting, and both drugs had the confounding characteristic of inhibiting MRP1 as well as P-gp. "In tumors where both pumps are playing a role, using two distinct blockers in combination might be very useful. But often its just a single pump thats active," said NCIs Fojo. And MRP1s prevalence throughout the body portends potential side effectsjust what oncologists administering maximal doses of cytotoxic drugs do not want.
Novartis and Vertex have curtailed active development of these drugs. The second-generation P-gp blockers were defeated above all by a factor that became clear to investigators only late in the drugs development: pharmacokinetic interactions. The agents interfered with the metabolism of cytotoxic compounds, slowing their clearance and increasing their toxicity to healthy tissues. To compensate, oncologists reduced dosages of the cytotoxics administered in late-phase trials. But each patients metabolism is different, therefore some patients were effectively receiving too much, and others too little, of their chemotherapy drugs.
New Drugs, New Trials
The early P-gp blockers low potency, poor specificity, short duration of action, and pharmacokinetic interactions bedeviled efforts to make the inhibitors a feasible addition to the clinic. But a new generation of P-gp blockerswhose attributes should allow rigorous testing of the hypothesis that P-gp reversal can restore, enhance, or prolong chemotherapeutic drug sensitivityis moving to center stage.
Two third-generation candidates, tariquidar (Xenova, Slough, U.K.) and zosuquidar (Eli Lilly, Indianapolis), are extremely potent and P-gp-specific. Neither drug blocks MRP1 or exhibits direct toxicity. Tariquidar is particularly long-acting: In one assay employing circulating "natural killer" cells that express P-gp in high amounts, "tariquidar completely blocked P-gp for at least 48 hours after a half-hour infusion," said the NCIs Bates. Chemotherapeutic drugs administered anytime during that blockade cannot be expelled by P-gp.
These agents do not appear to interfere with the metabolism of the chemotherapy drugs themselves. Tariquidar has been tested in clinical trials with paclitaxel, doxorubicin, and vinorelbine, best-selling representatives of three distinct categories of chemotherapeutic drugs (taxanes, anthracyclines, and vinca alkaloids) and shown not to necessitate any changes in chemotherapeutic dosing schedules.
Zosuquidar and tariquidar are in phase III trials in, respectively, AML and non-small-cell lung cancer (NSCLC), two diseases with very short median survival times (and the prospect of relatively fast results). Importantly, both trials employ P-gp blockers as first-line therapy in combination with chemotherapy, with the intention of destroying tumors before they evolve resistance mechanisms. Because the emergence of resistant clones may be prevented by the destruction of just a few P-gp-expressing cells, time to relapse and overall survivalnot tumor shrinkagewill be key endpoints.
The zosuquidar trial, led by Larry Cripe, M.D., director of clinical affairs at Indiana University Cancer Center, will focus on older patients, whose transformed leukocytes are generally known to be more likely to express high amounts of P-gp than those of younger patients. About 450 patients with AML will be randomly assigned to receive 6-hour infusions of either a placebo or zosuquidar plus the chemotherapy drugs daunorubicin (a P-gp substrate) and cytarabine. Results are expected in about 5 years, Cripe said. Numerous studies of AML patients published since 1995 have shown an association between P-gp expression on chemotherapy recipients tumors and clinical outcomes. However, daunorubicin is a substrate for both P-gp and MRP1. This could complicate attempts to reverse resistance by blocking P-gp, because MRP1 is fairly common on the leukocytes in which AML arises.
In the clinical trial of tariquidar, patients with NSCLC will receive a tariquidar infusion for about one-half hour preceding chemotherapy with paclitaxel and carboplatin in one 500-patient trial, and with vinorelbine in another, equal-sized study for poorer-performing patients. As with AML, MRP1 abounds in NCSLC tumors. But paclitaxela strong P-gp substratehas little affinity for MRP1. (MRP1s appetite for vinorelbine is not yet clear.) Final data should be available in 2005, said Graeme Boniface, Ph.D., director of research for oncology at QLT, Vancouver, B.C., the company to which Xenova licensed tariquidar for cancer indications.
There is much debate as to whether P-gp appears on NSCLC tumors frequently enough to provide statistically meaningful trial results. Arguing that the benefits of P-gp inhibitors are limited to tumors expressing P-gp, some authorities propose studies that enroll only patients whose tumors are already P-gp-positive. Fojo and others counter that current detection methods lack reliability, tend to underestimate the incidence of P-gp expression in NCSLC, and fail to distinguish between tumors with zero P-gp expression and those with low expression. But the latter tumors may still be prone to develop resistance and, therefore, may be responsive to co-administration of a blocking agent along with chemotherapy.
At the NCI, Fojo and Bates are developing better surrogate assays using radioactive dyes to diagnose P-gp expression and monitor P-gp inhibition in cells and tissues. An ultimate goal: to catalog each tumor cell for all mechanisms of resistance and tailor treatment accordingly with highly targeted agents.
Meanwhile, with the advent of highly specific P-gp inhibitors, the stage is now set to see if "tripping the goalie" will work. Given chemotherapys present failure rate, even a small but significant reduction in drug resistance may turn into thousands of lives saved.
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