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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online January 20, 2004 as doi:10.1096/fj.03-0462fje. |
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Laboratory of Tumor and Developmental Biology, University of Liège, Tour de Pathologie (B23), Sart-Tilman, B-4000 Liège, Belgium;
* Syntarga B.V., 6525 ED Nijmegen, The Netherlands; and
Department of Organic Chemistry, NSR-Center for Molecular Structure, Design and Synthesis, University of Nijmegen, The Netherlands
2Correspondence: Laboratory of Tumor and Developmental Biology, University of Liège, Tour de Pathologie (B23), Sart-Tilman, B-4000 Liège, Belgium. E-mail: agnes.noel{at}ulg.ac.be
SPECIFIC AIMS
As doxorubicin (Dox, Adriamycin®) suffers from severe dose-limiting side effects, nontoxic prodrugs of Dox were designed and synthesized for selective conversion into active drug at the tumor site by plasmin overproduced in many human tumors. Two prodrugs containing a conventional or an elongated self-elimination spacer linking a specific plasmin substrate to Dox were synthesized and evaluated for anti-cancer activity and systemic toxicity in syngeneic and xenograft tumor models.
PRINCIPAL FINDINGS
1. Plasmin-activated prodrugs exert in vitro cytotoxicity against uPA-producing cells
Since activation of plasminogen into plasmin mainly occurs by urokinase plasminogen activators (uPA) overproduced by cancer or stromal cells, both cells producing high levels of uPA (EF43.fgf-4 cells) and cells producing low levels of uPA (MCF7 cells) were used to determine ED50 values of Dox and prodrugs in vitro. The active parent drug Dox showed cytotoxic activity against both cell lines (ED50 
1 µM for EF43.fgf-4 cells and 5 µM for MCF7 cells), regardless of their ability to produce uPA. Both prodrugs showed cytotoxicity comparable to Dox upon incubation with EF43.fgf-4 cells, but a 4- to 5-fold reduced toxicity against low-level uPA-producing MCF7 cells. In EF43.fgf-4 cells, cytotoxicity was inhibited by the presence of aprotinin, a plasmin inhibitor.
2. In contrast to Dox, plasmin-activated prodrugs do not display systemic in vivo toxicity
In BALB/c mice, a daily dose of equimolar concentrations of parent Dox (4 mg/kg), ST-9802 (7.7 mg/kg) and ST-9905 (8.7 mg/kg) was given intraperitonally on 3 consecutive days. Treatment of mice with Dox induced a rapid and considerable weight loss (33.5% body weight loss, P<0.001) and led to death of all mice before the end of the experiment (Fig. 1
A, B). Similar toxic effects were observed in nude mice to which two series of 3 consecutive daily injections of Dox were administered (37% body weight loss, P<0.01) (Fig. 1C, D
). In sharp contrast, equimolar doses of both prodrugs did induce neither death of BALB/c mice and nude mice, nor body weight loss (Fig. 1)
.
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When given once daily to BALB/c mice on 3 consecutive days, maximum tolerated doses (MTD) of ST-9802 and ST-9905 were 23 mg/kg per injection (69 mg/kg cumulative) and 12 mg/kg per injection (36 mg/kg cumulative), respectively (data not shown). All Dox-treated animals (9 mg/kg cumulative) died the next day after the third i.p. injection whereas mice treated with prodrugs showed 100% survival at the end of the experiment. Body weight losses of prodrug-treated mice were negligible (<8% weight loss, P>0.05).
3. Plasmin-activated prodrugs display considerable anti-tumor activities
In tumor transplantation studies, the outward toxicity of Dox and a lack of prodrug toxicity were observed as in non-tumor-bearing mice. Dox treatment resulted in drastic inhibition of tumor development (87 and 79% inhibition of tumor growth in mice bearing EF43.fgf-4 or MCF7 tumors, respectively). At equimolar concentrations with respect to Dox, both prodrugs displayed potent anticancer activity in both tumor models (Fig. 2
A, B). ST-9905, which is cleaved faster by plasmin than ST-9802 because of its elongated linker, showed a significantly more pronounced anti-tumor efficacy than ST-9802 in EF43.fgf-4 tumors (68% tumor inhibition with ST-9905 vs. 43% with ST-9802) (Fig. 2A
). At equitoxic doses, both prodrugs induced a similar reduction of tumor growth as Dox (80% of tumor growth inhibition) (Fig. 2C
) but with negligible weight loss. At its MTD, ST-9905 was as efficient as Dox in inhibiting tumor growth and considerably more effective than ST-9802 tested at equimolar dose (10.5 mg/kg/injection; 31.5 mg/kg cumulative) (Fig. 2C
).
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The presence of active plasmin in both EF43.fgf-4 and MCF7 tumors was confirmed by immunoblotting on tumor extracts (Fig. 2D
).
4. Plasmin-activated prodrugs reduce tumor cell proliferation and tumor vascularization
Consistent with their rapid tumor growth, EF43.fgf-4 tumors were characterized by a rapid proliferation rate as assessed by their extensive and uniform in vivo incorporation of 5-bromodeoxyuridine (BrdU). Even at equimolar doses, Dox as well as prodrugs markedly decreased the percentage of positive cells (10% of BrdU-positive cells in Dox-, 35% in ST-9802-, and 20% in ST-9905-treated animals). Similar anti-proliferative effects were observed on MCF7 tumors treated with Dox or prodrugs. This marked in vivo antitumor efficacy of prodrugs was associated with a strong inhibition of angiogenesis as determined by immunostaining using anti-von Willebrand factor, anti-PECAM, or anti-type IV collagen antibodies.
CONCLUSIONS AND SIGNIFICANCE
The aim of the novel therapeutic approach presented here was to exploit tumor-specific active plasmin to selectively trigger release of cytotoxic drug at the tumor site. Our results demonstrate that the two plasmin-activated prodrugs 1) are activated by plasmin, 2) can be given systemically without inducing mice death and weight loss, 3) exert both anti-tumoral and anti-angiogenic activity, and 4) are potent anticancer agents against plasmin-generating tumors and tumors that do not produce plasmin by themselves. Of particular interest is the absence of systemic toxic effects of ST-9802 and ST-9905 in contrast to Dox. Indeed, although Dox administration into BALB/c mice or nude mice induced a rapid and considerable weight loss and led finally to death, equimolar amounts of both prodrugs affected neither weight nor survival of mice. At equimolar concentrations with respect to Dox, both prodrugs displayed potent anticancer activity in both tumor models. ST-9905, which contains an elongated releasable linker that induces a faster cleavage by plasmin as compared with ST-9802, showed a significantly more pronounced anti-tumor efficacy than ST-9802 in EF43.fgf-4 tumors (68% of tumor inhibition with ST-9905 vs. 43% with ST-9802).
When tested at their MTD (equitoxic doses), both prodrugs induced similar tumor inhibitory effects with respect to Dox, accompanied by only minor body weight loss, in sharp contrast to Dox. To the best of our knowledge, this is the first report of successful application of plasmin-activated prodrugs to target breast tumors or any other tumors in vivo. At its MTD, ST-9905 was as efficient as Dox in tumor growth reduction whereas an equimolar concentration of ST-9802 exerted only a partial antitumor efficacy; a 1.7-fold higher dose of ST-9802 did not induce higher anti-tumor efficacy than ST-9905. Thus, ST-9905 was more effective than ST-9802 when tested at equimolar concentration and at the MTD. In the case of ST-9905, the markedly higher anti-tumor efficacy may be a consequence of generation of higher tumor-specific Dox concentrations as a result of its longer spacer system. The data presented here validate in vivo the use of releasable linkers of sufficient length.
In in vitro cultures, the cytotoxicity of both prodrugs toward MCF7 cells was 70–80% lower in comparison to Dox. This is consistent with the fact that MCF7 cells produce minimal amounts of uPA and are poor activators of plasminogen into plasmin. However, the prodrugs substantially inhibited growth of MCF7 tumors in vivo. Western blot analysis of tumor extracts confirmed the presence of active plasmin. The efficacy of prodrugs in vivo is likely due to their conversion into active Dox in the presence of plasmin produced by activated endothelial cells and/or myofibroblasts that infiltrate the tumor. Stromal cells actively participate in tumor progression and represent an important source of matrix metalloproteinases (MMPs), serine proteases, and plasminogen. Our data demonstrate that plasmin-activated prodrugs are potent anticancer agents even against malignant cells that do not generate plasmin by themselves. The involvement of host cells to produce the proteases required for prodrug activation might limit the apparition of a drug-resistant phenotype through a down-regulation of plasmin production. Indeed, host cells are genetically stable and therefore less susceptible to develop drug resistance.
Treatment with plasmin-activated prodrugs markedly reduced angiogenesis in the two in vivo models tested, whereas Dox displayed a partial anti-angiogenic effect on EF43.fgf-4 tumors and even a proangiogenic effect on MCF7 tumors.
Altogether, our results demonstrate that selective chemotherapy with plasmin-activated prodrugs represents a novel therapeutic approach against cancer that interacts with at least two critical steps of tumor progression: growth and angiogenesis. Such prodrugs thus may exert both cytotoxic and cytostatic effects without systemic toxicity, limiting the side effects that hamper use of classical chemotherapeutic agents (Fig. 3
). In conclusion, these experiments support the clinical use of such plasmin-activated prodrugs in new therapeutic strategies against a large series of advanced human solid cancers. ST-9905 will be further evaluated in preclinical development.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0462fje; 
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2-antiplasmin and