Hyaluronidase induces a transcapillary pressure gradient and improves the distribution and uptake of liposomal doxorubicin (Caelyx) in human osteosarcoma xenografts

Abstract

Liposomal drug delivery enhances the tumour selective localisation and may improve the uptake compared to free drug. However, the drug distribution within the tumour tissue may still be heterogeneous. Degradation of the extracellular matrix is assumed to improve the uptake and penetration of drugs. The effect of the ECM-degrading enzyme hyaluronidase on interstitial fluid pressure and microvascular pressure were measured in human osteosarcoma xenografts by the wick-in-needle and micropipette technique, respectively. The tumour uptake and distribution of liposomal doxorubicin were studied on tumour sections by confocal laser scanning microscopy. The drugs were injected i.v. 1 h after the hyaluronidase pretreatment. Intratumoral injection of hyaluronidase reduced interstitial fluid pressure in a nonlinear dose-dependent manner. Maximum interstitial fluid pressure reduction of approximately 50% was found after injection of 1500 U hyaluronidase. Neither intratumoral nor i.v. injection of hyaluronidase induced any changes in the microvascular pressure. Thus, hyaluronidase induced a transcapillary pressure gradient, resulting in a four-fold increase in the tumour uptake and improving the distribution of the liposomal doxorubicin. Hyaluronidase reduces a major barrier for drug delivery by inducing a transcapillary pressure gradient, and administration of hyaluronidase adjuvant with liposomal doxorubicin may thus improve the therapeutic outcome.

Figure 1

Maximum reduction (%) in IFP 1 h after intratumoral injection of PBS (n=5), 150 U (n=6), 500 U (n=5), 1500 U (n=6) and 3000 U (n=6) hyaluronidase in o.t. tumours. Each value is the mean of n tumours, the bars represent s.e. ^*Data significantly different from untreated controls.

Figure 2

Interstitial fluid pressure response as a function of time after intratumoral injection of 150 U () (n=4), 500 U (Δ) (n=5), 1500 U (∇)(n=4) and 3000 U (□) (n=6) hyaluronidase in o.t. tumours. Interstitial fluid pressure is normalised to IFP before the injection. Each curve is the mean of n measurements.

Figure 3

Maximum reduction in IFP after intratumoral injection of hyaluronidase as a function of tumour volume. Each symbol represents one measurement, the solid line represents linear regression analysis and was found to be significant for all doses except 150 U.

Figure 4

Normalised pressure after (A) intratumoral and (B) i.v. injection of 1500 U hyaluronidase in s.c. tumours. Microvascular pressure (•) was not measured continuously, and the data are based on different time intervals for n=5 (intratumoral injection) and n=7 (i.v. injection) mice. The bars represent s.e. at some time points. The IFP (○) graph is the mean of n=2 measurements.

Figure 5

Tumour tissue hyaluronan (blue) in o.t. osteosarcoma xenografts relative to capillaries (red) and tumour cells (green) treated with PBS (A) and hyaluronidase (1500 U) (B, C).

Figure 6

Distribution of liposomal doxorubicin in osteosarcoma xenografts treated with liposomal doxorubicin alone (16 mg kg^{− 1}) (A) or liposomal doxorubicin combined with hyaluronidase (1500 U) (B). Representative images of doxorubicin (green) relative to capillaries (red) are presented from the rim to the centre of the tumour sections.

Figure 7

Doxorubicin fluorescence intensity profile in osteosarcoma xenografts treated with liposomal doxorubicin alone (16 mg kg^{− 1}) (○) or combined with hyaluronidase (1500 U) (•). Each symbol is the mean of 5–20 images per section and 10 sections per tumour in three tumours. Bars represent s.e. The increase in the doxorubicin uptake in the hyaluronidase-treated tumours is significant throughout the whole tumour (P<0.05).

Figure 8

Number of images as a function of fluorescence intensity for osteosarcoma xenografts given liposomal doxorubicin alone (16 mg kg^{− 1}) (□) or combined with hyaluronidase (1500 U) (▪). Each value is the mean of 5–20 images per section and 10 sections per tumour in three tumours. Bars represent s.e. ^*Data significantly different from Caelyx™ alone.

Figure 9

Intracellular distribution of doxorubicin (green) (A), DNA (red) (B) and colocalisation of doxorubicin and DNA (C) in osteosarcoma xenografts given liposomal doxorubicin alone (16 mg kg^{− 1}). Bar=20 μm.