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Targeted Delivery of Bio Active Compounds

#1759


Elaboration of New Approaches to Targeted Delivery of Biologically Active Compounds and Antisense Oligonucleotides

Tech Area / Field

  • BIO-CGM/Cytology, Genetics and Molecular Biology/Biotechnology
  • MED-DRG/Drug Discovery/Medicine
  • BIO-CHM/Biochemistry/Biotechnology

Status
8 Project completed

Registration date
27.12.1999

Completion date
14.04.2005

Senior Project Manager
Melnikov V G

Leading Institute
Research Center of Molecular Diagnostics and Theraphy, Russia, Moscow

Collaborators

  • Wadsworth Center / Oncofetal Antigen Laboratories, USA, NY, Albany\nMoffit Cancer Center & Research Institute, USA, FL, Tampa\nSouthern Research Institute, USA, AL, Birmingham\nColumbia University / College of Physicians & Surgeons, USA, New York\nBattelle Memorial Institute, USA, OH, Columbus

Project summary

Problem (Introduction)

The lack of medicinal drugs inducing selective damage of tumor cells is the basic challenge in the present-day chemotherapy of malignant tumors. An ideal antitumor drug is supposed to destroy cancer calls without any effect on the vital activity of normal cells. Extensive studies of targeted transport of cytotoxic drugs carried out in the past decades were largely focused on the delivery of antitumor drugs into malignant cells using antibodies against cancer-specific antigens. However, the use of immunoconjugates for the treatment of cancer patients was complicated by the risk of immune reactions.

Other possible objects for targeted delivery of antitumor drugs are specific receptors of growth factors or oncofetal proteins. In this case, the role of vector molecules (vehicles) can be played not only by antibodies to the corresponding receptors but also by their physiological ligands, viz., the growth factors and oncofetal proteins themselves. The latter have a number of advantages as vehicle molecules owing to a practically complete lack of immunogenic properties, high affinity for specific receptors and availability of cloned genes for the synthesis of recombinant proteins or peptides containing amino acid sequences necessary for the binding with corresponding receptors.

The successful solution of the problem of targeted delivery of medicamental preparations by receptor-mediated endocytosis is determined primarily by the choice of the vehicle. One of the best candidates for a vehicle effecting targeted delivery of medicinal drugs into tumor cells is the oncofetal protein (alpha-fetoprotein, AFP). AFP receptors are present on the surface of the overwhelming majority of tumor cells (independently of the tumor type) but are absent or present in insufficient amounts on the surface of normal cells.

The choice of a cytotoxic agent is also of crucial importance in the construction of targeted action drugs. This problem can be resolved by using two different approaches: (i) the use of already known chemotherapeutic agents and (ii) the use of highly efficient antitumor drugs whose clinical application is restricted due to their high nonspecific toxicity or for any other reason. The second approach makes use of new antibiotics of the enedyine series which manifest high antitumor activity but have not yet found wide acceptance in the clinical practice because of the side effects they produce. The use of AFP for targeted delivery of antisense oligonucleotides (ASON) into tumor cells is an alternative approach which also seems to hold great promise. However, until the present time the use of ASON was restricted due to their rapid digestion by endo- and exonucleases. This problem can be resolved through the synthesis of different modifications of oligonucleotides, among which phosphorothioate derivatives rank especially high. However, because of the high negative charge of ASON their entry into the cell is impeded, while their higher doses produce adverse side effects. The use of AFP as a vehicle for targeted delivery of ASON into tumor cells will help in: (i) reducing effective doses of ASON; (ii) circumventing the nonspecific toxicity of ASON and (iii) procuring highly specific effect of ASON.

State-of-the-art

Over the past six years, the researchers at RRCMDT were actively engaged in in-depth studies aimed at the synthesis of various types of AFP and its conjugates with a broad spectrum of antitumor drugs. These studies included the elaboration of methods for isolation of human AFP, the development of original procedures for the synthesis of miscellaneous AFP conjugates and their in vitro and in vivo testing. The high efficiency of the synthetic conjugates against tumor cells and their salient advantages over currently used antibiotics and free cytostatic agents were also demonstrated.

In addition, these studies showed the usefulness of AFP conjugates with esperamicin A1b (an antibiotic of the enedyine series) as a compound of choice. The use of these conjugates in vivo caused regression of malignant tumors in experimental animals: the average lifespan increased by more than 300% in comparison with the control group, while complete recovery was observed in 50% of animals. By virtue of its high antitumor activity, esperamycin can be used in very low doses; in mice with model tumors this dose was 6-20 mg per kg of body weight for one treatment course.

During the past few months, the Executive Organization has been engaged in extensive research into the synthesis of AFP conjugates with phosphorothioate derivatives of ASON.

Thus, a chemical synthesis of phosphorothioate derivatives of ASON to c-myc RNA whose activation is known to be critical for cell proliferation, was carried out Taking account of the fact that the majority of tumor cells are characterized by enhanced expression of this gene, an AFP conjugate with ASON to c-myc was synthesized and its high activity towards cells of various lines (Burkitt’s lymphoma, mammary carcinoma) was demonstrated.

Works on the Project will include the synthesis of AFP conjugates with ASON to mRNA of the following genes:


1. the protooncogen c-myc mRNA;
2. the bcl-2 gene encoding the antiapoptotic protein whose presence in tumor cells is usually associated with unfavorable prognoses in the treatment of cancer by conventional methods;
3. the multiple drug resistance gene (mdr1).

Besides, it is planned to synthesize AFP conjugates with ASON against the RNA component of telomerase. The latter is active in tumor cells; its inhibition results in the shortening of chromosomes and, ultimately, in cell death. All the conjugates and their variants will be tested in vitro and in vivo and the efficiency of inhibition of expression of the corresponding genes will be estimated using PCR and immunochemical methods.

Feasibility of the Project

RRCMDT disposes of all necessary equipment and highly skilled personnel for successful realization of the Project. The researchers have mastered the procedures for isolation of AFP and esperamicin, performed the synthesis of oligonucleotides and developed various synthetic procedures for preparation of AFP conjugates with esperamicin and ASON. The Center has a bank of cell lines and all necessary facilities for conducting animal studies. The results of preliminary testing of AFP conjugates and the pharmacokinetic data currently available permit one to anticipate a high probability of success in the implementation of works on the Project.

Final results

The works on the Project entail:


· synthesis of AFP conjugates with esperamicin;
· testing of AFPesperamicin conjugates on xenographic models of tumors and optimization of treatment schemes;
· study of feasibility of aerosolic application of AFPesperamicin conjugates;
· synthesis of AFP conjugates with ASON and selection of the most efficient variants of AFP ASON conjugates for further investigations;
· in vitro and in vivo testing of AFPASON conjugates on different models;
· study of mechanisms of cytotoxic effects of AFPASON conjugates and optimization of treatment schemes for animal studies;
· synthesis and studying of endocytosis of peptides containing AFP-receptor-binding site
· creation of the genetic construction containing AFP-related peptide and parnase, investigation of its antiproliferating activity in vitro.

Applicability of experimental results

Works on the Project are aimed at the synthesis and testing of an entirely new type of antitumor drugs which have no analogues in the world’s practice. The experimental results can be used for mass-scale production of antitumor preparations as well as for conducting clinical and preclinical trials. Further applications of the drugs may culminate in a successful recovery from cancer.


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