**3. Conclusion**

90 Prostate Cancer – Diagnostic and Therapeutic Advances

methotrexate-based peptide analoges were screened to identify PSMA selective substrates that are stable to unspecific hydrolysis in human and mouse plasma. Analogs containing α-linked or γ-linked glutamic or aspartic acids were most efficiently hydrolyzed by PSMA to release the cytolytic anti-metabolite methotrexate. As a consequence thereof, these analoges showed the highest cytotoxicity against PSMA-expressing prostate cancer cells (Mhaka et al., 2004). In a subsequent study, these peptides were coupled to a cytotoxic analogue of the plant toxin thapsigargin that induces apoptosis by inhibition of the endoplasmatic reticulum Ca-ATPase pump. PSMA hydrolysis of these peptide prodrugs led to a cytotoxicity against PSMA-positive prostate cancer cells that was 10- to 60-fold higher than against PSMAnegative ones. One of these prodrugs was also tested in mice with CWR22H xenografts and elicited tumor growth delay or tumor regressions following a single 3-day or 10-day course

The lack of specific delivery of photosensitizers, chemical compounds that can be excited by light of a specific wavelength for the destruction of tumor tissues, represents a significant limitation for photodynamic therapies (PDT). Therefore, a conjugate was generated containing the photosensitizer pyropheophorbide-a and a PSMA inhibitor for the treatment of prostate cancer. This construct demonstrated a high and specific cytotoxicity against LNCaP cells after irradiation, whereas PSMA-negative PC-3 cells remained unaffected. PDTmediated effects of the photodynamic conjugate were extensively studied and involved cell membrane permeabilization, rapid disruption of microtubules (α-/β-tubulin), microfilaments (actin), and intermediate filaments (cytokeratin 8/18) in the cytoplasm, activation of caspase-3, -8, and -9, Poly [ADP-ribose] Polymerase (PARP)-cleavage, and

Nanotechnology represents a new alternative for the treatment of prostate cancer. The production of nanoparticles enables the targeted delivery and controlled release of thousands of drug molecules per vehicle into the tumor cells and is a promising strategy to overcome the lack of specificity and limited efficacy of conventional chemotherapeutic

One of the first therapeutic nanoparticle against prostate cancer was a docetaxelencapsulated nanoparticle formulated with biocompatible and biodegradable poly(D,Llactic acid-co-glycolic acid)-block-poly(ethyleneglycol) copolymer (PLGA-b-PEG), which surface was derivatized with the anti-PSMA RNA-aptamer A10. With this construct, an enhanced cytotoxicity, compared to non-targeted nanoparticles that lack the aptamer, was shown. After a single intratumoral injection, the nanoparticle elicited a complete tumor reduction in 5/7 mice with LNCaP tumor xenografts of about 300 mm³ in size. The survival rate of these mice in a 109 days study was 100%, compared to 57% of mice treated with the non-targeted nanoparticle and to 14% of mice treated with docetaxel alone (Farokhzad et al., 2006). In a subsequent study, the cisplatin prodrug Pt(IV) was encapsulated in the anti-PSMA nanoparticle. Endocytosis was detected using fluorescence microscopy by colocalization of the encapsulated green fluorescent labeled cholesterol with early endosome marker EEA-1. In a series of *in vitro* cytotoxic assays, the IC50 value was determined as 0.03 µM for the nanoparticele compared to 0.13 µM for the non-targeted nanoparticle and to 2.4 µM

DNA fragmentation (Liu et al., 2009c; Liu et al., 2010a; Liu et al., 2010b).

of administration (Mhaka et al., 2006).

**2.3.8 Nanoparticles targeting PSMA** 

agents.

**2.3.7 Photodynamic therapy** 

In the last years, PSMA arouse increasing interest as a prognostic and diagnostic biomarker as well as an attractive target antigen for new therapeutic approaches against prostate cancer.

Anti-PSMA radioimmunoconjugates demonstrated efficient targeting of soft tissue and bone metastases and led to objective anti-tumor responses in a subset of patients. Moreover, high efficacy and tolerability of anti-PSMA immunotoxins was shown in many preclinical and clinical trials. The future strategy in this field is the recombinant production of immunotoxins, by which different limitations of chemically linked immunotoxins can be overcome.

Retargeting of cytotoxic lymphocytes *in vitro* and *in vivo* led to anti-tumor activities in different preclinical studies. These effects could be enhanced by the evocation of costimulatory signaling. Additionally, early vaccination trials showed that an anti-PSMA immune response can be generated without general toxicity in prostate cancer patients.

The development of PSMA-targeted prodrugs, photosensitizers and nanoparticles is still in an early stage, but such constructs also represent alternative therapeutics in the near future.

It has to be considered that most of the discussed clinical treatments were tested in patients with advanced prostate cancer. However, as shown in preclinical trials, these new therapeutic drugs seem likely to be more effective in patients with minimal residual disease or metastases after primary therapies.
