3/6/97
CONTACT: Rosanne Spector, (415) 723-6911 or via e-mail at [email protected]
PROTEIN PUMP KEEPS AIDS DRUG OUT
STANFORD -- The human body has several mechanisms for getting rid of foreign chemicals. Stanford researchers have now shown that one of the first lines of defense -- a versatile pump in the lining of the gut, called P-glycoprotein -- may prevent the absorption of saquinavir, one of the new generation of anti-HIV drugs.
Saquinavir belongs to a class of drugs known as protease inhibitors. In 1995, it became the first HIV protease inhibitor to gain approval by the Food and Drug Administration. Patients and doctors have found, however, that getting enough saquinavir out of the digestive system to reach HIV- infected T cells in the bloodstream is a difficult task.
There are many reasons why drugs may be poorly absorbed when taken by mouth. Some never manage to get into the cells lining the gut. But for saquinavir, the Stanford team's experiments on cultured cells suggest a different explanation: After the patient ingests saquinavir, gut cells take up the drug, but then P-glycoprotein may pump it back out again.
If this is true, then blocking the pump could increase the amount of drug reaching HIV-infected cells, the researchers propose.
The study was conducted by Dr. Terrence Blaschke, professor of medicine (clinical pharmacology), Dr. Branimir Sikic, professor of medicine (oncology and clinical pharmacology), oncology research biologist George Duran and postdoctoral fellow Carla Washington.
Washington presented the findings Thursday, March 6, in San Diego at the meeting of the American Society for Clinical Pharmacology and Therapeutics.
The researchers began with the observation that saquinavir can be put out of action by a second line of defense, the cytochrome P450 enzyme system, which modifies foreign chemicals. P450 and P-glycoprotein often tackle similar compounds, so Blaschke and Sikic decided to see if P- glycoprotein could recognize saquinavir.
They used two cancer cell lines: one that made P- glycoprotein and one that did not. The researchers found that the cells with P-glycoprotein could withstand higher doses of saquinavir (which has toxic effects on cells only at high levels), presumably because those cells were able to pump the drug out, keeping the level inside the cells relatively low.
"P-glycoprotein was originally discovered as a multi-drug transporter that produced resistance to chemotherapy in many cancers," said Sikic, whose laboratory studies the function of this protein. "It is also expressed in many normal tissues, and our clinical trials in cancer patients indicated that the pump has an important function in the disposition of many different drugs."
Saquinavir's effect on P-glycoprotein became even more apparent when, in another series of experiments, the investigators included several anti-cancer compounds that are known to be pumped out of gut cells by P-glycoprotein. These chemotherapy drugs are very toxic, and when saquinavir was present, the cultured cells with P- glycoprotein had difficulty ejecting them. With saquinavir keeping the pump occupied, the anti-cancer compounds accumulated inside the cells, killing them at lower doses than would otherwise be required.
In cultured cells lacking P-glycoprotein, however, these lower doses were sufficient to kill the cells even in the absence of saquinavir.
(The investigators said they do not anticipate using saquinavir to boost the action of anti-cancer drugs, because the saquinavir doses required in the experiment could not be used for this purpose in patients.)
To directly test the binding of saquinavir to P-glycoprotein, the researchers used a compound called azidopine, which is known to bind P-glycoprotein. They found that saquinavir could reduce the amount of azidopine binding to isolated cell walls containing P-glycoprotein.
These three lines of evidence provide a strong indication that saquinavir is recognized by P-glycoprotein, Blaschke said. "What we don't have," he said, "is direct evidence that saquinavir doesn't accumulate in cells because of P- glycoprotein."
Most recently, the researchers have found that another FDA- approved protease inhibitor, ritonavir, also binds to P- glycoprotein, but that indinavir, the third licensed protease inhibitor, does not. Despite its binding to P-glycoprotein, oral ritonavir seems to enter the bloodstream without difficulty, somehow evading detoxification systems such as P450, Blaschke said.
In the case of saquinavir, he speculates that blocking the action of P-glycoprotein might allow the drug to evade detoxification by the P450 system, perhaps by reducing repeated exposure of the drug to the P450 enzymes, which are also found in the gut lining. In fact, he said, some patients are already taking saquinavir and ritonavir in combination, and ritonavir does appear to raise their blood levels of saquinavir, perhaps by this mechanism. In addition, several drugs designed as P-glycoprotein blockers are in clinical trials for use with cancer chemotherapy.
Sikic and colleagues in the oncology and hematology divisions of the Department of Medicine are studying one of the most promising of the new, more specific inhibitors of P-glycoprotein, called PSC 833 or valspodar. "In addition to studying the role of these drugs in reversing multi-drug resistance in cancer patients," Sikic said, "we are very interested in their potential ability to increase the absorption of drugs."
Blaschke added, "We do not believe that this [P-glycoprotein transporter] is a mechanism for the onset of drug resistance in HIV disease, as the T cells infected by HIV are not known to make P-glycoprotein. But this should be further investigated."
He said he also wonders whether P-glycoprotein might be involved in maintaining the blood-brain barrier, which keeps many drugs from reaching effective levels in the brain. None of the current HIV protease inhibitors gets into the brain, and P-glycoprotein lining the blood vessels in the brain may be responsible for keeping them out, Blaschke speculates.
Support for the research comes from grants to Sikic from the National Cancer Institute and the American Cancer Society, as well as a National Institute of General Medical Science postdoctoral training grant and funding from the federal Adult AIDS Clinical Trials Group (to Blaschke).
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