supplementary DATA

FOR

Development of a novel prodrug of paclitaxel that is cleaved by prostate-specific antigen: an in vitro and in vivo evaluation study

Bakheet Elsadeka,d, Ralph Graeserb, Norbert Esserb, Cynthia Schäfer-Obodozieb, Khalid Abu Ajaja, Clemens Ungera, André Warneckea, Tahia Saleemc, Nagla El-Melegyc, Hafez Madkord, Felix Kratza,*

a Tumor Biology Center, Division of Macromolecular Prodrugs, Breisacher Strasse 117, D-79106 Freiburg, Germany

b ProQinase GmbH, Breisacher Strasse 117, D-79106 Freiburg, Germany

c Department of Biochemistry, Faculty of Medicine, Assiut University, Assiut, Egypt

d Department of Biochemistry, Faculty of Pharmacy, Al-Azhar University, Assiut, Egypt

1. Mass spectra of the key compounds 6 and prodrug 7:

ESI mass spectrum of H-Ser-Leu-PABC-paclitaxel 6 (previously called BE-108-01): MS (ESI, 5 kV, MeOH): m/z 1225.1 ([M+Na]+, 100), 1203.1 ([M+H]+, 83); HPLC analysis (λ = 254 nm) ~ 94% of peak area.

6

ESI mass spectrum of 7 (previously called BE-106-01): MS (ESI, 3 kV, MeCN): m/z 1027.2 ([M/2+2H]2+, 100); HPLC analysis (λ= 254 nm) > 95% of peak area.

7

2. Albumin binding studies

Fig. 1S: Chromatograms of incubation studies of prodrug 7 with human serum albumin (HSA) and human plasma at 37 oC. Chromatograms of HSA, human plasma and prodrug 7 are introduced as references. A new peak for HSA7 (rt ~ 22 min) appears and the peak of prodrug 7 (rt ~ 30 min) decreases over time and disappears after 1 h.

3. Orientating toxicity studies

In order to estimate the maximum tolerated dose (MTD) of our novel prodrug 7, we injected intravenously two groups of healthy male C.B-17/SCID mice (3 animals each) with 12 mg/kg and 24 mg/kg paclitaxel equivalents of prodrug 7. The prodrug was injected on day 1, 8 and 15. The body weight, as an indicator for the drug toxicity, was recorded three times per week (see Table 1S and Fig. 2S). On day 24, the toxicity study was terminated and animals were sacrificed.

Table 1S: Orientating toxicity studies for 7 in SCID mice

Group / Treatment / Application / Mortality / Animal
Number
Route / Scheme
1 / Prodrug 7 / 12 mg/kg paclitaxel equivalents / i.v. / on days 1, 8 and 15 / 0 / 3
2 / Prodrug 7 / 24 mg/kg paclitaxel equivalents / i.v. / on days 1, 8 and 15 / 0 / 3

Fig. 2S: Body weight curves under treatment with prodrug 7 in healthy SCID mice at 12 and 24 mg/kg paclitaxel equivalents:

Based on the results of these toxicity studies it was decided to choose a dose of 3 × 24 mg/kg paclitaxel equivalents of prodrug 7 and to compare this dose to the MTD of conventional paclitaxel (3 × 12 mg/kg) for the present study.

4. In vitro proliferation assays:

Figure 3S: Dose response curves of paclitaxel, doxorubicin, H-Ser-Leu-PABC-paclitaxel (6) and HSA7 in the LNCaP LLN cell line as determined with luciferase assay.

5. Serum PSA levels

Table 2S: PSA levels (ng/mL) as determined nephelometrically in the serum of animals treated with 3 × 12 mg/kg paclitaxel, 3 × 24 mg/kg paclitaxel equivalents of prodrug 7 and the negative control group.

Animal No / PSA level for Vehicle (d 44) / PSA level for paclitaxel (d 44) / PSA level for prodrug 7 (d 44)
1 / 168.2 / 5.6 / 66.0
2 / 54.6 / 19.4
3 / 178.2 / 33.7 / 18.0
4 / 121.9 / 27.8 / 0.7
5 / 263.4 / 48.7
6 / 45.6 / 103.7 / 19.6
7 / 252.5 / 224.7 / 66.6
8 / 126.7 / 28.9 / 2.5
Mean ± SEM / 148.9 ± 28.2 / 92.8 ± 34.7 / 30.1 ± 9.4

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