Tumor-Targeting Drug Delivery

The drug discovery program includes tumor-specific drug delivery systems, “guided molecular missiles” in the fight against cancer. Despite the significant progress in the development of cancer detection, prevention, surgery and therapy, there is still no common cure for this disease. In addition, the long-standing problem of chemotherapy is the lack of tumor-specific treatments. Traditional chemotherapy relies on the premise that rapidly proliferating cancer cells are more likely to be killed by a cytotoxic agent. In reality, however, cytotoxic agents have very little or no specificity, which leads to systemic toxicity, causing undesirable severe side effects. Therefore, various “molecularly targeted cancer therapies” have been developed for use in specific cancers, including tumor-targeting drug delivery (TTDD). In general, a TTDD system consists of a tumor recognition moiety and a cytotoxic warhead connected through a “smart” linker to form a conjugate. When a multi-functionalized nanomaterial is used as the vehicle, a “Trojan Horse” approach becomes possible for mass delivery of cytotoxic warheads to maximize the efficacy. The Ojima Laboratory has been making an excellent progress in the novel molecular approaches to the design and discovery of “guided molecular missiles” for tumor-targeting chemotherapy.

Taxoids as Potent Cytotoxic Agents for Tumor-Targeting Drug Delivery

One of the key problems with conventional chemotherapy is its toxicity: In addition to its beneficial characteristics of killing cancer cells, anticancer drugs also destroy healthy tissue resulting in systemic toxicity. Improvement in the potency and anti-MDR activities of anticancer drugs has provided efficient in treatment of aggressive and resistant tumors, although systemic toxicity persists and plays an important role in overall efficacy.

A Possible solution is Tumor-Targeting Drug Delivery which combines powerful anti-cancer compounds with tumor-targeting molecules. Paul Ehrlich (14 March 1854 – 20 August 1915) was a German scientist who won the 1908 Nobel Prize in Physiology or Medicine. He is noted for his work in hematology, immunology, and chemotherapy. Ehrlich predicted autoimmunity calling it "horror autotoxicus". He coined the term “chemotherapy" and popularized the concept of a “magic bullet". He is credited with the first empirical observation of the blood-brain barrier and the development of the first antibacterial drug in modern medicine.

ImmunoGen has developed technology known as “tumor-activiated prodrugs,” (TAPs), in which the anticancer agents are linked chemically to certain types of monoclonal antibodies. Those antibodies specifically bind to antigens that are known to be expressed predominantly on the cancer cell surface. The anit-cancer drug is inactive until it enters the tumor cell to which it is targeted to by the monoclonal antibody. In this way, the anticancer drugs are exclusively delivered to the cancer cells, leaving the normal cells intact. We have designed and synthesized novel taxanes used as cytotoxic agents in TAPs. Biological assays have since shown very impressive results.

S. Jaracz, J. Chen, L. V. Kuznetsova, and I. Ojima, “Recent Advances in Tumor-targeting Anticancer Drug Conjugates”, Bioorg. Med. Chem., 13, 5043-5054 (2005)

Tumor-targeting Molecules

S. Jaracz, J. Chen, L. V. Kuznetsova, and I. Ojima, "Recent Advances in Tumor-targeting Anticancer Drug Conjugates", Bioorg. Med. Chem., 13, 5043-5054 (2005).

Protein-Mediated TTM Internalization

Ojima, I. "Guided Molecular Missiles for Tumor-Targeting Chemotherapy: Case Studies Using the 2nd-Generation Taxoids as
Warheads", Acc. Chem. Res. 2008, 41, 108-119.

Chen, S., Zhao, X., Chen, J., Chen, J., Kuznetsova, L., Wong, S. S., & Ojima, I. "Mechanism-based tumor-targeting drug delivery system. Validation of efficient vitamin receptor-mediated endocytosis and drug release." Bioconjugate Chemistry 21, no. 5 (2010): 979-987.

Seitz, J. D., Ojima, I. Drug Delivery in Oncology: From Basic Research to Cancer Therapy. Ed. Kratz, F., et. al. In Press

First Generation mAb-Taxoid Drug Conjugate

Ojima, I. "Guided Molecular Missiles for Tumor-Targeting Chemotherapy: Case Studies Using the 2nd-Generation Taxoids as
Warheads", Acc. Chem. Res. 2008, 41, 108-119.

New Self-Immolative Linkers for Taxoid Conjugates

Ojima, I. "Use of fluorine in the medicinal chemistry and chemical biology of bioactive compounds—A case study on fluorinated taxane anticancer agents." Chem. Bio. Chem. 5, 628-635(2004).

Ojima, I. "Guided Molecular Missiles for Tumor-Targeting Chemotherapy: Case Studies Using the 2nd-Generation Taxoids as
Warheads", Acc. Chem. Res. 2008, 41, 108-119.

Metabolic Stability Assessment of Tumor-Targeted Drug Delivery Systems by ¹⁹F NMR

Novel biotin-linker-taxoid conjugates were synthesized with strategically incorporated fluorine substituents to observe disulfide linker cleavage by ¹⁹F NMR. Ring substitution of fluorine on the linker and formulation with excipients significantly altered the rates of disulfide exchange and thiolactonization. BLT-S-F6, with improved solubility and heightened sensitivity, demonstrated strong stability in blood plasma, but readily cleaved and released the taxoid when exposed to glutathione levels found in the cytosol.

Time-resolved ¹⁹F NMR observation of thiolactonization of [2.5 mM] probe 1 in 30% DMSO in D₂O beginning 1 hour after the addition of 6 equivalents of GSH at 25 °C at intervals of 15 min (128 scans/spectrum). This ¹⁹F NMR experiment disclosed that the "self-immolation" of the disulfide linker proceeded in two steps, generating the mechanistically anticipated thiolate 3-A as detectable transient species/intermediate, prior to thiolactonization to release a drug. It also suggests that the introduction of a fluorine para to a disulfide linkage had a profound effect on the rate of linker cleavage as well as thiolactonization. 

Left: ¹⁹F NMR spectral overlay of drug release of [200 μM] solution of BLT-S-F6 (2) in 86% blood plasma, 10% D₂O, 2% ethanol, and 2% Tween 80 at 37 °C without supplemental GSH at 0, 24, and 48 h (1024 scans/spectrum). The result implies that the putative half-life of drug conjugates and TTDDSs using this self-immolative linker unit and taxoids as warheads in this formulation would be longer than one week in human blood plasma. Right: Time-resolved ¹⁹F NMR observation of drug release of [200 μM] BLT-S-F6 (2) in 86% blood plasma, 2% ethanol, and 2% Tween 80 in D₂O 30 min after the addition of 100 equivalents of GSH at 37 °C at intervals of 1 h (1024 scans/spectrum). >98% of probe 2 disappeared and the corresponding amount of fluorotaxoid 4 appeared within 10 hour at 37 °C. The half-life of probe 2 with 100 equivalents of GSH in blood plasma was calculated to be ~3 h. This experiment allows us to estimate the half-life of this self-immolative disulfide linker in the cytosolic compartments following RME.

J. D. Seitz, J. G.Vineberg, L. Wei, J. F. Khan,B. Lichtenthal, C.-F. Lin and I. Ojima, "Design, synthesis and application of fluorine-labeled taxoids as 19F NMR probes for the metabolic stability assessment of tumor-targeted drug delivery systems" J. Fluor. Chem., 171 (2015), 148-161.

Fluorescence-Labeled Probes

Ojima, I. "Guided Molecular Missiles for Tumor-Targeting Chemotherapy: Case Studies Using the 2nd-Generation Taxoids as
Warheads", Acc. Chem. Res. 2008, 41, 108-119.

Chen, S., Zhao, X., Chen, J., Chen, J., Kuznetsova, L., Wong, S. S., & Ojima, I. "Mechanism-based tumor-targeting drug delivery system. Validation of efficient vitamin receptor-mediated endocytosis and drug release." Bioconjugate Chemistry 21, no. 5 (2010): 979-987.

Theranostic Vitamin−Linker−Taxoid Conjugates

Recently, we have designed and synthesized novel tumor-targeting theranostic conjugates 1 and 2, which consist of biotin as the tumor-targeting module, SB-T-1214 as the cytotoxic agent, a selfimmolative disulfide linker for drug release, 1,3,5-triazine as the splitter module, ethylene glycol oligomers to increase aqueous solubility, and either a fluorine-labeled prosthetic in 1 for potential ¹⁸F-PET imaging in vivo or an FITC tether in 2 for internalization and drug-release studies in vitro. This TTDDS exhibited very high specificity to BR+ cancer cells.

CFM images and flow cytometry analysis of L1210FR (BR+), MX-1 (BR+), ID8 (BR+), L1210 (BR−), and WI38 (BR−) cell lines after incubation with conjugate 2 (5 uM) (upper row) or probe 5 (5 uM) (lower row) at 37 oC for 3 h.

Vineberg, G., Wang, T., Zuniga, E., Ojima, I., Design, Synthesis, and Biological Evaluation of Theranostic Vitamin− Linker−Taxoid Conjugates, J. Med. Chem., ASAP 2015.

Drug Release

Ojima, I. "Guided Molecular Missiles for Tumor-Targeting Chemotherapy: Case Studies Using the 2nd-Generation Taxoids as
Warheads", Acc. Chem. Res. 2008, 41, 108-119.

Chen, S., Zhao, X., Chen, J., Chen, J., Kuznetsova, L., Wong, S. S., & Ojima, I. "Mechanism-based tumor-targeting drug delivery system. Validation of efficient vitamin receptor-mediated endocytosis and drug release." Bioconjugate chemistry 21, no. 5 (2010): 979-987.

Internalization and Drug Release of Biotin-CNT-Linker-Taxoid-Fluorescein

CFM images of L1210FR cells treated with biotin-CNT-taxoid-fluorescein incubated in the absence (A) and in the presence (B) of GSH Et ester. The latter shows the presence of a microtubule network, polymerized by taxoid, after the disulfide bonds had been cleaved by the GSH ethyl ester.

Advantages of CNT-based DDS:

• Amplification of tumor-targeting drug delivery via the “enhanced permeability and retention” (EPR) effect associated with nano-scale materials.

• Ability to exploit multivalent recognition of overexpressed receptors on the surfaces of the tumor cells.

• Increased drug loading capacity

• Capability of controlled drug release inside tumor cells.

 J. Chen S. Chen, X. Zhao, L. Kuznetsova, S. S. Wong, I. Ojima, Functionalized single-walled carbon nanotubes as rationally designed vehicles for tumor-targeted drug delivery. J. Am. Chem. Soc. 130, 16778-16785 (2008).

Asymmetric Bow-Tie Dendrimer-based (ABTD) Drug Delivery System

Dendrimer is a class of tree-like polymer with well-defined and shaped structure and good biocompatibility. It has been used as a macromolecular carrier to deliver anticancer drugs in tumor-targeted drug delivery. However, traditional dendrimer-based drug delivery systems directly decorate a dendrimer with different functionalities resulting in a mixture unable to be isolated to single pure components. To solve this problem, an advanced strategy is designed in Ojima Group by fully functionalizing different generations PAMAM dendrimer bearing cleavable cystamine core, followed by the cleavage of the fully functionalized dendrimers to generate two half dendrons. Then the two half dendrons could be re-coupled through a suitable linker to construct an ABTD versatile platform, which can be converted to a series of conjugates through an orthogonal click reaction. Each intermediate can be isolated as a single component with determined molecular weight. This ABTD system is a combination of active and passive targeting strategies. The tumor-targeting moiety (TTM) is recognized by its receptor overexpressed on the surface of cancer cells, and multiple TTMs can possess multi-binding effect to enhance the receptor-mediated endocytosis (RME) process for active targeting. Passive targeting replies on enhanced permeability and retention (EPR) effect which is typical for nano-sized particle. In general, this ABTD drug delivery system consists of sixteen polyethylene glycolylated (PEGylated) TTM on generation 3 half dendron, coupling through a PEGylated bis(maleicimido) linker to generation 1 half dendron with four PEGylated cytotoxic warheads each with/without a fluorescent probe and connected through a self-immolative smart disulfide linker. Excellent progress have been made to prove ABTD system is highly efficient for delivering anticancer drugs in tumor-targeted drug delivery.

Synthetic Strategy for Construction of Asymmetric Bow-Tie Dendrimer (ABTD) Platform: Instead of attaching different functionalities on one dendrimer molecule resulting in a mixture, in Ojima group we developed the synthetic strategy to generate dendrimer-based conjugate as determined and repeatable single component macromolecule. Different generations of dendrimer bearing cleavable core are fully functionalized by one type but different functionality, and cleaved to form half dendrons, followed by re-coupled through a suitable linker to construct a dendrimer-based platform. And we have developed purification method for isolating each macromolecular intermediate as single component with good purity.

Model Structure of Asymmetric Bow-Tie Dendrimer (ABTD) Conjugates: A versatile asymmetric bow-tie dendrimer (ABTD) platform is designed and a series of ABTD conjugates has been successfully synthesized. A PEGylated biotin (tumor-targeting moiety in blue) is functionalized on each arm of half dendron of generation 3 dendrimer, a PEGylated linker-taxoid (warhead in red) with or without fluorescent probe (imaging probe in green) is functionalized on each arm of half dendron of generation 1 dendrimer, and connected through a PEGylated bis(maleimido) linker to afford a novel nano-sized drug delivery system. This dendrimer-based system has shown a multi-binding effect due to the multiple tumor-targeting modules and high efficiency for delivering warhead in tumor-targeted drug delivery.

Associated Publications:

1. “Design and synthesis of tumor-targeting theranostic drug conjugates for SPECT and PET imaging studies”, Tao Wang, Jacob G. Vineberg, Tadashi Honda and Iwao Ojima, Bioorg. Chem. 76, 458-467 (2018). PMID: 29287255 

2. "Design, Synthesis and Biological Evaluations of Asymmetric Bow-Tie PAMAM Dendrimer-based Conjugates for Tumor-Targeted Drug Delivery", Tao Wang,Yaozhong Zhang, Longfei Wei, Yuhan G. Teng,Tadashi Honda and Iwao Ojima, ACS Omega, 3(4), 3717–3736 (2018).PMC5928494

3. “Nanoemulsion Formulation of a Novel Taxoid Prodrug SBT-1214 Conjugated with Omega-3 Fatty Acid Inhibits Prostate Cancer Stem Cell-Induced Tumor Growth”, G. Ahmad, R. El-Sadda, G. Botchkina, I. Ojima, J. Egan, and M. M. Amiji, Cancer Lett., 406, 71-80 (2017). PMC5591776

4. “High Capacity Poly(2-oxazoline) Micelles for 3rd  Generation Taxoids: preparation, in vitro and in vivo evaluation”, Z. He, A. Schulz, X. Wan, J. Seitz, H. Bludau, D. B. Darr, C. M. Perou, R. Jordan, I. Ojima, A. V. Kabanov, R. Luxenhofer, J. Control. Release 208, 67-75 (2015). PMID: 25725361 

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