In Vitro and In Vivo Metabolism of CT2103

When incubated in the presence of human HT-29 colon cancer cells or murine RAW macrophages, CT-2103 is taken up by the cells and increased levels of free paclitaxel are found in the medium than in control cultures without cells. When the washed cellular fractions were tested for extractable Taxanes, increasing amounts of paclitaxel 2' monoglutamate and free paclitaxel were observed over several days (Figure 4). These observations suggest that the polymeric conjugate is taken up by the cells, and the PG backbone is cleaved by proteolysis and is followed by hydrolysis of the monoglutamate to yield free paclitaxel. To determine which proteolytic enzymes might be involved in this process, CT-2103 was exposed to a variety of proteases under cell free conditions. Of the enzymes tested, only cathepsin B was capable of degrading CT-2103 to paclitaxel-diglutamates16. The enzyme responsible for cleavage to the paclitaxel-monoglutamate was not identified. The 2' paclitaxel monoglutamate is unstable and breaks down spontaneously to paclitaxel and glutamic acid.

Figure 4. Accumulation of Paclitaxel Monoglutamate in HT-29 Cells In Vitro

To determine if paclitaxel monolglutamate was detectable as a metabolite in vivo, a series of tumours explants were extracted and assayed 24 hours after a single intravenous dose of CT-2103 (Figure 5A). As seen in Figure 5B, increasing amounts of the paclitaxel-monoglutamate were detected over several days in HT-29 colon cancers. Similar data were obtained from analysis of liver and spleen, suggesting that reticuloendothelial cells are also able to break down CT-2103 by a similar mechanism17.

B1S LL2 HCT-15 LoVo HT-29 CoU-320 HCT-116

■ Paclitaxel 2'-m on og I utamyl-paclitaxel

Figure 5A. Extractable Metabolites in Tumour Tissue from Mice with Various Tumours 24 Hours After a Single Dose of CT-2103

B1S LL2 HCT-15 LoVo HT-29 CoU-320 HCT-116

■ Paclitaxel 2'-m on og I utamyl-paclitaxel

Figure 5A. Extractable Metabolites in Tumour Tissue from Mice with Various Tumours 24 Hours After a Single Dose of CT-2103

Shrpost tw posi (n=4) 48 hr post 72 hr posl SChirpoat

Time

■ Paclitaxel 2'-monoglutamyl-paclitaxel

Figure 5B. Changes in CT-2103 Metabolites in HT-29 Tumours with Time After a Single Dose (5 Animals/Group)

Shrpost tw posi (n=4) 48 hr post 72 hr posl SChirpoat

Time

■ Paclitaxel 2'-monoglutamyl-paclitaxel

Figure 5B. Changes in CT-2103 Metabolites in HT-29 Tumours with Time After a Single Dose (5 Animals/Group)

Note: The heights of the bars do not represent absolute values since only a paclitaxel standard was used. Later studies with authentic standards determined that the relative values are about 50% of the 2'-monoglutamyl-paclitaxel bar heights.

Taken in sum, these data suggest that the CT-2103 is taken up by tumour tissues and cells within the reticuloendothelial system. Free paclitaxel is slowly released following cleavage of the PG backbone by cathepsin B. In circulation, CT-2103 does not release paclitaxel in appreciable quantities. An important parameter for a successful polymeric conjugate is a slow rate of release only outside of circulation. This spares normal organs high levels of exposure to free paclitaxel15.

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