U researcher develops new way to test pharmaceutical drugs

U researcher develops new way to test pharmaceutical drugs

By Veronica Pineda

Approving a new drug takes a minimum of 500 million dollars and years of clinical research. Despite this large investment, about 73 percent of pharmaceuticals drugs fail clinical trials, and 10 percent are recalled after FDA approval due to unforeseen harmful side effects.

What’s the flaw? One possible reason could be that pharmaceutical testing overlooks the harmful side effects that show up on unexpected organ systems.

University of Utah scientist, Shannon Gaukler, believes her lab has developed a drug testing methodology called the Organismal Performance Assay (OPA) that may be capable of revealing drug toxicities that are often missed by other pharmaceutical tests.

The OPA utilizes semi-natural enclosures to test the performance of wild mice. Measures of performance include reproductive success, survivorship, and male dominance.

“I come from an ecology background, and this is what animals in the environment do. They compete for mates and resources that are essential for survival,” said Gaukler.

           

Pharmaceutical safety and efficacy are assessed in both in vivo and in vitro studies. In vivo studies are conducted in animals, typically inbred strains, to determine if the drug causes cancer, birth defects, or genetic mutation. In vitro studies are usually conducted in tissue culture, cells from an organism grown in a flask that are used as the experimental model instead of an animal.

 One major downside to in vitro testing is that this approach ignores the synergy, or interaction, between the many physiological systems in the body. The drug might be designed to affect one body system, though the drug could affect various physiological systems.

“If you determine that a particular drug is safe for the heart, (it) does not mean that it will be safe for other organs and organ systems,” said Gaukler. “A lot of drug failures are due to toxicities that emerge in unexpected organ systems.”

One way that the pharmaceutical approval process could be improved is through the use of the OPA. The OPA utilizes genetically diverse wild mice that compete for limited resource in semi-natural enclosures. Since humans are genetically diverse, it is best to test in genetically diverse animals. Typically, preclinical trials are conducted in genetically inbred strains that are housed in a stress-free caged environment.

“By housing our wild mice in semi-natural environments, we are challenging mice to be mice,” she said.

Gaukler has used the OPA to test paroxetine (Paxil), a selective serotonin reuptake inhibitor (SSRI), and an antidepressant. At the moment, Paxil is available on the market and is suspected of causing birth defects in babies born to mothers who take the drug during pregnancy.

GlaxoSmithKline* now warns pregnant women against taking the drug. Studies reveal that women who took Paxil during the first three months of pregnancy were about one and a half to two times more likely to have a baby with a heart defect, such as murmurs, than women who received other antidepressant medication or women not on antidepressant medication. The FDA approved Paxil after successful preclinical and clinical trials.

Paxil was incorporated into rodent food so that each mouse would ingest approximately 30 mg/kg/day, which is about 10 times the human therapeutic dose. Breeding pairs are exposed to the drug in a caged environmental. The offspring produced by these breeders are the test individuals that go into the OPA.

Gaukler found that Paxil litters were significantly skewed towards female offspring (p=0.0021) and that the offspring weighted significantly less (p<0.0001) than litters in the control treatment. Gaukler also found that pregnancy was significantly delayed (p=0.05) in the Paxil breeders.

Once the offspring are approximately two months old, they are released into semi-natural enclosures. Each population consists of 24 individuals: eight males, four from the control treatment and four from the Paxil treatment; and 16 females, eight from the control treatment and eight from the Paxil treatment.

Each enclosure consists of six territories. Four of the territories contain dark nesting sites, in which males compete for. The remaining two territories contain open nesting areas in which the losers are then forced into.

The males constantly fight to either obtain or defend the optimal territories, while females continually gestate and nurse new young.

“Unlike caged animals used in preclinical studies, mice competing in these semi- enclosures require high performance from most of their physiological systems to establish social dominance and become successful individuals,” said Gaukler.

Paxil- exposed males were significantly less dominant (p<0.0001) and had approximately 50 percent fewer sons (p=0.0236) than control males. Paxil-exposed females had approximately 25 percent fewer offspring (p=0.1014) that control females.

However, there were no significant differences in terms of survivorship between treatments Gaukler said.

“These results tell us that Paxil is really impacting the overall fitness of these animals. We do not know the mechanisms that are responsible for these fitness declines, but they could be determined in future studies,” she said.

One possible mechanism of fitness declines could be how Paxil interferes with serotonin levels. Serotonin, known as the happy drug, impacts a region of the brain called the hypothalamus, a gland within the endocrine system.

The endocrine system is responsible for regulating hormones in the body, including testosterone, estrogen, and progesterone. Alterations to these hormone levels could explain why males have reduced dominance and why females have a reduced number of offspring, said Gaukler.

In addition to Paxil, Gaukler has also used the OPA to test the safety of Baycol (cerivastatin), a drug that was prescribes to reduced cholesterol.

Gaukler has detected significant fitness declines in Baycol-exposed mice as well.

The results from these two studies suggest that the OPA is a superior approach to assessing the safety of pharmaceutical drugs according to Gaukler.

“If OPA’s were implemented as a tool in preclinical testing, there is the potential to save millions of dollars and reduce human suffering by identifying drug toxicities early in development and prior to clinical trials,” said Gaukler.

     

Drug Delivery Design: Industry versus Academia

By Krystal Brown

How often do you take ibuprofen? How often do you think about how it works to provide relief?

As a common over-the-counter drug, ibuprofen inhibits cyclo-oxygenase enzymes in tissues to diminish their inflammatory response. Though ibuprofen has been used for more than 50 years to ease pain and stiffness, it would be worthless without an effective delivery system. Oral drug delivery provides a convenient method of drug administration whose ease promotes patient comfort and compliance.

According to Professor David Grainger, chair of the department of pharmaceutics and pharmaceutical chemistry at the University of Utah, 86 percent of drugs on the market are taken orally, mainly relying on drug dissolution in the stomach or upper intestine and then passive absorption through cell membranes to enter the bloodstream. Using long-established methods, several chemical and physical drug benchmarks are used to predict whether a drug will be efficiently absorbed orally.

Due to their long history of use, little fundamental research is currently done on oral delivery methods; consequently, many standard measurement methods for oral drug delivery, dosing and uptake, have remained largely unchanged.

Professor Grainger stated that researchers competing for federal grants are expected to focus on new, cutting edge delivery methods. Oral delivery vehicles and drug delivery strategies are often considered low innovation, often not supported by federal monies, leaving academia without research and training mechanisms in an area of high priority with the pharma industry. 

One way around this is to develop new methodologies that improve current standard measurement capabilities.

One such standard, the partition coefficient, estimates how well a drug molecule will insert into cell membranes by measuring its equilibrium partitioning in solution between immiscible bulk phase water and octanol. Despite widespread use of this water/octanol model, its significant divergence from cell membrane properties makes the corresponding partition coefficient a purely correlative measure, often only one of several variables determining a drug’s cell entry potential. 

Dr. Grainger cited this “poor correlation between drug in vitro properties and in vivo efficacy” as a major problem for applying basic research tools to actual drug delivery design. Synthetic lipid bilayers are a superior cell membrane model to the water/octanol system and can be customized to incorporate different types and ratios of lipids, cholesterol, proteins, and ligand molecules for modeling drug uptake. 

Despite many desirable properties, the challenges associated with studying such lipid membrane interfaces have led to the continued preference of the water/octanol system in pharmaceutical industry; however, research in the Professor John Conboy group in the department of chemistry at the University of Utah aims to change that. 

Recently, Trang Nguyen of the Conboy group has demonstrated the use of deep ultraviolet–visible sum-frequency generation (UV-vis SFG) to measure the partition coefficients of several drug molecules, including ibuprofen, in lipid bilayers.

As a coherent laser technique, UV-vis SFG has an inherent surface sensitivity and utilizes the native electronic transitions of the molecule of interest. This allows the drug partitioning into lipid bilayers to be monitored without chemical modification and with low limits of detection—two major obstacles in studying these interfaces. This enables a kind of retrofit to existing drug delivery designs seeking cell membrane absorption, allowing researchers to evaluate possible drug molecules in screens with more biologically relevant information.

With this, the Conboy group has provided a new method for assaying one property long-recognized as important to oral drug delivery efficacy. Nonetheless, the pharmaceutical field is changing to offer more significant challenges to delivering new drug classes. Ten years ago, most of the top 10 drugs on the market were small molecules taken orally; however, Professor Grainger says that by 2014, eight of the top 10 drugs on the market are predicted to be proteins—biologic drugs currently incapable of effective oral delivery and thus delivered via injection.

Since many of these drug activities will still involve cell membrane penetration, the lipid bilayer model can still offer valuable information. Additionally, biologics are expensive and often suffer from stability and shelf-life issues. Their interactions with lipid membranes could offer hints for new methods to stabilize and preserve the protein-based drugs in their formulations using membranes. 

Researchers in the Conboy group have already used UV-vis SFG to quantify protein-ligand interactions in model bilayers. This is an example of how fundamental research methods may continue to have relevance to the developing pharmaceutical industry.