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Insight & Intelligence : Nov 15, 2012
Slaying the Fungus Among Us
Meningitis outbreak aggravates concerns about amphotericin B, as study begins shedding light on how the antifungal works.
More than a half-century after it was first used on patients, amphotericin B (AmB) made news again last month after it was recommended as part of a combination therapy for many of the patients who contracted noncontagious fungal meningitis from injections of steroids compounded by the New England Compounding Center.
According to CDC’s Interim Treatment Guidance for Central Nervous System and Parameningeal Infection Associated With Injection of Contaminated Steroid Products, updated November 8, “Providers should strongly consider giving liposomal amphotericin B in addition to voriconazole to patients who present with severe disease, and patients started initially on voriconazole monotherapy who do not improve or who experience clinical deterioration.” CDC added that liposomal AmB “may also be considered as an alternative to voriconazole in patients who are unable to tolerate voriconazole.”
With the advent of voriconazole and other newer drugs, from the -azole class to the echinocandin class, amphotericin drugs have been much less frequently used now than in decades past. “The reason is because we have less toxic alternatives now. Their use has declined significantly in the past 10 to 20 years,” Helen W. Boucher, M.D., director, infectious diseases fellowship program and associate professor of medicine at Tufts Medical Center’s Division of Geographic Medicine and Infectious Diseases, told GEN.
“However, amphotericin is still valuable in several cases, such as the recent meningitis outbreak. For these rare molds, it’s still valuable in treatment, especially when it’s used in combination,” Dr. Boucher added. “It’s useful in Cryptococcus, which is another fungal infection that infects many people throughout the world, but fortunately not so many in the U.S. anymore because of all our good HIV therapies. It’s useful in a couple of other rare fungal infections.”
Dosages Increase with Infections
The meningitis outbreak and resulting advice to use AmB in combination with voriconazole is likely to aggravate concerns, most recently voiced in a study published last month in Nature Scientific Reports about the rise seen over the past 20 years in the frequency of invasive fungal infections. Those infections have been followed by corresponding increases in illnesses and deaths —and more worrisome, to increased dosages in the use of AmB, following by increased resistance to the antifungal and complications such as kidney failure.
At such doses, about half of patients suffer from some form of kidney poisoning; 15% of patients on AmB in a 1999 study (Wingard, et. al., Clinical Infectious Diseases) were forced into kidney dialysis. Less commonly but more frighteningly, AmB use can result in complete failure of the kidneys, liver, or heart. Kidney toxicity explains why two decades ago the lipid forms of AmB were developed, but they only reduce nephrotoxicity.
“The blood vessels in the kidney and the cell membranes of the kidney tubules seem to be particularly susceptible to amphotericin; the drug damages the membranes of these cells and causes changes to the body’s sodium levels. Both directly and indirectly, therefore, the drug causes a constriction of the renal blood vessels and thereby makes the kidney less efficient in removing unwanted waste from the blood,” the study’s corresponding author, David Barlow, Ph.D., of King’s College London’s (KCL) Institute of Pharmaceutical Science, remarked.
Dr. Barlow, who is head of KCL’s pharmaceutical chemistry teaching section and also a reader in computational & molecular biophysics, joined two research colleagues from his institute and from France’s Institut de Laue Langevin in the study, which featured results from neutron diffraction studies of AmB’s incorporation within lipid-sterol membranes.
They noted that combating the problems associated with AmB through development of new antifungal drugs or antimycotics will require detailed knowledge of the fungus’ molecular mechanism, which remains to be understood.
Searching for Structure
“While it is generally thought that the drug forms intra-membrane pores through its preferential interaction with ergosterol in fungal cell membranes, it is clear, too, that the drug can also form pores in cholesterol-containing mammalian cell membranes, which is why it can cause toxicity in patients. There is no direct structural evidence to support the idea of AmB-sterol pores, however, nor any structural characterization of the proposed complexes formed with sterols,” Dr. Barlow and colleagues noted.
What in the structure of ergosterols allows AmB to react more readily with ergosterol to form the membrane pores than with cholesterol?
“We don’t yet have the answer to that question, and that is one of the reasons why we’re carrying out this research,” Dr. Barlow said.
In the study, Dr. Barlow and colleagues used neutron diffraction of oriented lipid-sterol multi-layers to determine the structures of AmB-perturbed lipid-sterol membranes and—more specifically—to determine the differences in the drug’s interactions with synthetic human and fungal cell membranes, to help establish which if any of the various models proposed for its interaction with membranes is correct.
Researchers modeled human and fungal cell membranes with layers of lipids combined with either cholesterol or ergosterol. The team introduced deuterium, a heavier isotope of hydrogen, to either the membrane model or the drug, tagging that part of the system to follow it during the interaction.
The team found that while “barrel-like” structures formed in both membranes upon the introduction of AmB, the barrels penetrated more deeply into the fungal membranes than human membranes at a low dose, which may explain why AmB can open up pores more readily in fungal cells than in human cells. But at higher doses, the pores pass right across both types of membranes, resulting in damage to healthy tissue.
Tilt Toward Fungi?
The difference could be as simple as the angle at which AmB enters the fungal membranes, which appears less tilted than when it inserts into human membranes. “Sadly, our neutron diffraction profiles do not provide any substantive clue as to the nature of any interaction between AmB and cholesterol or ergosterol, and we are unable, therefore, to determine whether the drug and sterols are arranged head-to-head or head-to-tail,” the research group wrote.
The group will work to improve the resolution of the analysis in order to identify the exact part of the drug that forms the barrel structures. “We need to get more samples in which our model cell membranes are more ordered, and we also need to be able to perform the same kind of experiments using deuterated sterols and/or deuterated drugs—to be able to ‘highlight’ exactly how the molecules are oriented in the membranes,” Dr. Barlow said.
That new knowledge should also yield clues to aid in discovery of new antifungal drugs. To help ensure that happens, Congress ended its partisan squabbling long enough earlier this year to pass a fifth authorization of the Prescription Drug User Fee Act (PDUFA V), incorporating incentives meant to stoke antifungal development.
“We hope that the provisions in the new FDA legislation will be helpful in two main ways,” Dr. Boucher said. “One is in the provision of an extra five years of exclusivity. The other is in the ability to designate certain products that are in development as qualified infectious diseases products (QIDP) designated products that meet an unmet need in infectious disease. That guarantees a shorter review time with FDA, six months instead of a year. And we’ve heard from many sponsors, people who develop drugs, that they see that as a very big plus. We hope that will flow into the antifungal space.”
Until those new drugs emerge, research efforts like those of Dr. Barlow’s group will be key to better understanding, then counteracting, the side effects of AmB by the time the next meningitis outbreak occurs.
[CDC's Interim Treatment Guidance for Central Nervous System and Parameningeal Infection Associated With Injection of Contaminated Steroid Products is available at http://www.cdc.gov/hai/outbreaks/clinicians/guidance_cns.html]
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