Baas B, Kindt K, Scott A, Scott J, Mikulecky P and Hartsel SC Activity and Kinetics of Dissociation and Transfer of Amphotericin B from a Novel Delivery Form AAPS PharmSci 1999; 1 (3) article 10 (https://www.pharmsci.org/scientificjournals/pharmsci/journal/99_10.html).

Figures and Tables

Figure 1.The first six singular value decomposition vectors for the time dependent amplitude, column 1, x axes = time (seconds), y axes = amplitude of the corresponding basis spectrum; basis spectra, column 2, x axes = wavelength units (nm), y axes = absorption amplitudes of the independent basis spectra; and singular values, column 3, diagonal matrix elements that describe the magnitudes of the contributions of the outer products of the previous column vectors. These values were obtained from an SVD of a spectral family gathered from the time-dependent redistribution of 100 mM HAmB-DOC diluted 11:1 into 1.1 mg/ml LUV composed of 10 mole percent ergosterol/egg PC at 37°C.

Figure 2.Reconstituted spectra of HAmB-DOC redistribution corresponding to the first two vectors for the SVD in Figure 1. Inset is the raw data output collected logarithmically from 0.005 to 200 seconds from the stopped-flow diode array detector.

Figure 3.Photo and air oxidation loss of Amphotericin B from the two drug delivery systems. No attempt was made to remove dissolved oxygen or minimize light exposure. a) t = 0 seconds and t = 500-second spectra and difference spectrum of AmB-DOC, 9.1 µ'b5M, 1 cm pathlength, 0.5 mm optical slit exposed to a 150-W Xenon lamp output; b) t = 0 seconds and t = 500-second spectra and difference spectrum of HAmB-DOC, 9.1 µM, 1 cm pathlength, 0.5 mm optical slit exposed to a 150-W Xenon lamp output; c) t = 0 seconds and t = 500-second spectra and difference spectrum of AmB-DOC, 1.6 µM, 1 cm pathlength, 0.5 mm optical slit exposed to a 150 W-Xenon lamp. Notice that in all three cases only the blue-shifted, self-associated form of AmB is lost, leaving behind the monomer.

Figure 4.Basis spectra and concentration profiles produced from the Pro-K global fitting procedure of a 200-second data set produced from an 100-µM AmB-DOC solution diluted 11:1 into 1.1 mg/ml LUV composed of 10 mole percent ergosterol/egg PC at 37°C. The fit of the 3 SVD reconstituted spectra were fit to a sequential first-order scheme, A A2 M, where A is the initial self-associated form of AmB-DOC, A2 is an intermediate state formed from A, and M is a constrained basis spectrum produced by complete association of AmB with ergosterol-containing LUV.

Figure 5.Basis spectra and concentration profiles produced from the global fitting of a 200-second data set produced from a 100-mM HAmB-DOC solution diluted 11:1 into 1.1 mg/ml LUV composed of 10 mole percent ergosterol/egg PC at 37°C. The fit of the two SVD reconstituted spectra were fit to a sequential first-order scheme, A→A2→M, where A is the initial self-associated form of HAmB-DOC, A2 is an intermediate state formed from A, and M is a constrained basis spectrum produced by complete association of AmB with ergosterol-containing LUV.

Figure 6.Examples of single-fitted kinetics to show the quality of the fit at the most intense monomer-like wavelength(s) are shown here. The four fits correspond to the four samples of AmB-DOC and HAmB-DOC globally fitted to obtain the data in Table 1. The blue lines correspond to AmB-DOC, while the red lines are HAmB-DFOC data. Black lines underlying these traces represent the individual fits. All data were collected at 37°C and pH 7.4. a) AmB-DOC and HAmB-DOC dissociating in buffer. Samples were rapidly diluted from 100 µM to 3.8 µM; b) AmB-DOC and HAmB-DOC dissociating in a suspension of 1.1 mg/ml LUV composed of 10 mole percent cholesterol/egg PC samples were rapidly diluted from 100 µM to 9.1 µM; c) AmB-DOC and HAmB-DOC dissociating in a suspension of 1.1 mg/ml LUV composed of 10 mole percent ergosterol/egg PC. Samples were rapidly diluted from 100 µM to 9.1 µM; d) AmB-DOC and HAmB-DOC dissociating into 6:1 diluted (with PBS) whole bovine serum. Samples were rapidly diluted from 100 µM to 9.1 µM.

Table 1.

Figure 7.Fluorescence detected pH changes induced by AmB-DOC and HAmB-DOC in a suspension of 1.1 mg/ml LUV composed of 10 mole percent ergosterol/egg PC with a 6.67:1 K+ gradient. The decrease in fluorescence is due to K+/H+ exchange and demonstrates electrogenic K+ currents caused by AmB channel structures. The currents were detected for both systems at final (AmB) of 0.25 and 2.0 µM. The direction and magnitude of the antibiotic channel-mediated currents suggest a high degree of selectivity of K+ over Cl-. Valinomycin at 0.1 µM was used as a potassium-selective control.

Figure 8.Fluorescence-detected ion currents induced by AmB-DOC, HAmB-DOC, and AmB from a 1-mM DMSO stock solution in a suspension of 1.1 mg/ml LUV composed of 10 mole percent cholesterol/egg PC with a 6.67:1 K+ gradient. The currents were detected for both systems at final (AmB) of 2.0 µM. The direction and magnitude of the antibiotic channel-mediated currents again demonstrates a high degree of selectivity for K+ over Cl-. Valinomycin at 0.1 µM was used as a potassium-selective control. In this experiment, a striking reduction in activity is noted for the HAmB-DOC preparation as compared to the other preparations.