Compound in Grapefruit Impedes HCV Infection 4-30-08

BOSTON -- A natural compound found in grapefruit may provide a new treatment for hepatitis C, researchers here found.

Naringenin, a flavonoid, reduced the secretion of hepatitis C virus from infected cells by 80%, Yaakov Nahmias, Ph.D., of Harvard, and colleagues reported in the May issue of Hepatology.

The compound was effective at concentrations that were non-toxic in human hepatocytes and in mice engineered for severe combined immunodeficiency, they said.

"The possible reduction of HCV viral load by inhibiting viral secretion could allow uninfected cells to regenerate," they said, "potentially increasing the overall rate of viral clearance."

The standard treatment for HCV -- interferon-alpha-2a and ribavirin -- is not well tolerated by patients, according to the researchers, and alternative strategies are needed.

It has long been known that HCV associates with lipoproteins in the blood and recent studies have shown that secretion of the virus is dependent on the expression of apolipoprotein B, which is used in the assembly of very low-density lipoprotein.

This suggests, the researchers said, that HCV is being secreted while bound to very low-density lipoprotein.

Therefore, they said, compounds that have been previously shown to influence lipoprotein assembly and secretion, like naringenin in grapefruit, could possibly inhibit HCV secretion.

To test their hypothesis, the researchers used a model of the lifecycle of HCV infection.

First, using cultured cells, they confirmed that the HCV core protein was bound to apolipoprotein B in infected cells.

Next, to explore whether HCV was actively secreted while bound to very low-density lipoprotein or if the binding occurred outside the cell, the researchers analyzed the secretion of HCV and apolipoprotein B in response to oleate, which upregulates secretion, and insulin, which down-regulates it.

There was significant upregulation in response to oleate for both HCV (P=0.0073) and apolipoprotein B (P=0.0023) and significant downregulation with insulin for the viral protein (P=0.0223) and the lipid-binding protein (P=0.0073).

The ability of the secreted virus to infect healthy cells was significantly increased with oleate and significantly decreased with insulin (P<0.01 for both).

The researchers also found that silencing apolipoprotein B production and reducing its secretion by 69% significantly decreased the secretion of the HCV core protein by 75% (P=0.0002) and HCV RNA by 69% (P=0.0015).

"These results suggest the HCV is being actively secreted by the cells, perhaps while bound to [very low-density lipoprotein]," the researchers said.

Next, the researchers treated the infected cells with the grapefruit flavonoid naringenin at doses of 200, 1,000, and 5,000 µM.

The compound significantly inhibited the secretion of HCV core protein (P=0.0001) and RNA (P=0.0006) at a concentration of 200 µM.

The ability of the secreted virus to infect healthy cells was reduced by 79% (P=0.0018) by naringenin.

Also at this concentration, naringenin reduced the activity of microsomal triglyceride transfer protein by 58% (P=0.0012) and the secretion of apolipoprotein B in human hepatocytes by 60% (P=0.007).

At this concentration, the viability of the hepatocytes did not differ significantly from the controls.

In mice engineered for severe combined immunodeficiency, concentrations of about 200, 1,000, and 5,000 µM of naringenin did not increase mortality. Alanine aminotransferase levels remained unchanged, but levels of aspartate aminotransferase appeared to increase, remaining below 100 U/L at the highest dose.

Concentrations of 200 and 1,000 µM of naringenin also lowered triglycerides in the mice.

The results strongly support the hypothesis that "HCV might 'hitch a ride' on the lipoprotein-cholesterol lifecycle," the researchers said.

"The concept of supplementing HCV patients' diets with naringenin is appealing," they continued, but the poor absorption of the compound by the intestines would most likely necessitate intravenous delivery in any therapies.