Liquid Shark Cartilage Extract With Antiangiogenic Properties

^-941 is a liquid shark cartilage extract prepared by washing and scrubbing fresh shark cartilage under water to remove any residual tissue. The cleaned cartilage is then homogenized in demineralized water and stirred for 1 h to allow extraction of water-soluble molecules. After centrifugation, the liquid fraction is ultrafiltrated and the fraction below 500 kDa is retained. The ultrafiltrate is further filtered through a 0.22-^m membrane, dispensed into sterile glass vials, and stored at -20°C until use (30-32).

The anti-angiogenic potential of ^-941 was studied in a variety of in vitro and in vivo assays. Among them, this cartilage liquid extract was tested for its ability to inhibit blood vessel formation in the ex ovo chick chorioallantoid membrane assay. This assay is based on the development of a vascular system in the vitelline membrane, which is, upon opening of the egg shell, directly accessible for observation. When placed onto the vitelline membrane, antiangiogenic substances can inhibit angiogenesis. Methylcellu-lose disks containing ^-941 were placed on the external border of the vascular perimeter of the vitelline membrane where the angiogenic process is the most active. The effect of the ^-941-containing disk on angiogenesis was always compared to that of the control disk containing an equimolar amount of NaCl (Fig. 1). As shown in Fig. 2, ^-941 inhibits in a dose-dependent fashion the growth of capillaries in the chorioallantoid membrane.

Concentration (fj.g/dlsc)

fig. 2. Antiangiogenic activity of ,42-941. Dosc-response inhibition of blood vessel formation by ,£-941 (EVT).

fig. 2. Antiangiogenic activity of ,42-941. Dosc-response inhibition of blood vessel formation by ,£-941 (EVT).

Fig. 3. Antimetalloproteinase activity of ¿93-941. The enzymatic activity of gelatinase is inhibited by ^-941 in a dose-response manner.

Concentration (mg/ml)

Fig. 3. Antimetalloproteinase activity of ¿93-941. The enzymatic activity of gelatinase is inhibited by ^-941 in a dose-response manner.

These results indicates that the liquid shark cartilage extract contains compounds that block angiogenesis. In addition to its ability to inhibit angiogenesis, the potential presence of metalloproteinase inhibitors in ^-941 was explored based on previous reports of the presence of such molecules in cartilage extracts. A collagenase inhibition assay was developed using a fluorogenic peptide substrate. This test led to the demonstration that the cartilage extract induces a dose-dependent inhibition of MMP-2 activity (Fig. 3) (33). Further in vitro assays are under investigation to determine the mechanism of action through which ^-941 exercises its antiangiogenic and anticollagenolytic activities.

The possibility that ^-941 may retain its antiangiogenic properties in tumor-bearing animals and potentially stop or slow down cancer progression or inflammatory phenomena was examined in vivo. Among the various animal models for solid tumors where 941 have been tested, the results obtained with the DA3 adenocarcinoma model are the

Concentration (fj.g/dlsc)

60% inhibition

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