Novel Taxoid-Based Tumor-Targeting Drug Conjugates

Abstract

A long standing problem of conventional cancer chemotherapy is the lack of tumor specificity. Tumor-targeting drug delivery systems have been explored to overcome this problem. These systems combine a powerful cytotoxic anticancer agent with a tumor-targeting molecule via a suitable linker to form highly efficacious drug-conjugates. These conjugates can deliver potent cytotoxic drugs specifically to tumors and cancer cells with minimal systemic toxicity. This article describes the design, development and application of novel taxoid-based tumor-targeting drug-conjugates, which possess excellent specificity and efficacy in vitro and in vivo.

INTRODUCTION

Traditional chemotherapy relies on the premise that rapidly proliferating tumor cells are more likely to be destroyed by cytotoxic agents than normal cells. However these cytotoxic agents have little or no specificity, which leads to systemic toxicity causing undesirable side effects such as hair loss, damage to liver, kidney and bone marrow. Various drug delivery systems have been investigated over the past few decades to address this problem. These systems take advantage of the morphological and physiological differences between the tumor and normal tissues. Rapidly growing cancer cells require nutrients and vitamins, hence overexpress tumor-specific receptors. These receptors can be used as targets to deliver cytotoxic agents specifically to cancer cells through receptor-mediated endocytosis. Moreover, the physiological characteristics of tumor and cancer cells can be exploited to selectively accumulate and release an anticancer agent inside them.¹

Tumor-targeting drug-conjugates typically consist of a tumor-targeting module (TTM) connected to an anticancer agent directly or through a suitable linker (Figure 1). This conjugate should be nontoxic and stable in blood circulation to minimize systemic toxicity and should be effectively internalized inside the target tumor cells. Upon internalization, the conjugate should efficiently release the anticancer agent without loss of potency.^{2, 3} We describe here a concise overview of the design, synthesis and biological evaluation of our novel taxoid-based tumor-targeting drug conjugates.

Figure 1.

Tumor-targeting modules

OMEGA-3 POLYUNSATURATED FATTY ACID CONJUGATES

Omega-3 polyunsaturated fatty acids (PUFA) such as linolenic acid (LNA), arachidonic acid (AA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are naturally-occurring compounds found in vegetable oils, cold-water fish and meat. DHA is a nutritional additive approved by the FDA in the US and is considered safe for humans.^{3, 4} Perfusion studies have shown that some PUFAs are taken up more rapidly by tumor cells than normal cells. In addition some omega-3 PUFAs have shown anticancer activity against various cancer cell lines in both clinical and preclinical trials. It has also been shown that PUFAs are readily incorporated into the lipid bilayer of tumor cells which disrupts the morphology of the cell and presumably influences the susceptibility of the tumor cells to anticancer agents.⁵ DHA-paclitaxel which is currently in Phase III clinical trials was shown to have better stability and efficacy than paclitaxel in some studies but would not be effective against multidrug-resistant (MDR) tumors that overexpress P-glycoprotein (Pgp).⁶ New generation taxoids exhibit 2–3 orders of magnitude better activity than paclitaxel against MDR cancer cell lines. Thus, PUFA conjugates which bear a new generation taxoid should be more efficacious than DHA-paclitaxel against drug-resistant cell lines.^{7, 8}

Novel PUFA-taxoid conjugates were synthesized and assayed in vivo for their efficacy against different drug-resistant and drug-sensitive human tumor xenografts in severe combined immune deficiency (SCID) mice. Several of these conjugates led to a complete regression of the tumor in all surviving mice with minimal systemic toxicity. For example, DHA-SB-T-1214 led to a complete regression of the highly drug-resistant DLD-1 colon tumor xenograft in 5 of 5 mice without recurrence of tumor growth for more than 190 days after treatment as well as appreciable systemic toxicity. DHA-SB-T-1213 and DHA-SB-T-1216 delayed the tumor growth of A121 ovarian tumor xenografts for more than 186 days and caused complete regression in all surviving mice ⁷. The excellent efficacy of the PUFA–taxoid conjugates against drug-resistant and drug-sensitive human tumor xenografts provides bright prospect in cancer chemotherapy and warrants further preclinical and clinical development of these conjugates.

MONOCLONAL ANTIBODY CONJUGATES

Cancer cells overexpress certain antigens on the cell surface and these tumor-specific antigens can be used as a biomarker to differentiate tumor tissues from normal tissues.^{1, 9, 10} Certain monoclonal antibodies (mAb) have high binding specificity to tumor-specific antigens and can be used as drug delivery vehicles to carry a payload of cytotoxic agents specifically to the tumor site. The mAb-drug conjugate is internalized upon binding to the tumor antigen via receptor-mediated endocytosis (RME) and the payload is released inside the cancer cell. Mylotarg^® was the first mAb–drug immunoconjugate approved by the FDA in 2000 for the treatment of acute myelogenous leukemia (AML) and several other mAb-drug conjugates are currently in clinical trials.^{1, 9}

The efficacy of mAb-drug immunoconjugates depends not only on the specificity of the mAb and the potency of the cytotoxic drug, but also on the linker which connects the mAb to the drug. We have successfully conjugated a highly cytotoxic C-10 methyldisulfanylpropanoyl taxoid to different immunoglobin G class mAbs, recognizing the epidermal growth factor receptor (EGFR), through a disulfide-containing linker.¹¹ Among these conjugates, immunoconjugate KS61–SB-T-12136 exhibited remarkable tumor-specific antitumor activity in vivo against A431 squamous tumor xenografts in SCID mice resulting in complete inhibition of tumor growth in all the treated mice with no noticeable toxicity and there was no trace of cancer cells at the end of the experiment.¹¹ The disulfide linker employed in this first-generation mAb-taxoid conjugates was found to be stable in blood plasma, but efficiently cleaved inside the tumor by glutathione or other intracellular thiols to release taxoid warhead, SB-T-12136H. However, the taxoid released through linker cleavage had a modification at C10 to introduce the disulfide linker moiety, which resulted in 8–10 times reduced potency compared to the parent taxoid.^{8, 11} Accordingly, mechanism-based second-generation disulfide linkers which would release unmodified parent taxoids were devised for more efficacious conjugation of taxoids to tumor-targeting modules.

NOVEL SELF-IMMOLATIVE DISULFIDE LINKERS

Second-generation mechanism-based bifunctional disulfide linkers can be generally used to connect a warhead to one end and a tumor-targeting module (TTM) to the other end. This self-immolative disulfide linker module can release a taxoid warhead efficiently inside cancer cells by taking advantage of 1,000 times higher concentration of glutathione in tumor as compared to that in blood plasma.¹² When the TTM module navigates the drug-conjugate to the target receptors on the tumor surface, the whole conjugate is internalized via RME. Then, an intracellular thiol-triggered cascade drug-release takes place through thiolactonization (Figure 2) and the released potent anticancer drug attacks its target protein, i.e., microtubules for taxoids.⁸ To promote the thiolactonization process, a phenyl moiety was strategically placed to direct the cleavage of the disulfide bond by intracellular thiol (e.g., glutathione), generating a thiophenolate or sulfhydrylphenyl species which attacks the ester linkage to the drug molecule (Figure 2). The validity of this self-immolative drug-release mechansim has been proven in a model system using fluorine-labeling and monitoring by ¹⁹F NMR spectroscopy² as well as in a real system with cancer cells using fluorescence-labeling and confocal fluorescence microscopy (CFM).⁸ These self-immolative disulfide linkers have been successfully incorporated to various tumor-targeting drug conjugates and their efficacy evaluated in cancer cells.⁸

Figure 2.

Drug-release mechanisms of self-immolative disulfide linker

VITAMINS AS TUMOR-TARGETING MODULES

Biotin and folic acid are essential vitamins involved in fatty acid metabolism and nucleotide synthesis, respectively. Cancer cells need these vitamins to maintain their rapid proliferation and thus overexpress these vitamin receptors on the cancer cell surface.^{13, 14} The strategic incorporation of either biotin or folic acid into a drug-conjugate, bearing a self-immolative disulfide linker coupled with a potent taxoid, ensures tumor-targeting delivery of the drug-conjugate and internalization via RME.

A biotin-linker-SB-T-1214 conjugate labeled with a fluorescence probe was designed and synthesized to study the internalization mechanism, drug release and drug binding to the target protein by means of CFM (Figure 3).⁸ The internalization mechanism of the drug-conjugate in L1210FR (leukemia) cells was confirmed to be RME based on its clear temperature dependence and its almost complete blockage by the pretreatment of the cells with excess biotin. The drug release mechanism was confirmed by using biotin-linker-coumarin conjugate, which was a fluorogenic probe, hence the observation of fluorescence provides evidence for the self-immolation of disulfide linker and release of free coumarin, which is the model for a taxoid. The drug release was further confirmed by the observation of the binding of a fluorescently labeled taxoid to microtubules (Figure 3). Because of a short incubation time for CFM analysis, glutathione ester was added to accelerate the cleavage of disulfide linkage in this experiment, which in turn confirmed the glutathione-triggered drug release.⁸

Figure 3.

CFM images of L1210FR cells treated with biotin-linker-SB-T-1214-fluorescein: (a) internalization of whole drug-conjugate; (b) binding of fluorescent taxoid to microtubule network after the drug release via self-immolation of the linker.

Furthermore, unlabeled biotin-linker-SB-T-1214 conjugate was assayed in vitro against L1210 (mouse lymphocytic leukemia), L1210FR (folate and biotin receptors overexpressed L1210 leukemia) and WI38 (normal human lung fibroblastoma) cells to examine the efficacy of the biomarker-specific targeting of the conjugate. The IC₅₀ values of the conjugate against L1210FR, L1210 and WI38 cell lines were 8.80 nM, 522 nM and 570 nM, respectively. The results clearly indicate the high biomarker-specificity of the drug-conjugate, which is consistent with the RME-based internalization and drug release observed by CFM and fluorocytometry using fluorescent probes.

SINGLE-WALLED CARBON NANOTUBE AS UNIQUE VEHICLE FOR TUMOR-TARGETING DRUG DELIVERY

Single-walled carbon nanotubes (SWNTs) have been attracting substantial interest in their potential as unique drug delivery vehicles.^{15, 16} Functionalized SWNTs can bear multiple units of TTM and drug molecules, which can penetrate the cell membrane and accumulate in the cytoplasm, wherein the drug molecules are released.^15–17 Thus, we designed and synthesized a novel biotin-SWNT-linker-taxoid conjugate for mass-delivery of payloads to cancer cells, wherein the enhancement of internalization via RME was also expected through multivalent binding of TTM to the vitamin receptors (Figure 4).¹⁸ Functionalization of SWNT begins with the oxidation of the SWNT with concentrated H₂SO₄ and HNO₃. The resulting carboxylic acid groups were converted to amines via condensation with diamines. Then, the amine termini were connected to biotin as TTM. The side wall of the SWNT was functionalized through 1,3-dipolar cycloaddition of azomethine generated in situ, bearing an amine group. These amines were conjugated to the linker-taxoid units.¹⁸

Figure 4.

CFM images of L1210FR cells treated with biotin-SWNT-SB-T-1214-fluorescein. (A) internalization of whole SWNT-conjugate; (b) binding of fluorescent taxoid to microtubule network after the drug release.

The internalization via RME as well as drug release and binding to the target protein (i.e., microtubules) of fluorescein-labeled biotin-SWNT-taxoid conjugate was investigated using CFM, and the results were fully consistent with those for the biotin-linker-taxoid(fluorescein) conjugate mentioned above (Figure 4). The cytotoxicity assay of the biotin-SWNT-taxoid(fluorescein) conjugate against L1210FR, L1210 and WI38 cell lines (IC₅₀ 0.36, >50, and >50 μg/mL respectively) has revealed excellent biomarker-specificity and substantially enhanced potency attributed to mass delivery of the taxoid warheads inside the cancer cells.¹⁸ assuring the merit of the “Trojan Horse” strategy in tumor-targeting drug delivery.

CLOSING REMARKS

Tumor-targeting drug-conjugates, which show bright prospect in cancer chemotherapy, have been successfully designed and constructed. These novel drug-conjugates consist of tumor-targeing modules (PUFAs, mAb’s and biotin), mechanism-based self-immolative disulfide linkers and warheads (new generation taxoids). The PUFA-taxoid conjugates and the mAb-taxoid conjugates have exhibited remarkable efficacy against human tumor xenografts in animal models with minimal systemic toxicity. CFM studies using fluorescent and fluorogenic probes have unambiguously confirmed the designed internalization of drug-conjugates via RME and drug release via glutathione-triggered self-immolation of the disulfide linker. The use of functionalized SWNTs as drug delivery vehicle for tumor-targeting drug conjugates bearing multiple-warhead and multiple-targeting modules has shown highly promising results on the benefit of mass-delivery of anticancer drug molecules to cancer cells with high specificity.