Pharmacokinetic Properties of Fluorescently Labelled Hydroxypropyl-Beta-Cyclodextrin

Abstract

2-Hydroxypropyl-beta-cyclodextrin (HPBCD) is utilized in the formulation of pharmaceutical products and recently orphan designation was granted for the treatment of Niemann-Pick disease, type C. The exact mechanism of HPBCD action and side effects are not completely explained. We used fluorescently labelled hydroxypropyl-beta-cyclodextrin (FITC-HPBCD) to study its pharmacokinetic parameters in mice and compare with native HPBCD data. We found that FITC-HPBCD has fast distribution and elimination, similar to HPBCD. Interestingly animals could be divided into two groups, where the pharmacokinetic parameters followed or did not follow the two-compartment, first-order kinetic model. Tissue distribution studies revealed, that a significant amount of FITC-HPBCD could be detected in kidneys after 60 min treatment, due to its renal excretion. Ex vivo fluorescent imaging showed that fluorescence could be measured in lung, liver, brain and spleen after 30 min of treatment. To model the interaction and cellular distribution of FITC-HPBCD in the wall of blood vessels, we treated human umbilical vein endothelial cells (HUVECs) with FITC-HPBCD and demonstrated for the first time that this compound could be detected in the cytoplasm in small vesicles after 30 min of treatment. FITC-HPBCD has similar pharmacokinetic to HPBCD and can provide new information to the detailed mechanism of action of HPBCD.

Keywords: HUVECs; endocytosis; fluorescence; hydroxypropyl-beta-cyclodextrin; pharmacokinetics.

Figure 1

Plasma concentration curves of FITC-HPBCD after i.v. administration in mice. Sampling times 1, 15, 30 and 60 min (A and B; n = 6) and 1, 15, 45 and 60 min (C and D; n = 6). A and C followed the first-order elimination kinetic, while B and D did not follow it.

Figure 2

FITC-HPBCD content in the supernatant of the tissue homogenates of mice. FITC-HPBCD was administered i.v. and after 60 min of treatment significant fluorescence intensity could be measured only in the supernatant of kidney homogenates (*** p < 0.001).

Figure 3

Representative in vivo fluorescence images of FITC-HPBCD treated mice. FITC-HPBCD (0.5 and 2.5 mg) was injected i.v. and images were recorded at 5, 15 and 30 min after injection. Untreated mice can be seen on the left and FITC-HPBCD treated mice can be seen on the right in each image. FITC-HPBCD distributed over the entire body of mice and the in vivo fluorescence decreased as a function of time.

Figure 4

Representative ex vivo fluorescence images of the organs of FITC-HPBCD treated mice after 30 min treatment. Brains were examined separately from other organs after 30 and 60 min of treatment and imaged from beneath. The organs of untreated mice can be seen on the left in the images. Kidneys showed the highest fluorescence intensities in both doses (0.5 and 2.5 mg). In the case of 2.5 mg dose, lung also had elevated intensity. At 30 min the brains of the FITC-HPBCD treated animals had higher fluorescence than the controls, while after 60 min there was no difference between the fluorescence of the brains.

Figure 5

Fluorescence microscopic images of FITC-HPBCD treated (A) and untreated (B) human umbilical vein endothelial cells (HUVECs). Endocytotic green vesicles can be observed in the cytoplasm (A), while some autofluorescent vesicles can be seen in the control cells (B). (Green—FITC-HPBCD, blue—cell nuclei).

Figure 6

Flow cytometric analysis of FITC-HPBCD uptake in HUVECs. Incubation of HUVECs for 30 min with 50 µM of FITC-HPBCD significantly increased the cellular fluorescence and a following 60 min washing with HBSS decreased it. (Data are expressed as means ±SD, n = 3, significance is expressed as **p < 0.01).