Variability in Polyene Content and Cellular Toxicity
Study Objective: To evaluate the toxicity of amphotericin B deoxycholate formulations.
Design: In vitro experiment.
Setting: University research center.
Material: Human mononuclear THP-1 cells.
Intervention: The human mononuclear cells were exposed in vitro for 2 hours to the following deoxycholate formulations of amphotericin B, in 2.5- and 5-µ/ml concentrations: Apothecon, Pharmacia, Sigma, Gensia, Pharma-Tek, and VHA.
Measurements and Main Results: Toxicity of the amphotericin B formulations were assessed by measuring interleukin (IL)-1
expression in an in vitro model. Amphotericin B content was measured by enzyme-linked immunosorbent assay (ELISA), and amphotericin A and B contents were assessed by spectrophotometry. Endotoxin contamination was evaluated in all reagents. Expression of IL-1
from Sigma, Pharmacia, and Pharma-Tek formulations was increased approximately 250%, 50%, and 25%, respectively, compared with amphotericin A. Amphotericin B content of Sigma, Pharmacia, Pharma-Tek, and Gensia formulations, as measured by ELISA, was increased approximately 450%, 200%, 200%, and 100%, respectively, compared with Apothecon. This variation could not be explained by differences in amphotericin A or B content as measured by spectrophotometry.
Conclusion: Amphotericin B is obtained from a fermentation plant and manufactured as a pharmaceutical at different facilities. Both previous clinical observations and the current in vitro evaluation revealed significant differences among the formulations. Likely, other polyenes or pyrogenic toxins in differing amounts are in these formulations, thus explaining the variability in toxicity observed among the formulations.
Systemic antifungal therapy often is required to treat mycotic infections in immunocompromised patients, such as those with malignancies, organ transplants, or acquired immunodeficiency syndrome. Although several new antifungal agents are being developed, amphotericin B remains the most effective agent for treating a broad range of systemic fungal infections. Ever since amphotericin B was introduced in 1955, its clinical efficacy has not substantially diminished. However, patients undergoing amphotericin B therapy continue to experience significant infusion-related and chronic adverse effects. Amphotericin B infusion-related adverse effects include thrombophlebitis, nausea, vomiting, headache, myalgias, arthralgias, fever, shaking chills or rigors, dyspnea, and anaphylaxis. Although the frequency and severity of these adverse effects vary among patients, we recently recognized atypical reactions within patients. Infusion-related reactions are typically more severe in the first few days of therapy but attenuate with prolonged therapy. We observed new-onset infusion-related reactions in patients who were several days into therapy and previously had tolerated the infusion of Apothecon brand amphotericin B. On further investigation it was discovered that the development of infusion-related reactions corresponded to a change in the generic supplier of amphotericin.
An in vitro evaluation of these observations was initiated because of the ethical concerns about performing a clinical study and the large patient sample required. Our objective was to evaluate these clinical observations by using two in vitro tests of amphotericin B products administered at our institution. Interleukin (IL)-1
expression from mononuclear cells appears to be a good marker of infusion-related reactions. Our first hypothesis was that different generic sources of amphotericin B vary significantly in their ability to cause IL-1
expression. Our second hypothesis was that the differences in IL-1
expression are due to variability in the quantity of contamination in the commercial formulations. Two common types of contamination were evaluated: endotoxin and amphotericin A.
Study Objective: To evaluate the toxicity of amphotericin B deoxycholate formulations.
Design: In vitro experiment.
Setting: University research center.
Material: Human mononuclear THP-1 cells.
Intervention: The human mononuclear cells were exposed in vitro for 2 hours to the following deoxycholate formulations of amphotericin B, in 2.5- and 5-µ/ml concentrations: Apothecon, Pharmacia, Sigma, Gensia, Pharma-Tek, and VHA.
Measurements and Main Results: Toxicity of the amphotericin B formulations were assessed by measuring interleukin (IL)-1
expression in an in vitro model. Amphotericin B content was measured by enzyme-linked immunosorbent assay (ELISA), and amphotericin A and B contents were assessed by spectrophotometry. Endotoxin contamination was evaluated in all reagents. Expression of IL-1
from Sigma, Pharmacia, and Pharma-Tek formulations was increased approximately 250%, 50%, and 25%, respectively, compared with amphotericin A. Amphotericin B content of Sigma, Pharmacia, Pharma-Tek, and Gensia formulations, as measured by ELISA, was increased approximately 450%, 200%, 200%, and 100%, respectively, compared with Apothecon. This variation could not be explained by differences in amphotericin A or B content as measured by spectrophotometry.
Conclusion: Amphotericin B is obtained from a fermentation plant and manufactured as a pharmaceutical at different facilities. Both previous clinical observations and the current in vitro evaluation revealed significant differences among the formulations. Likely, other polyenes or pyrogenic toxins in differing amounts are in these formulations, thus explaining the variability in toxicity observed among the formulations.
Systemic antifungal therapy often is required to treat mycotic infections in immunocompromised patients, such as those with malignancies, organ transplants, or acquired immunodeficiency syndrome. Although several new antifungal agents are being developed, amphotericin B remains the most effective agent for treating a broad range of systemic fungal infections. Ever since amphotericin B was introduced in 1955, its clinical efficacy has not substantially diminished. However, patients undergoing amphotericin B therapy continue to experience significant infusion-related and chronic adverse effects. Amphotericin B infusion-related adverse effects include thrombophlebitis, nausea, vomiting, headache, myalgias, arthralgias, fever, shaking chills or rigors, dyspnea, and anaphylaxis. Although the frequency and severity of these adverse effects vary among patients, we recently recognized atypical reactions within patients. Infusion-related reactions are typically more severe in the first few days of therapy but attenuate with prolonged therapy. We observed new-onset infusion-related reactions in patients who were several days into therapy and previously had tolerated the infusion of Apothecon brand amphotericin B. On further investigation it was discovered that the development of infusion-related reactions corresponded to a change in the generic supplier of amphotericin.
An in vitro evaluation of these observations was initiated because of the ethical concerns about performing a clinical study and the large patient sample required. Our objective was to evaluate these clinical observations by using two in vitro tests of amphotericin B products administered at our institution. Interleukin (IL)-1
expression from mononuclear cells appears to be a good marker of infusion-related reactions. Our first hypothesis was that different generic sources of amphotericin B vary significantly in their ability to cause IL-1
expression. Our second hypothesis was that the differences in IL-1
expression are due to variability in the quantity of contamination in the commercial formulations. Two common types of contamination were evaluated: endotoxin and amphotericin A.
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