Preserving enhancement in freeze-dried contrast agent ST68: Examination of excipients.

The perfluorcarbon (perfluorobutane) ultrasound contrast agent ST68, composed of sonicated mixtures of non-ionic surfactants, is stable in solution for only a few weeks at 4 degrees C. Freeze-drying critically diminished ST68's ability to reflect ultrasound (its echogenicity). A method of incorporating specific lyoprotectants before lyophilization was investigated. Reintroduction of perfluorobutane to the protected freeze-dried sample, followed by reconstituting with water preserved echogenicity. Glucose, trehalose, sucrose, and mannitol were tested at 100mM and in vitro echogenicity data was collected from samples with dose concentrations of 50-300microl/l. Glucose was found to be the best lyoprotectant providing an average (n=3) maximum peak enhancement of 23.2+/-1.2dB in vitro, measured at 5MHz, 684kPa, and a pulse repetition frequency (PRF) of 100Hz (p<0.05 over freeze-dried ST68 control) and 20.8+/-0.8dB in vivo in New Zealand white rabbits at 5MHz and a PRF of 6.7kHz. Pulse inversion harmonic US images of a rabbit kidney, pre- and post-contrast injection (0.1ml/kg), showed excellent enhancement and clear vascular delineation, similar to that of the original agent. For the first time this contrast agent can be successfully freeze-dried yielding a longer self-life without the need for refrigeration.