In vivo evaluation of a novel albumin-binding prodrug of doxorubicin in an orthotopic mouse model of prostate cancer (LNCaP).

PSA, which is overexpressed in prostate carcinoma, represents a molecular target for selectively releasing an anticancer agent from a prodrug formulation. In this study, we report on the in vivo antitumor efficacy of an efficacious albumin-binding prodrug of doxorubicin (PSA9) that incorporates p-aminobenzyloxycarbonyl (PABC) as a self-immolative spacer in addition to the heptapeptide, Arg-Ser-Ser-Tyr-Tyr-Ser-Leu, which serves as a substrate for PSA. The prodrug is cleaved very efficiently by PSA releasing H-Ser-Leu-PABC-doxorubicin and subsequently doxorubicin in PSA-positive cell lysates and prostate tumor homogenates as the final cleavage product. PSA9 at 3 × 6 mg kg(-1) doxorubicin equivalents (intravenous) was compared with conventional doxorubicin at equitoxic doses (at 3 × 3 mg kg(-1); intravenous) in an orthotopic mouse model of prostate cancer using LNCaP lentiviral luciferase-neomycin cells transduced with luciferase. Whereas doxorubicin did not show any efficacy against the primary tumor or metastases, the prodrug reduced the primary tumor by 30-50% and circulating PSA levels, and in addition, showed a pronounced reduction in lung and bone metastases by ∼77% and ∼96%, respectively, and a positive trend regarding the activity against liver and lymph-node metastases compared with control and doxorubicin-treated animals. The incorporation of PABC as a self-immolative spacer together with a PSA substrate demonstrates superior antitumor effects over doxorubicin attributed to an efficient cleavage by PSA releasing doxorubicin as the final active agent in prostate tumor homogenates. Using this approach for developing effective prodrugs against prostate cancer, is worthy of further preclinical optimization.

INNO-206, the (6-maleimidocaproyl hydrazone derivative of doxorubicin), shows superior antitumor efficacy compared to doxorubicin in different tumor xenograft models and in an orthotopic pancreas carcinoma model.

The (6-maleimidocaproyl)hydrazone derivative of doxorubicin (INNO-206) is an albumin-binding prodrug of doxorubicin with acid-sensitive properties that is being assessed clinically. The prodrug binds rapidly to circulating serum albumin and releases doxorubicin selectively at the tumor site. This novel mechanism may provide enhanced antitumor activity of doxorubicin while improving the overall toxicity profile. Preclinically, INNO-206 has shown superior activity over doxorubicin in a murine renal cell carcinoma model and in breast carcinoma xenograft models. In this work, we compared the antitumor activity of INNO-206 and doxorubicin at their respective maximum tolerated doses in three additional xenograft models (breast carcinoma 3366, ovarian carcinoma A2780, and small cell lung cancer H209) as well as in an orthotopic pancreas carcinoma model (AsPC-1). INNO-206 showed more potent antitumor efficacy than free doxorubicin in all tumor models and is thus a promising clinical candidate for treating a broad range of solid tumors.

A new liposomal formulation of Gemcitabine is active in an orthotopic mouse model of pancreatic cancer accessible to bioluminescence imaging.

Despite its rapid enzymatic inactivation and therefore limited activity in vivo, Gemcitabine is the standard drug for pancreatic cancer treatment. To protect the drug, and achieve passive tumor targeting, we developed a liposomal formulation of Gemcitabine, GemLip (Ø: 36 nm: 47% entrapment). Its anti-tumoral activity was tested on MIA PaCa-2 cells growing orthotopically in nude mice. Bioluminescence measurement mediated by the stable integration of the luciferase gene was employed to randomize the mice, and monitor tumor growth. GemLip (4 and 8 mg/kg), Gemcitabine (240 mg/kg), and empty liposomes (equivalent to 8 mg/kg GemLip) were injected intravenously once weekly for 5 weeks. GemLip (8 mg/kg) stopped tumor growth, as measured via in vivo bioluminescence, reducing the primary tumor size by 68% (SD +/- 8%; p < 0.02), whereas Gemcitabine hardly affected tumor size (-7%; +/- 1.5%). In 80% of animals, luciferase activity in the liver indicated the presence of metastases. All treatments, including the empty liposomes, reduced the metastatic burden. Thus, GemLip shows promising antitumoral activity in this model. Surprisingly, empty liposomes attenuate the spread of metastases similar to Gemcitabine and GemLip. Further, luciferase marked tumor cells are a powerful tool to observe tumor growth in vivo, and to detect and quantify metastases.

An overview of Plasmodium protein kinases.

Protein kinases are key regulators of many biochemical processes in eukaryotic cells. Malaria parasites, in spite of all their peculiarities, are not likely to represent an exception in this respect. Over the past few years, several genes encoding Plasmodium protein kinases have been cloned and characterized; these molecular studies extend previous data on kinase activities in parasite extracts. Here, Barbara Kappes, Christian Doerig and Ralph Graeser present available data on this topic, with an emphasis on cloned protein kinase genes, and discuss the potential outcome of such research in the context of drug development.

Characterization of a mitogen-activated protein (MAP) kinase from Plasmodium falciparum.

A mitogen-activated protein (MAP) kinase gene, PfMAP, from Plasmodium falciparum was recently identified. We expressed this gene in Escherichia coli to test whether it encodes a functional MAP kinase. Recombinant PfMAP kinase autophosphorylates on both the tyrosine and threonine residues within the TXY motif, and readily phosphorylates myelin basic protein as exogenous substrate. This identifies the PfMAP gene product as a true member of the growing family of MAP kinases. Wild-type PfMAP kinase expressed in COS-7 (SV40 transformed African green monkey kidney) cells seemed to induce apoptosis in these cells. Western blots and immunoprecipitations indicated that the kinase is expressed during the growth of the parasite in the red blood cell as three major forms: truncated forms with apparent molecular masses of 40 kDa and 80 kDa, and as a protein of approximately 150 kDa. The 40 kDa form is present throughout the intraerythrocytic development, whereas the two larger forms are only detected in mature parasites. The 40 kDa and 80 kDa forms are tyrosine phosphorylated, indicating that they represent the active forms of the PfMAP kinase. The total PfMAP kinase activity constantly increases with the maturation of the parasite.

Plasmodium falciparum protein kinase 5 and the malarial nuclear division cycles.

In the course of our studies on cell cycle regulation mechanisms of Plasmodium falciparum, we investigated expression pattern, kinase activity, and localization of PfPK5, a putative malarial member of the family of cyclin-dependent protein kinase (cdks). The kinase was immunoprecipitated from parasites of selected stages and from parasites blocked with the cell-cycle inhibitor aphidicolin. An elevated kinase activity of PfPK5 from aphidicolin-blocked cells suggested that the enzyme might be implicated in the regulation of the parasite's S-phase. To further investigate this hypothetical function, parasite cultures were treated with the specific cdk inhibitors flavopiridol and olomoucine, which act on PfPK5 in vitro at similar concentrations as on other cdks. When applied during the nuclear division cycles of the parasite, both drugs markedly inhibited the DNA synthesis, as predicted from our proposition that PfPK5 is necessary to activate or maintain the parasite S-phase. Immunolocalization studies provide further evidence for this potential role of PfPK5.

Isolation and expression of a gene specifying a cdc2-like protein kinase from the human malaria parasite Plasmodium falciparum.

A partially redundant oligonucleotide based on conserved protein sequences of cdk and cdc2-like proteins was used to isolate from genomic libraries of Plasmodium falciparum fragments of chromosome XIII carrying a 288-residue open-reading frame encoding a protein kinase sharing 57-58% identity with yeast p34cdc2. Based on sequence data, base composition and the striking similarity with other cdk and related proteins, four intervening sequences were identified. Their removal in vitro allowed expression of the gene, designated PfPK5, in Escherichia coli, the resulting product having kinase activity against casein and histone H1. Western blotting using a polyclonal antibody raised against the expressed protein showed that the kinase was located in the parasite's cytosol and was present in approximately constant amounts throughout the intra-erythrocytic asexual reproductive stage of the life cycle. The PSTAIRE region of the PfPK5 protein differs at three sites from that of p34cdc2, and the gene failed to complement cdc2/28 yeast mutants. However, Western blotting showed that the gene was not expressed in yeast, so this does not eliminate the possibility that it is the malarial version of cdc2.