Tracking Key Targets

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BY DEBORAH KOMLOS

Better disease detection can lead to better drugs. Researchers working on biomarker discovery hope to make that so by understanding how disease changes with drug intervention.

Biomarkers, or biological markers, come in various forms, depending on the needs of research projects. In the context of drug R&D, they can be generally defined as measured characteristics that indicate a “normal” health state, a diseased state, or changes as a result of drug therapy. Examples of such markers include monitoring disease progress via biochemical blood traits, or clinical outcomes of treatment, such as survival rate.

The past year has seen big interest in biomarkers, particularly in proteomics, says Clarissa Desjardins, PhD, executive vice-president of Corporate Development at Caprion Pharmaceuticals Inc. (Montreal, QC).

“There’s been a shift because pharmaceutical companies aren’t doing so well, their productivity is declining, they have to find a way to discover drugs cheaper, faster. So biomarkers are one way that they’re going to try to streamline,” Desjardins says. “The technology at the same time has just come to the fore, you know, right when you needed it.”

An expert in cell fractionation and organelle isolation, Caprion has a proprietary proteomics platform, CellCarta™, involving protocols such as 1- and 2-D gel profiling, Desjardins says, as well as more sensitive capabilities using 2-D liquid chromatography and mass spectrometry. Among the firm’s current industry collaborations are broad discovery-based biomarker projects in pain and inflammation.

“All of our studies are designed, for example, group A, B, C: A is control, B is disease, C is drug-treated,” Desjardins says, adding that some of the drugs Caprion is using are considered gold standards in the industry and do not necessarily belong to the companies the firm is working for. “They want to see what each of the proteomics signatures in those three conditions looks like because it has never, in fact, been looked at before.”

The question, she says, is how many proteins are changed by the disease model, and then, how many return to their normal state with the drug. Desjardins says it’s all about rationalizing the discovery process. “What proteomics signatures give you is almost a digital representation of phenotype,” she adds.

The Profile Says

Toronto, Ont.-based SYN X Pharma Inc. is also using protein profiling for biomarker discovery. With a focus on Syndrome X, which is associated with risk factors indicating predisposition to heart disease and Type II diabetes, SYN X is addressing unmet medical needs, says George Jackowski, PhD, chairman, CEO and CSO.

Jackowski gives the example of insulin resistance, which he says is going to be a major health concern in the future. “There’s a need to diagnose these individuals early and the drugs that are out there actually will work for them, but you need to identify them (those individuals),” he says.

SYN X’s proprietary Proteomics Discovery Platform™ involves various procedures, including electrophoresis, orthogonal chromatography and HPLC. “Once we discover the biomarker, we create the antibodies and the reagents to measure that particular molecule in the blood and we develop a diagnostic test that goes into a point-of-care, which can be done in a physician’s office,” Jackowski says.

The firm’s protein identification work also involves SELDI (surface-enhanced laser desorption and ionization) mass spectrometry using Ciphergen Biosystems Inc. (Fremont, CA) technology, allowing detection of markers on the picogram-per-millilitre level, Jackowski says. Another strength of SYN X, he says, is that in nine months or less it can develop a diagnostic test based on its serum markers. One such achievement, Jackowski mentions, is an Alzheimer’s disease biomarker diagnostic that SYN X developed and has licensed to Ortho-Clinical Diagnostics Inc. (Raritan, NJ).

Determining drug efficacy earlier by monitoring disease progression is a giant step, Jackowski says.

“Recently, we have been working with looking at our Alzheimer’s patients and we can tell what drug they’re on based on their protein profiles in the blood,” he says. “So I think that’s a whole new future. I think it’s going to be very important for diagnostics for patients, but also, I think, for drug development — the pharma companies with their models as they’re screening their drug choices, to see whether they (the profiles) are going to change.”

Jackowski recalls how 10 years ago pharma companies had a different mentality, mainly wanting to position and sell a drug. “They didn’t want to limit their market because they would have less sales,” he says. “So people who did not necessarily need the drug would be getting the drug, but as long as it’s efficacious, there were no bad side-effects on that drug. What they realized in the last few years is they’re not going to segment the market if they give the right drug to the right patient at the right time.”

Customizing Needs

Desjardins predicts that many useful biomarkers will be integrated onto current diagnostic platforms over the next 10 years, progress that is especially required for oncology and diseases of the central nervous system, which she says are “universally appreciated as really needing biomarkers.

“Right now in oncology it’s a really terrible situation where you’re looking for tumours to reappear . . . This is horrible for these people who are just waiting, all the anxiety of just not knowing,” she says. “Eventually, we’ll have biomarkers that will be able to say, A-ha, something has just appeared, let’s go aggressively to treat this early stage tumour because we detected it early.”

Likewise, for illnesses such as dementia, by the time a patient presents to a doctor’s office, “you’ve already lost a significant proportion of neurons,” Desjardins says. “Now there’s nothing to bring them back. All you can hope to do is stabilize the disease at that stage.”

Biomarkers are therefore very desirable from an ethical standpoint, she says. While timing of diagnosis is critical, so is quality of testing, a detail that Miraculins Inc. (Winnipeg, MB) has found particularly important in its work on prostate cancer. The firm has established its proof of principle and expects to validate a diagnostic product by the fourth quarter of 2004.

“There are tests available, but they have a huge rate of false positives,” says Miraculins’ research and development manager Doug Barker, PhD regarding PSA tests used to detect prostate cancer. Among the approximately 25 million such tests done annually in the U.S., Barker says a proportion of patients are deemed “at risk” for cancer, and of those, one million are subjected to prostate biopsies. Of those, he says, 75 per cent are found to not have cancer. One condition that is commonly misdiagnosed as prostate cancer, he adds, is benign prostatic hyperplasia.

“Ideally what we’ll be able to do is develop a system that is not only more accurate than current diagnostics, but it’s also less invasive,” he says, adding that it would be more comfortable than the commonly used digital rectal exam or even drawing blood, which can deter those who dislike needles.

Miraculins is using mass spectrometric methods through a research collaboration with the University of Manitoba (Winnipeg, MB). “Generally speaking, with proteomics, you’re looking for a very small needle in a very, very large haystack . . . What a lot of companies do is use robotics and high throughput methodologies, essentially to go through the haystack more quickly,” Barker says. “What we’re trying to do is reduce the size of the haystack, as it were. So, by trying to focus on likely targets, we’re trying to use the resources that we have as efficiently as we can.”

A challenge, however, is finding a valid therapeutic target that is also a disease biomarker. “One example would be, say you have a protein that’s being modified post-transcriptionally; if that modification changes, that may result in the disease state,” Barker says. “But it may well be that the change to that protein may not be the root cause of the disease; it may be a change in the protein that’s modifying the second protein.”

Critical Percentages

Uncertainty in timing and extent of drug effect is exactly what Dr. Jack Uetrecht, PhD is researching at the Leslie Dan Faculty of Pharmacy, University of Toronto (Toronto, ON). Uetrecht is looking to understand the mechanisms of idiosyncratic drug reactions (IDRs): unexpected adverse reactions that occur in a small proportion of patients taking a drug.

Even though their incidence might be one in 10,000 individuals or less, IDRs total a large number when one multiplies the incidence by the number of people taking drugs and the number of drugs they take, Uetrecht says. IDRs can affect any organ, but the skin, liver and bone marrow are most commonly targeted. They can also lead to autoimmune reactions resembling lupus.

Uetrecht says that IDRs are a significant problem clinically and for drug development because they cannot be predicted.

“I started out as a chemist and a physician and there was a lot of circumstantial evidence, though in most cases not proof, that reactive metabolites of drugs, rather than the drug itself, were responsible for the reaction. And so we did a lot of work on identifying reactive metabolites,” Uetrecht says.

He notes, however, that formation of a reactive metabolite does not mean that it is involved in an IDR. This can be due to one of two reasons, he explains. One is that the reactive metabolite needs to chemically bind host proteins in vivo. Another possibility is that the reactive metabolite has to cause some sort of cell damage or cell stress, which then acts as a stimulus to the immune system. “So if that hypothesis is correct,” he says, “there ought to be a marker that shows that the drug is causing some sort of cell stress.”

To find this evidence, the team is using animal models to look for patterns upon drug treatment — such as increase in glutathione S-transferase (GST) activity, which indicates that a cell is responding to an oxidative stress.

“Having been in this business a long time, I suspect that there will be patterns,” Uetrecht says. “And if it’s 10 patterns, we can live with that. If it’s 100, it’s getting tricky. If it’s thousands, then we’re almost back to square one.”

What does all this mean for drug development? Uetrecht says it’s always going to be risk versus benefit.

“Almost any drug can cause an idiosyncratic reaction,” he says. “It’s more a matter of what’s the incidence and how bad are they . . . If this is another antihistamine, you’re not going to accept very many people dying. If it’s a drug to treat cancer, then you will accept quite severe side-effects.” Uetrecht and his group are working with drugs that are already known to cause IDRs, such as sulphonamides — antibiotics that can give a range of reaction from a mild skin rash to the most severe form called toxic epidermal necrolysis, in which large portions of the skin peel off, and the mortality rate is 30 per cent. Being able to be more certain of drug safety could “revolutionize drug development,” he says, saving enormously on time and money.

But Uetrecht recognizes the complexities involved. “I’m still skeptical that you could do these tests in the test tube because we’re talking about an immune response, which you’re never — assuming they are immune-mediated, which I think is a reasonable assumption in most cases — you’re never going to duplicate it in a test tube,” he says.

A Biomarker Bandwagon

Biomarker use in drug development is being encouraged by the U.S. Food and Drug Administration (FDA), says Lawrence Lesko, PhD, director of the Office of Clinical Pharmacology and Biopharmaceutics, Center for Drug Evaluation and Research, FDA (Rockville, MD). In April, he says, the FDA released an industry guidance titled Exposure-Response Relationships — Study Design, Data Analysis, and Regulatory Applications, which promotes biomarker use in clinical trials. “Biomarkers traditionally have been used for efficacy because this is really the goal of drug development, to get a drug that works,” Lesko says. “So, the number of applications or the number of INDs that are coming through with biomarkers has been tending toward increasing, which kind of makes sense because if you’ve looked at drug development, you know that it’s not as prolific as it was in the past.” To lower the high failure rate in drug development, Lesko says firms are looking at using biomarkers to help eliminate candidates earlier that show less market potential. Selecting more suitable trial patients is also an advantage of biomarkers, he says.

Biomarkers also benefit the regulators, Lesko explains, because they help in the review of data submitted by companies to analyse the relationship between pharmacokinetic and pharmacodynamic properties, or concentration and response. “Without that information,” he says, “it becomes difficult to figure out whether these patients are different, at different doses, and biomarkers are a nice tool to allow for better regulatory decisions — also, I think, better labels, as a result.”

Lesko points out that biomarkers are not a new phenomenon. For instance, biomarker guidances for AIDS were used 10 years ago for CD4 (T-cell) counts and viral load. “Biomarkers were used to identify prognosis of disease; this was the basis of drug approval . . . And here we are in 2003, and biomarkers are still valuable, as they were then,” he says. “There haven’t been very many new biomarkers at the high level of those AIDS biomarkers in the last 10 years. But there’s so much excitement about genomics to generate new biomarkers that will be presumably better and more useful.”

Lesko is referring to technologies, such as microarrays, that are producing potentially thousands of biomarkers comparing diseased versus normal tissue. “The struggle at this point is to say, well, what do these differences mean and which ones of these many differences are the most important,” Lesko says. “That’s where I believe that field currently is — the up-regulation and down-regulation of different genes that compose a microarray. And if we can figure that out, then those are really powerful.”