ScienceDaily (June 3, 2010)

Tests on 379 individuals who abstained from caffeine for 16 hours before being given either caffeine or a placebo and then tested for a range of responses showed little variance in levels of alertness.

The study, published online in the journal of Neuropsychopharmacology, reports that frequent coffee drinkers develop a tolerance to both the anxiety-producing effects and the stimulatory effects of caffeine. While frequent consumers may feel alerted by coffee, evidence suggests that this is actually merely the reversal of the fatiguing effects of acute caffeine withdrawal. And given the increased propensity to anxiety and raised blood pressure induced by caffeine consumption, there is no net benefit to be gained.

Peter Rogers, from the University of Bristol's Department of Experimental Psychology and one of the lead authors of the study, said: "Our study shows that we don't gain an advantage from consuming caffeine -- although we feel alerted by it, this is caffeine just bringing us back to normal. On the other hand, while caffeine can increase anxiety, tolerance means that for most caffeine consumers this effect is negligible."

Approximately half of the participants were non/low caffeine consumers and the other half were medium/high caffeine consumers. All were asked to rate their personal levels of anxiety, alertness and headache before and after being given either the caffeine or the placebo. They were also asked to carry out a series of computer tasks to test for their levels of memory, attentiveness and vigilance.

The medium/high caffeine consumers who received the placebo reported a decrease in alertness and an increase in headache, neither of which were reported by those who received caffeine. However, their post-caffeine levels of alertness were no higher than the non/low consumers who received a placebo, suggesting caffeine only brings coffee drinkers back up to 'normal'.

The authors also found that the genetic predisposition to anxiety did not deter coffee drinking. In fact, people with the gene variant associated with anxiety tended to consume slightly larger amounts of coffee than those without the variant, suggesting that a mild increase in anxiety may be a part of the pleasant buzz caused by caffeine.

This research was funded by a grant from the Biotechnology and Biological Sciences Research Council (BBSRC), UK.

ScienceDaily (June 3, 2010)

When loaded with an anticancer drug, a delivery system based on a novel material called nanosponge is three to five times more effective at reducing tumor growth than direct injection.

"Effective targeted drug delivery systems have been a dream for a long time now but it has been largely frustrated by the complex chemistry that is involved," says Eva Harth, assistant professor of chemistry at Vanderbilt, who developed the nanosponge delivery system. "We have taken a significant step toward overcoming these obstacles."

The study was a collaboration between Harth's laboratory and that of Dennis E. Hallahan, a former professor of radiation oncology at Vanderbilt who is now at the Washington University School of Medicine. Corresponding authors are Harth and Roberto Diaz at Emory University, who was working in the Hallahan laboratory when the studies were done.

To visualize Harth's delivery system, imagine making tiny sponges that are about the size of a virus, filling them with a drug and attaching special chemical "linkers" that bond preferentially to a feature found only on the surface of tumor cells and then injecting them into the body. The tiny sponges circulate around the body until they encounter the surface of a tumor cell where they stick on the surface (or are sucked into the cell) and begin releasing their potent cargo in a controllable and predictable fashion.

Targeted delivery systems of this type have several basic advantages: Because the drug is released at the tumor instead of circulating widely through the body, it should be more effective for a given dosage. It should also have fewer harmful side effects because smaller amounts of the drug come into contact with healthy tissue.

"We call the material nanosponge, but it is really more like a three-dimensional network or scaffold," says Harth. The backbone is a long length of polyester. It is mixed in solution with small molecules called cross-linkers that act like tiny grappling hooks to fasten different parts of the polymer together. The net effect is to form spherically shaped particles filled with cavities where drug molecules can be stored. The polyester is biodegradable, so it breaks down gradually in the body. As it does, it releases the drug it is carrying in a predictable fashion.

"Predictable release is one of the major advantages of this system compared to other nanoparticle delivery systems under development," says Harth. When they reach their target, many other systems unload most of their drug in a rapid and uncontrollable fashion. This is called the burst effect and makes it difficult to determine effective dosage levels.

Another major advantage is that the nanosponge particles are soluble in water. Encapsulating the anti-cancer drug in the nanosponge allows the use of hydrophobic drugs that do not dissolve readily in water. Currently, these drugs must be mixed with another chemical, called an adjuvant reagent, that reduces the efficacy of the drug and can have adverse side-effects.

It is also possible to control the size of nanosponge particles. By varying the proportion of cross-linker to polymer, the nanosponge particles can be made larger or smaller. This is important because research has shown that drug delivery systems work best when they are smaller than 100 nanometers, about the depth of the pits on the surface of a compact disc. The nanosponge particles used in the current study were 50 nanometers in size. "The relationship between particle size and the effectiveness of these drug delivery systems is the subject of active investigation," says Harth.

The other major advantage of Harth's system is the simple chemistry required. The researchers have developed simple, high-yield "click chemistry" methods for making the nanosponge particles and for attaching the linkers, which are made from peptides, relatively small biological molecules built by linking amino acids. "Many other drug delivery systems require complicated chemistry that will be difficult to scale up for commercial production, but we have continually kept this in mind," Harth says.

The targeting peptide used in the animal studies was developed by the Hallahan laboratory, which also tested the system's effectiveness in tumor-bearing mice. The peptide used in the study is one that selectively binds to tumors that have been treated with radiation.

The drug used for the animal studies was paclitaxel (the generic name of the drug Taxol) that is used in cancer chemotherapy. The researchers recorded the response of two different tumor types -- slow-growing human breast cancer and fast-acting mouse glioma -- to single injections. In both cases they found that it increased the death of cancer cells and delayed tumor growth "in a manner superior to know chemotherapy approaches."

The next step is to perform an experiment with repeated injections to see if the nanosponge system can stop and reverse tumor growth. Harth is also planning to perform the more comprehensive toxicity studies on her nanoparticle delivery system that are required before it can be used in clinical trials.

Additional participants in the study were Ralph J. Passarella, Daniel E. Spratt, John G. Phillips, Hongmei Wu and Li Zhou from the Vanderbilt University Medical Center and Alice E. van der Ende and Vasanth Sathiyakumar from Vanderbilt's Department of Chemistry.

The research was supported by grants from the Department of Defense, National Science Foundation and National Institutes of Health.

ScienceDaily (June 7, 2010)

Duke University researchers have devised a method to dry and preserve proteins in a glassified form that seems to retain the molecules' properties as workhorses of biology.

They are exploring whether their glassification technique could bring about protein-based drugs that are cheaper to make and easier to deliver than current techniques which render proteins into freeze dried powders to preserve them.

Duke engineer and chemist David Needham describes this glassification process as "molecular water surgery" because it removes virtually all the water from around a dissolved protein by almost magically pulling the water into a second solvent.

"It's like a sponge sucking water off a counter," said Needham, a professor of mechanical engineering and materials science at Duke's Pratt School of Engineering, who has formed a company called Biogyali ("gyali" means glass in Greek) to develop the innovation. That firm has also applied to patent the idea of turning proteins into tiny glass beads at room temperature for drug delivery systems.

A report by Needham, graduate student Deborah Rickard and former graduate student P. Brent Duncan online in the Biophysical Journal describes how his team carefully controlled water removal during glassification by releasing single tiny droplets of water-dissolved protein into the organic solvent decanol with a micropipette.

Preliminary evaluations by his senior scientist David Gaul and a team of undergraduate students showed that four test proteins undergoing such procedures retained all or most of their original activity when water was restored. His group has received about $1 million from the National Institutes of Health grants for the research.

Having devised a way to turn proteins into glassy microbeads measuring only about 26 millionths of a meter in diameter, Needham hopes those can be directly injected into the body for use as "biologic" drugs.

His group's early research shows high concentrations of such tiny beadlets would not be as viscous as proteins dehydrated into the normal powder form, which tend to clog up syringes, he said.

These microbeads might also be packaged for slow time-release by surrounding them with a polymer that would biodegrade over time, though how to do that has not been resolved yet, he added.

In collaborations with Duke's Brain Tumor Center and Comprehensive Cancer Center, the researchers are seeking additional funding to do initial evaluations on glassified forms of three molecules with drug potential.

One, known as O6-AMBG, can help the cancer drug Temozolomide work better when infused into brain tumors. A second, Lapatinib, is designed to knock out other molecules that help cancer cells grow in the breast and elsewhere. The third, shepherdin, also targets breast cancers.

Their discovery of protein glassification grew out of a basic exploration of a general question: What can dissolve in what?

Needham's research group found, for example, that air and the organic liquid chloroform will both dissolve in water at about the same rate. It also found that water will dissolve in decanol, a substance it cannot even mix with in large quantities.

These experiments, and the theory underlying them, are described in a second report led by Needhams's graduate student Jonathan Su, now published online in the Journal of Chemical Physics ( http://link.aip.org/link/?JCP/132/044506 ).

"Mixing" and "dissolving" are not the same thing, Needham said. "A good example of a suspended mixture is salad dressing, where oil and water are mixed but oil does not appreciably dissolve in water, nor water in oil."

They next tried a more complex variation of a familiar high school experiment which dissolves so much salt in water that some begins coming back out of the solution as a crystal.

In this case, after dissolving the salt in water, Needham's group then inserted a microbubble of that solution into immiscible decanol in a microscopic chamber. The water itself then dissolved into the decanol and left behind the salt, which also crystallized.

According to his group's Biophysical Journal report, while decanol has practically no tendency to dissolve in water, water has a high probability of dissolving in decanol, allowing the latter to be used as a "drying" agent to remove the former.

"So then we asked: what if we did the same thing with the protein albumin?" Needham said. "I expected to maybe get crystallized albumin," Needham recalled. "But, in just a few minutes, we instead formed a glassified microbead of protein on the tip of a micropipette, at a high density just a bit more dense than water itself. That protein glass is not a crystal. It's really a solid liquid."

Many proteins can be coaxed into forming crystals, solids created by repeating three dimensional patterns of atoms as surrounding water is removed. On the other hand, Needham said he was not really surprised that his protein samples instead formed into glasses, which are more unorganized assemblage of molecules that can still "flow" over very long time scales.

The water loss in his process is apparently too rapid for the molecules of big and irregular proteins to reorganize into a crystal form in such a short time, he explained.

Careful studies by his graduate student Rickard found that the decanol removed all the water that is not bound up in the proteins' molecular structures. And the remaining "bound" water was insufficient to support the growth of bacteria and fungi. Storing proteins as microbeads could thus preserve them.

Proteins are currently dried into clumpy, irregular powders by several industrial processes -- usually freeze-drying -- to protect them from such microbe damage. Drying also avoids the chemical breakdowns that can also occur when proteins are kept in solution. "But in the freeze-drying process itself, some very sensitive biologic drugs can also get damaged," Needham said.

Freeze-drying proteins into solids is also slower and more expensive than glassifying them, he added. And the resulting "flaky" powder is harder to handle than glassified beads.

Glassification "is a fast process," said Gaul, a senior research scientist in Needham's lab. Unlike freeze-drying, "we can dry particles within minutes, if not seconds, and don't need any specialized equipment."