Special Delivery

(From DukeMed Magazine)

By Dennis Meredith

A cloud of gelatinous capsules swirls into the bloodstream from the tip of a comparatively colossal hypodermic needle. At a thousandth of the diameter of a human hair, the capsules spreading through the circulation are nearly a hundred times smaller than the blood cells that stream alongside them. Yet tiny as they are, these submicroscopic capsules bear the stamp of human design–— their surfaces are a waxy patchwork not found in nature, and their interiors are loaded with the chemotherapeutic agent doxorubicin.

The purpose of these singular spherules becomes dramatically apparent when they drift into a slightly warmer region of the bloodstream–— the vessels inside a tumor being gently heated with an external microwave device. In a matter of seconds, perforations open in the melting capsule membranes, and the capsules' deadly cargo leaks through the vessel walls into the surrounding cancerous tissue. Once inside, the anti-cancer drug wreaks havoc on the tumor, snarling the tumor cells' genetic material and shriveling the mass to a tiny, enfeebled lump.

To materials scientist David Needham, PhD, and cancer biologist Mark Dewhirst, DVM, PhD, this synergy of heat and the cleverly engineered capsules called liposomes offers a highly promising way to ameliorate a central problem in chemotherapy: the fact that, while anticancer drugs are hazardous to cancers, they are only slightly less so to healthy tissue. The drug doxorubicin, for example, may efficiently jam the genetic machinery of rapidly dividing cancer cells, but it is also highly toxic to heart tissue–— all too often causing congestive heart failure and disrhythmias in chemotherapy patients. Such cardiac toxicity limits how much of this drug can be administered to patients.

Needless to say, a method of ensuring that doxorubicin and other cancer drugs would be unleashed preferentially at the site of a tumor would constitute a godsend for chemotherapy. And the best solution to date appears to be the marriage of two existing cancer therapies–— liposomes and hyperthermia–— that turn out to be dramatically more effective together than alone.

A natural clinical marriage
Liposomes, created in the 1960s, were first used simply as models to study biological membrane structures, since, like cell membranes, they self-assembled from lipid molecules.

In the '70s, though, "There was the realization that these little bags could actually carry drugs, and perhaps make them less toxic," says Needham. But the pathway from concept to clinical benefit was long and torturous. "A huge industry was set up around the promise of encapsulating drugs and getting them to tumors," he says. "One of the first products that came out was a doxorubicin-containing liposome. Lo and behold, it wasn't as toxic to the heart–but instead of heading for the tumor site, it stayed in circulation for only half an hour or so, and then lodged in the liver and spleen."

So, basically–— despite additional technical advances such as increasing the longevity of liposomes in the bloodstream–— there rested the technology of liposomes for the next two decades: they were less toxic than free drug, but not much more efficacious in treating cancer.

Then there was another promising therapy, hyperthermia–— temporarily elevating the temperature above normal in part of the body. During the 1980s, Dewhirst and his colleagues had launched a multidisciplinary research program to study the potential role of heat in killing cancers, based on intriguing human trials and laboratory studies. For example, seven positive phase III trials showed that hyperthermia improved the effectiveness of radiation therapy. However, there were little clinical data to support the use of hyperthermia with chemotherapy. And, says Dewhirst, "It turned out that, mainly for technical reasons, we were often not able to reach the temperatures necessary to maximize the synergy between hyperthermia and radiation or chemotherapy."

In the 1990s, however, Dewhirst and Needham began the collaboration that would combine their areas of expertise. They started by exploring how liposomes circulated in tumors, quite literally by peering through a window. The window happened to be installed in the skin of a rat–— a system developed by Dewhirst in which a small transparent pane fitted over a tumor could give researchers a front-row seat at seeing how drugs and other treatments affected tumor physiology.

Studies in rats with human tumor xenografts showed that at 40 degrees Celsius (104 degrees Fahrenheit) pores began to open up in the blood vessels, allowing liposomes to leak into tumor tissue. The researchers found that turning up the heat could speed the rate of leakage: for some tumors, in fact, increasing the temperature to 42 degrees caused liposomes to accumulate in the tumor 50 times faster than in non-heated controls.

Importantly, says Dewhirst, the experiments showed that hyperthermia dramatically improves liposome delivery to tumors within a temperature range that is readily achievable with current technology.

A new “melting” liposome
Prompted by Dewhirst's suggestion to somehow use the modest heating achievable clinically in combination with some form of heat-activated chemotherapy, Needham began to research ways to engineer thermally sensitive liposomes to melt within just that small temperature window. In his basic studies, he had mastered–— indeed greatly improved–— the art of the micropipette technique used to study liposome properties, in particular the properties of their lipid membranes. In this technique, a micromanipulator is used to precisely position a micropipette to suck a liposome onto its tip. By measuring the liposome's deformation and other properties under known forces, Needham can determine its mechanical and thermal properties–— information essential to the design of lipid-based drug delivery and releasing systems.

When a liposome is cooled from its liquid state, it freezes into a waxy material, with a grain structure that resembles a soccer ball with stitch-like defects. It was already known that these defects allowed small molecules to leak through the liposome membrane when the membrane was heated to its melting point, albeit relatively slowly. In his studies, though, Needham had discovered that adding small amounts of a molecule called lysolipid to the liposome's two-molecule-thick membrane created additional stable pore-like defects in the stitch regions of the melting "liposome soccer ball." Needham hypothesized that these pores would allow a drug encapsulated inside the liposome to leak out faster and more completely.

"We showed in test-tube experiments that, indeed, that's what happened," says Needham. "When we heated the membranes, the stitches melted and the trapped drugs cascaded from the liposomes in seconds–— 20 to 50 times faster than with standard liposomes."

Dramatic proof of the innovative "melting" liposomes' clinical promise first came in mouse studies reported in the April 2000 issue of Cancer Research. Working with human cancers implanted into mice, the researchers compared the effects of a full set of combinations of different liposome preparations, free drug, and heating.

"The only formulation that gave us permanent, long-term control of these tumors was David's low-temperature-sensitive liposomes," says Dewhirst. After applying just one treatment of the drug-laden liposomes, heating for one hour, and then measuring the tumor size, the researchers found that a stunning 17 out of 20 mice showed complete disappearance of the tumor without regrowth out to 60 days.

Needham describes the novel liposome as "a Trojan horse for cancer." In these lysolipid liposomes, he says, "All the drug came out in the bloodstream right in the tumors' food supply–— where the most active cells were growing–— and killed them." Also highly intriguing, says Needham, is new evidence from the laboratory of Duke biomedical engineer Fan Yuan that the cancer drug might also destroy blood vessels feeding the tumor, in effect sealing off the pathway by which the drug might leak back into the circulatory system. The effect is to entomb the tumor with the very drug that destroys it. "The treatment may well deliver a double whammy, killing the tumor cells and producing a dramatic anti-vascular effect that shuts down the blood supply that would normally feed it," says Needham.

Testing the therapy
Dewhirst and his colleagues recently launched a series of clinical studies to explore the effectiveness of hyperthermia and liposomes in combination in treating human cancers. In the first trial, a 2000 phase I study of women with inoperable locally advanced breast cancers, the researchers injected doxorubicin-containing liposomes intravenously while heating the women's breasts by beaming microwaves through a specially designed water bath. The researchers found that by using liposomes as "neoadjuvant" therapy–— that is, therapy given prior to other treatments–— they were able to stop tumor growth and, in the majority of cases, shrink the tumors substantially. The shrinkage led to "downstaging," meaning that the patients could now undergo either lumpectomy or mastectomy, followed by radiation and other adjuvant chemotherapy. While the lengthy approvals process for clinical trials necessitated the use of earlier-generation liposomes in this study, similar trials testing the new, more effective "melting" liposome are planned.

The researchers are also studying the effects of Needham's liposomes in spontaneous tumors in dogs–— research led by Jeannie Poulson, DVM, PhD, also of Radiation Oncology, working with veterinarians at North Carolina State University and Colorado State Veterinary College. "These are pets with soft tissue sarcomas in which the liposomes are being used as neoadjuvants to shrink the tumor before surgery and/or radiation therapy," says Dewhirst. So far, the treatments have shown considerable effectiveness, he says.

The new liposome technology has also been licensed to Celsion Corp.–— a company specializing in hyperthermia treatment–— which has launched phase I clinical trials of the liposomes to treat prostate cancer. Those trials are at Roswell Park Cancer Institute, in Buffalo.

Such testing, however, is only the beginning of a long road of clinical trials, emphasizes Dewhirst. The need to meet government regulatory requirements and to fund and organize consortia for large-scale clinical trials means that the liposome technology will not likely enter common clinical use for at least five years, although the therapy will be available to patients participating in clinical trials.

Also, he says, extensive basic studies will be needed to explore the potential use of liposomes for delivering other anticancer drugs. Needham and Dewhirst say they aim to secure such funding and coordinate multi-center trials that may ultimately bring these two technologies into clinical use.

Dewhirst believes liposome treatment could potentially be used to impact a range of cancers, including breast, pancreatic, colorectal, ovarian, cervical, head/neck and brain. Such treatments could significantly improve survival, he believes.

"While this therapy aims strictly at local control of tumors, and not at treating metastases, many studies have now shown that if you improve local control, you improve survival," he says. "And to a certain extent that has to do with the fact that you prevent the onset of metastases because you stop the tumor before it can spread.

"There is also a flip side to this issue, that failure to control local disease often leads to death in the absence of metastasis," he adds. "Many of the cancers that we hope to treat with liposomes either do not metastasize, or spread slowly, so we believe that this therapy will ultimately prove an important weapon in the oncologist's armamentarium."