TECHNOLOGY
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F Positron Emission Tomography Advances Brain Imaging
Noninvasive technique uses labeled fluorodopa to gain new insights into chemistry of dopamine in the brain of parkinsonian patients
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Ron Dagani, C&EN Washington
A radioactive isotope of a corrosive and deadly element is coming into its own as an important medical tool, helping researchers to look, safely and noninvasively, into the malfunctioning brain of patients with neurologic disorders. Such "live-action" peeks are leading to a clearer understanding of brain dysfunctions and may point to dramatic new treatments. The isotope is fluorine-18. The technique is positron emission tomography (PET). And one goal, among many, is a cure for the shaking palsy, also known as Parkinson's disease. Patients with this malady have difficulty doing anything that requires a moderate amount of muscular control, such as w a l k i n g , eating, speaking, and writing. Parkinson's disease causes the body to assume a permanent stoop and the head and limbs to shake uncontrollably. Complete rigidity and dementia may appear in the final stages of the disease. These symptoms are blamed on a deficiency of dopamine or dopamine activity in the brain. Dopamine is a n e u r o t r a n s m i t t e r , one of many chemicals used by nerve cells in the brain to communicate with each other. Normally, cells in a brain structure called the substantia nigra supply another brain structure, the striatum, with a steady supply of dopamine. The striatum apparently 26
August 15, 1988 C&EN
PET image of a brain slice in a normal human being (left) shows high uptake of [*8F]fluorodopamine in the putamen (pink areas in center). In a patient with moderately severe Parkinson's disease, the uptake is markedly reduced (right) needs dopamine to carry out its function of controlling bodily movements. But for some unknown reason, the dopamine-producing cells may begin to fail, reducing the river of dopamine to a trickle, and thus effectively crippling the striatum. Parkinson's disease has no known cure. But patients have been able to obtain some relief by taking tablets of the drug levodopa or L-dopa [(-)-3-(3,4-dihydroxyphenyl)-L-alanine], which is metabolized to dopamine in the brain. Unfortunately, the drug has serious limitations and side-effects, and symptomatic relief is only temporary. The search for superior drug therapies so far has been discouraging. In the past few years, medical researchers have been exploring a different, perhaps more natural, way of replenishing the brain's supply of dopamine. They have trans-
planted dopamine-producing cells into the brain of parkinsonian patients. The cells are usually taken from the patient's own adrenal glands, located next to the kidneys. Two patients with severe symptoms who received grafts of adrenal tissue in Mexico reportedly showed dramatic improvements. But such results, which are controversial, have so far not been replicated in patients elsewhere, says neurologist W. R. Wayne Martin, associate director of the PET project at the University of British Columbia's University Hospital in Vancouver, Canada. Controlled studies in more patients will need to be carried out to evaluate the usefulness of this approach, he says. The quest for better treatments— and p e r h a p s preventatives—for Parkinson's disease will depend on gaining a better understanding of the brain chemistry of dopamine.
Dopamine metabolism has been charted to some extent in animal brains. Now, thanks to recent advances in radiofluorination and positron emission tomography, researchers are extending their studies into the functioning h u m a n brain. Access to the brain's dopamine pathways has been gained through the use of 6-fluoro-L-dopa tagged with fluorine-18. This dopa derivative crosses the blood /brain barrier into the brain, where it is converted into 6-[ 18 F]fluorodopamine. The tagged d o p a m i n e analog serves as a beacon inside the dark recesses of the brain because fluorine-18 emits positrons. A positron will travel no more than a few millimeters in tissue before it slams into an electron, its antimatter twin. The resulting annihilation produces two gamma-ray p h o t o n s , which shoot off in nearly opposite directions. These distinctive photon pairs are picked up by coincidence detectors mounted on the PET scanner's assembly, which encircles the subject's head. A computer uses the detection data to reconstruct the spatial distribution of a n n i h i l a t i o n events. From this, it creates crosssectional images of the radionuclide's distribution in the brain. Thus, metabolically active regions of the brain can easily be distinguished from inactive ones. Before the advent of PET, studies of brain metabolism in humans had to be done either indirectly or after the fact: Researchers could make their measurements on samples of cerebrospinal fluid removed by needle from the base of the spine, or
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COOH
HO 6-[18F]Fluoro-L-dopa
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^V-CH 2 CH 2 NH 2
6-[18F]Fluorodopamine
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Garnett: a fantastic step on tissues removed during autopsy. To be able to look at chemistry within the brain of a live, unmedicated subject is a "fantastic step," marvels E. Stephen Garnett, director of nuclear medicine at ChedokeMcMaster Hospitals in Hamilton, Ontario. Using PET, Garnett and his coworkers have shown—for the first time in live human beings—that dopamine accumulates in the striatum and that parkinsonian patients have a deficiency of this neurotransmitter. That much was known before, from studies on cadavers. The striking feature of Garnett's work is that he has been able to pinpoint the specific area of the striatum that is damaged first in Parkinson's disease. The striatum is composed of two smaller structures called the caudate nucleus and the putamen. Histologically, the two tissues are the same, Garnett says. But after examining some 200 Parkinson's patients, his group has discerned a basic pattern: The putamen is damaged early, but the caudate nucleus remains unharmed until very late in the disease. "And if we see the patients early enough," he points out, "that damage is in a very specific area within the putamen." These results, he adds, support the hypothesis that the putamen is associated with the control and reg-
ulation of movement, which is affected early, while the caudate is associated with cognition, which is the last to go. Garnett's PET evidence also has some implications for the treatment of Parkinson's disease. In one of the current experimental approaches, dopamine-producing cells are transplanted into the caudate nucleus, which is relatively easy to reach by surgery. But the finding that the putamen is affected much earlier would suggest that neural grafts might be more successful if placed in the putamen, even though it is less accessible. "That makes a great deal of sense," says Martin, a member of Donald B. Calne's movement disorder group at Vancouver's University Hospital. Still, he adds, putting the implant into the head of the caudate also makes sense because "anything you implant there comes in direct contact with the cerebrospinal fluid." And that ensures wider distribution through the brain. In any case, he says, "the answers are not yet in." One key factor in the success of the Chedoke-McMaster workers was the possession of a PET scanner with the requisite resolution to distinguish between the two parts of the striatum. They had to build the device themselves to achieve that resolution, according to Garnett. The other factor was the availability of an 18F-labeled dopa derivative. Most of the tracers presently used in brain imaging contain carbon-11, also a positron emitter. But researchers are steadily moving away from n C tracers, where possible, and switching to 18F labels in-
Caudate nucleus, putamen are deep within the brain Caudate nucleus
Amygdaloid body
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Technology
Human breast tumors imaged for first time using PET Although the human brain has yielded the flashiest images from positron emission tomography (PET), medical researchers are coming to realize that this elegant, noninvasive technique might also aid in the fight against breast cancer. At Washington University School of Medicine, St. Louis, researchers have imaged human breast tumors for the first time using PET and a fluorine18-labeled estrogen. Their results sug-
stead, says radiochemist/pharmacologist Giinter Firnau, a colleague of Garnett's at Chedoke-McMaster. One reason for the switch is convenience. Compounds containing U C must be prepared, purified, and used quickly because the isotope's half-life is only 20 minutes. The rush can put a real burden on the synthetic chemist, Firnau says. 18 F, by contrast, offers a considerably longer half-life—110 minutes. Given the choice of labeling with either n C or 18 F, chemists tend to choose the latter, Firnau notes. 18F's longer halflife, though, makes it unsuitable for multiple back-to-back studies on the same subject. Another factor in fluorine- 18's favor has been the recent development of improved methods to incorporate the isotope into a wide variety of compounds. For example, acidic functions on molecules, such as amine, hydroxyl, and thiol groups, can be used as attachment sites for fluoroalkyl groups. To fluorinate deactivated rings, fluoride ion can be used to displace a nitro group. And for electron-rich rings such as dopa's, the reagent of choice seems to be dilute fluorine gas in anhydrous hydrogen fluoride. 28
August 15. 1988 C&EN
gest that PET scans may one day help physicians diagnose breast cancer and predict whether it will respond to hormone therapy. Because hormone therapy is relatively innocuous compared with other cancer therapies, oncologists often try it first after the diagnosis has been made. But if the tumor doesn't have a high enough concentration of estrogen receptors, it's not going to respond well, and the patient may have wasted four to six months on less-effective therapy, says Michael J. Welch, professor of radiation chemistry at Washington University's Mallinckrodt Institute of Radiology. Excised tumor tissue can be assayed in vitro for estrogen receptors, but the test—besides requiring surgery—is not always accurate. Preliminary results soon to be published in the journal Radiology by Welch and his coworkers suggest that a PET scan may be able to provide a more
It may be heresy, Firnau admits, to expose easily oxidizable substrates such as dopa to molecular fluorine (a powerful oxidizer) and hydrogen fluoride (a very strong acid)—but it works. Raman Chirakal, a synthetic radiochemist working with Firnau and Garnett, fluorinates dopa using dilute fluorine gas (0.5% F2 in neon) in liquid hydrogen fluoride containing boron trifluoride at about - 7 0 °C. The fluorine gas is first labeled with 18F produced by bombarding neon-20 atoms with deu-
reliable indication of the estrogenreceptor level in breast tumors. With support from the National Institutes of Health and the Department of Energy, the Washington University group measured the uptake of the 18F tracer in 10 female patients who were undergoing evaluation for breast tumors. The results showed a high correlation between tumor uptake of the tracer (as revealed by PET) and the level of estrogen receptors in the tumor (as determined later by in-vitro assay). The goal of the work, Welch explains, is to be able to predict whether metastatic tumors—those that have traveled to other sites in the body—will respond to hormone therapy. Metastases, he points out, can't always be biopsied. His group is now checking to see whether uptake of the tracer by metastatic tumors correlates with their response to hormone therapy. The fluorine-18 PET images the St.
terons at McMaster's tandem Van de Graaff accelerator. Hydrogen fluoride was chosen as the solvent because it promotes electrophilic fluorination of phenols. It may be difficult to work with, but no other solvent system works as well, Chirakal finds. A milder but less efficient fluorination reagent that Chirakal also has used is acetyl hypofluorite (CH3COOF). This reagent was first characterized a few years ago and it, like dilute fluorine in hydrogen
Chedoke-McMaster's Firnau (left) and Chirakal
Multicolored bull's-eyes reveal high uptake of 18F-labeled estradiol In a breast cancer tumor and In the liver, which clears the tracer from the body. Lower uptake Is seen In a lymph node metastatic tumor
fluoride, is rapidly becoming more popular in fluorine chemistry. Both reagents fluorinate the phenyl ring of dopa at all three possible positions, although the 6-fluoro isomer predominates. High-performance liquid chromatography on a semipreparative scale is used to isolate the desired isomer in about 50% radiochemical yield (for the F 2 /HF route). Other groups have developed regiospecific routes to 6-[18F]fluorodopa that involve the cleaving of a carbon-silicon or carbon-mercury bond at the C-6 position with acetyl [ 18 F]hypofluorite. In 6-fluorodopa and most other fluorinated tracers, the fluorine atom substitutes for a hydrogen. In most cases, the fluorinated position is chosen to be one that was biologically inactive in the parent molecule, says Alfred P. Wolf, who heads the PET group at Brookhaven National Laboratory. This is done, he explains, because "you don't want the fluorine to interfere with the natural biological process." In the case of 6-fluorodopa, however, the 18F does interfere—but in a subtle and desirable way, says Firnau. The presence of the fluo-
rine atom suppresses a metabolic pathway that would otherwise interfere with the evaluation of brain metabolism using PET. Normally, dopa enters the bloodstream and some of it is methylated in the liver. The metabolite, 3-O-methyldopa, is carried to the brain along with the dopa. But the fluorine atom at C-6 inhibits the methylating liver enzyme. Thus, explains Firnau, 6-[18F]fluorodopa becomes the only significant source of fluorine-18 in the brain, where it is metabolized to 6-[ 18 F]fluorodopamine. All the other biological properties of dopa are preserved. This feature of 6[18F]fluorodopa makes it "an almost perfect tracer" for probing dopamine metabolism in the brain, Firnau says. 6-[18F]Fluorodopa was first used in humans in 1983. It has now become one of a handful of fluorine-18 PET tracers that are "vigorously used throughout the world," says Wolf. In fact, notes Firnau, 6-[18F]fluorodopa probably is the second most important PET tracer after 2-[18F]fluoro-2deoxy-D-glucose. The latter compound, first applied to human studies in 1976, is widely used to measure glucose metabolism in the brain.
Louis group has gotten are "by far the most impressive images" of breast tumors ever obtained, Welch tells C&EN. That's largely due to the tracer the researchers used— 16a- [ 18F] f luoroestradiol-17/3. Its synthesis, now carried out robotically, grew out of a long-term collaborative effort between Welch's group, which includes Mark A. Mintun, Barry A. Siegel, Carla J. Mathias, James W. Brodack, and Andrea H. McGuire, and a group at the University of Illinois, Urbana, headed by organic chemist John A. Katzenellenbogen. The compound is prepared by using [18F]fluoride ion to displace, in the key step, a trifluoromethanesulfonate group at C-16 of the estrogen skeleton. Before the 18F tracer became readily available, the researchers had tried to image human breast tumors using an estradiol analog tagged with bromine-77, a gammaemitter. But, says Welch, "the images were awful."
In the past few years, additional F tracers have emerged as useful tools for studying brain activities. For example, researchers are using 3-N-[ 18 F]fluoroalkylspiperones to measure the density of dopamine receptors in the brain. These receptors, most of which are clustered in the caudate nucleus and putamen, are thought to play a key role in neurologic and psychiatric disorders. Schizophrenia, for example, is believed to involve excessive dopamine activity—either too much dopamine being formed or the dopamine receptors over-responding to it, according to Garnett. N-alkylspiperones have been used to control hallucinations and other symptoms in chronic schizophrenics. Now, says Wolf, 18F-labeled analogs of these drugs are enabling researchers to probe biochemical pathways in the brain, noninvasively, in an effort "to connect the subjective symptoms that one sees in the schizophrenic with the biochemistry that's going on in the brain." Such studies, like those on Parkinson's patients, weren't possible a few years ago. "That's really the power of PET," says Wolf. D 18
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