Melflufen (melphalan flufenamide, chemical name L-melphalanyl-p-L-fluorophenylalanine ethyl ester hydrochloride, previously called J1) is a derivative of the classical alkylating agent melphalan. Melflufen is potentiated by hydrolytic cleavage by aminopeptidase N (APN), leading to high intracellular concentrations of alkylating moieties and subsequent cell death. Increased APN expression is associated with the malignant phenotype of several human cancers, including acute myeloid leukemia, lymphoma and ovarian cancer, and plays a functional role in tumor angiogenesis. Therefore investigations of melflufen activity in these malignancies as well as detailed studies of inhibition of angiogenesis are interesting. The aim of this project was to investigate the cytotoxic and antiangiogenic effect, in vitro and in vivo, of melflufen, compared to melphalan and other cytotoxic drugs used in the clinic.
We showed that melflufen was more effective than its parental drug melphalan in lymphoma, AML and ovarian cancer in cell lines as well as in primary patient samples. An improved in vitro therapeutic index was demonstrated by an increased cytotoxic activity in the patient samples compared to normal peripheral blood mononuclear cells (PBMCs). Furthermore, melflufen in combination with cytarabine was synergistic in an AML cell line in a sequence-dependent manor. Melflufen was shown effective in several animal models using lymphoma, AML and ovarian cell xenografts (single drug or in combination), including an intraperitoneal ovarian xenograft. Finally, we demonstrated that melflufen had antiangiogenic properties in several different models.
Lymphomas are malignant tumours arising from cells in the lymphatic system. They are classified as B-cell lymphomas, T-cell lymphomas and Hodgkin lymphoma (HL). Of the B-cell lymphomas, one of the most common is diffuse large B-cell lymphoma (DLBCL). Many patients with lymphomas can be successfully treated however patients who relapse or are refractory have a poor prognosis, warranting further investigations to identify potential targets and develop novel drugs.
Picropodophyllin (PPP), a potent and selective inhibitor of IGF-1R, inhibits malignant cell growth with low or no toxicity on normal cells in preclinical models. In paper I, we investigated the potential benefits of using PPP against DLBCL and found that the anti-tumor effects of PPP might possibly be explained by IGF-1R-unrelated mechanism(s). However, the inhibitory effects of PPP on lymphoma cells together with its low toxicity in vivo makes it a promising drug candidate for treatment. Melflufen, a derivative of melphalan, is currently being evaluated in a clinical phase I/II trial in relapsed or refractory multiple myeloma. In paper II, we confirmed previous reports of superior potency of melflufen over melphalan. Being active in cell lines and primary cultures of lymphoma cells as well as in a xenograft model in mice, melflufen considered being a candidate for further evaluation in treatment. bAP-15, a novel inhibitor of proteasome activity, inhibits ubiquitin specific peptidase 14 (USP14) and ubiquitin carboxyl-terminal hydrolase L5 (UCHL5). In paper III, we investigated the activity of b-AP15 in DLBCL and HL cell lines and compared the results to standard drugs used in treatment. Results showed inhibition of the proteasome and growth inhibition/cytotoxicity with IC50-values in the micromolar range. Treatment failure and lack of clinical benefit of proteasome inhibitors like bortezomib in DLBCL patients inspired us investigating for possible new targets, with major focus on proteasome inhibitors in DLBCL. In paper IV, we suggested that UCHL5 and/or USP14, as new targets for proteasome inhibitors in DLBCL, be further evaluated.
The findings in this thesis suggest that PPP, Melflufen and b-AP15 are potential candidates for clinical drug development and UCHL5 and/or USP14 are new potential targets for proteasome inhibitors in DLBCL.