DEVELOPMENT OF A NEW SYNTHETIC SCHEME FOR VINYL DIETHERS AND SYNTHESIS OF PRODRUGS FOR TARGETED DELIVERY AND VISIBLE/NIR LIGHT-TRIGGERED RELEASE

Tumor specific drug delivery has become increasingly interesting in cancer therapy, as the use of chemotherapeutics is often limited due to severe side effects. Targeted chemotherapy for cancer treatment offers a great potential advantage in tumor treatment due to greater specificity of delivery, which leads to an increased dose of the cytotoxin delivered to the malignant cells relative to healthy cells in the rest of the body. There are two general directions in anticancer drug delivery that focus on achieving a high local drug concentration specifically in the cancerous tissue while reducing its uptake in healthy cells. i) Site-specific delivery can be achieved by conjugating the drug or coating the delivery vehicle (liposomes, micelles, etc.) with ligands or antibodies that target overexpressed receptors in the tumor tissue. This could also possibly be used to direct a drug away from the body sites that are sensitive to the toxic action of the carried drug (site avoidance). ii) By incorporating an active and site-specific release mechanism within the prodrug or delivery vehicle, it is possible to increase the release and therapeutic efficacy of the cytotoxic agent. A chemotherapeutic drug delivery system designed to combine these two principles (site–specific targeting and site-specific triggering) will fulfill Paul Ehrich’s vision of a magic bullet in the treatment of diseases there by overcoming the selectivity problems of conventional chemotherapy. The folate receptor (FR) is a potentially useful biological target for the management of human cancers. Owing to the overexpression of the FRs on the surface of malignant cells, conjugation of the cytotoxic agent to folic acid (FA) via suitable spacers has demonstrated the enhanced selective drug delivery to the tumor site. Furthermore the degree of over-expression has been found to correlate with the stage of tumor growth. Various FA-conjugated prodrugs and folate targeted delivery vehicles have been synthesized and are currently in preclinical and clinical trials. Biotin (vitamin B7) is also an essential cellular micronutrient responsible for various normal cellular functions, and its receptors are overexpressed in various cancer cell lines. It has been suggested that the sodium dependent multivitamin transporter (SMVT) is responsible for the uptake of biotin. It has also been indicated that there has been a higher expression of SMVT in several lung, renal, colon and breast cancer cell lines than FR. Several biotinylated anticancer agents have also been used in the selective delivery to cell lines overexpressing the SMVTs. Most of the folate and biotinylated conjugates are equipped with release mechanisms that rely on intrinsic activating agents such as small changes in temperature, pH differences, enzyme and an external activating tool such as light triggered release (light of shorter wavelengths). The use of light of longer wavelengths with better tissue penetration to active release of the drugs is warranted, which then will lead to the search for novel singlet oxygen photocleavable linkers. To address this issue, our lab has screened various olefins and identified vinyl diether linker as a potential singlet oxygen-mediated photocleavable linker that can be cleaved with a fast rate and in the presence of a photosensitizer (core-modified porphyrin). The reported synthetic schemes were for symmetric molecules with lengthy reaction steps and some reaction conditions not being functional group tolerant. Herein, the chemical synthesis (synthetic scheme) and kinetic studies of vinyl diether and some nitrogen and sulphur analogs will be described. The potential application of the vinyl diether linker to the synthesis of the biological active molecule was not achieved due to high instability of the linker in the presence of light. Hence we turned our attention to the alternative linker, the aminoacrylate linker, which could be synthesized with high yields and was functional group tolerant. A multifunctional drug delivery system constituted of the folate (or biotin) linked to the photosensitizer (phthalocyanine) by PEG and combretastatin A-4 linked to the photosensitizer by a photolabile linker (aminoacrylate) was designed and synthesized, and biological activity of six conjugates were determined. The conjugates were prepared through straightforward and versatile synthetic routes. The evaluation of cell specificity was examined using colon 26 cells that overexpress both the folate receptors and biotin receptors. Three of the conjugates (FA-PEG2K-PC-CA4, Biotin-PEG897-PC-CA4 and FA-PEG897-PC-CA4) exhibited high specificity in the in vitro test conducted and in vivo imaging using the colon 26 cells. Preliminary in vivo PDT indicated tumor shrinkage after the irradiation with two of these conjugates: FA-PEG2K-PC-CA4 and Biotin-PEG897-PC-CA4. (Data not included in the thesis) The ability of the multifunctional prodrugs of optical imaging and treatment by a combination of PDT and local chemotherapy could possibly lay the foundations for further development for the clinical management of FR and biotin receptor overexpressing tumors

Ubiquitin Receptor RPN13 Mediates the Inhibitory Interaction of Diphenyldihaloketones CLEFMA and EF24 With the 26S Proteasome

The proteasome is a validated target in drug discovery for diseases associated with unusual proteasomal activity. Here we report that two diphenyldihaloketones, CLEFMA and EF24, inhibit the peptidase activity of the 26S proteasome. The objective of this study was to investigate interaction of these compounds with the proteasome and identify a putative target within the protein components of the 26S proteasome. We employed standard fluorogenic peptide-based proteasome activity assay for trypsin-like, chymotrypsin-like, and caspase-like activities of human purified 26S proteasome in cell-free conditions. GFPu-1 and HUVEC cells were used as proteasome reporter cells. Direct binding studies used purified 19S, 20S, 26S, and recombinant RPN13-Pru for interaction with biotinylated analogs of CLEFMA and EF24. The reaction mixtures were subjected to horizontal gel electrophoresis, streptavidin-blotting, pull-down assays, and immunoblotting. The identity of the interacting protein was determined by 2D gel electrophoresis and LC-MS/MS. Drug affinity responsive target stability technique was utilized to examine if CLEFMA binding confers protection to RPN13 against thermolysin-catalyzed proteolysis. We found that trypsin-and chymotrypsin-like activities of the 26S proteasome were reduced significantly by both compounds. The compounds also reduced the proteolytic activity in GFPu-1 and HUVEC cells, resulting in accumulation of ubiquitinated proteins without affecting the autophagy process. From direct binding assays a 43 kDa protein in the 26S proteasome was found to be the interacting partner. This protein was identified by tandem mass spectroscopy as regulatory particle subunit 13 (RPN13), a ubiquitin receptor in the 19S regulatory particle. Furthermore, binding of CLEFMA to RPN13 did not protect latter from thermolysin-mediated proteolysis. Together, this study showed diphenyldihaloketones as potential proteasome inhibitors for treatment of diseases with perturbed proteasome function. The results also unraveled RPN13 as a unique target of CLEFMA and EF24. As a result, these compounds inhibit both trypsin-like and chymotrypsin-like proteasome activities

Surface Modification of Liposomes by a Lipopolymer Targeting Prostate Specific Membrane Antigen for Theranostic Delivery in Prostate Cancer

Prostate specific membrane antigen (PSMA) is a marker for diagnosis and targeted delivery of therapeutics to advanced/metastasized prostate cancer. We report a liposome-based system for theranostic delivery to PSMA-expressing (PSMA+) LNCaP cells. A lipopolymer (P3) comprising of PSMA ligand (PSMAL), polyethylene glycol (PEG2000), and palmitate was synthesized and post-inserted into the surface of preformed liposomes. These P3-liposomes were loaded with doxorubicin and radiolabeled with 99mTc radionuclide to study their theranostic characteristics. Differential expression of PSMA on LNCaP and PC3 cells was confirmed by immunoblotting as well as by uptake of PSMAL labeled with 18F radionuclide. We found that the uptake of 99mTc-labeled P3-liposomes by LNCaP cells was >3-fold higher than 99mTc-labeled Plain-liposomes; the amount of doxorubicin delivered to LNCaP cells was also found to be >3-fold higher by P3-liposomes. Cell-based cytotoxicity assay results showed that doxorubicin-loaded P3-liposomes were significantly more toxic to LNCaP cells (p < 0.05), but not to PSMA-negative PC3 cells. Compared to doxorubicin-loaded Plain-liposomes, the IC50 value of doxorubicin-loaded P3-liposomes was reduced by ~5-fold in LNCaP cells. Together, these results suggest that surface functionalization of liposomes with small PSMA-binding motifs, such as PSMAL, can provide a viable platform for specific delivery of theranostics to PSMA+ prostate cancer

Stability study on an anti-cancer drug 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid (CLEFMA) using a stability-indicating HPLC method

CLEFMA, 4-(3,5-bis(2-chlorobenzylidene)-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid, is a new chemical entity with anti-cancer and anti-inflammatory activities. Here, we report its stability in solution against stress conditions of exposure to acid/base, light, oxidant, high temperature, and plasma. The identity of the degradation products was ascertained by mass and proton nuclear magnetic resonance spectroscopy. To facilitate this study, we developed and validated a reverse phase high performance liquid chromatography method for detection of CLEFMA and its degradation. The method was linear over a range of 1–100 µg/mL; the accuracy and precision were within acceptable limits; it was stability-indicating as it successfully separated cis-/trans-isomers of CLEFMA as well as its degradation product. The major degradation product was produced from amide hydrolysis at maleic acid functionality caused by an acidic buffer, oxidant (3% hydrogen peroxide), or temperature stress (40–60 °C). The log k-pH profile showed that CLEFMA was most stable at neutral pH. In accelerated stability study we found that the shelf-life (T90%) of CLEFMA at 25 °C and 4 °C was 45 days and 220 days, respectively. Upon exposure to UV-light (365 nm), the normally prevalent trans-CLEFMA attained cis-configuration. This isomerization also involved the maleic acid moiety. CLEFMA was stable in plasma from which it could be efficiently extracted by an acetonitrile precipitation method. These results indicate that CLEFMA is sensitive to hydrolytic cleavage at its maleic acid moiety, and it is recommended that its samples should be stored under refrigerated and light-free conditions, and under inert environment

Far-Red Light Activatable, Multifunctional Prodrug for Fluorescence Optical Imaging and Combinational Treatment

We recently developed “photo-unclick chemistry”, a novel chemical tool involving the cleavage of aminoacrylate by singlet oxygen, and demonstrated its application to visible light-activatable prodrugs. In this study, we prepared an advanced multifunctional prodrug, Pc-(L-CA4)₂, composed of the fluorescent photosensitizer phthalocyanine (Pc), an SO-labile aminoacrylate linker (L), and a cytotoxic drug combretastatin A-4 (CA4). Pc-(L-CA4)₂ had reduced dark toxicity compared with CA4. However, once illuminated, it showed improved toxicity similar to CA4 and displayed bystander effects <i>in vitro</i>. We monitored the time-dependent distribution of Pc-(L-CA4)₂ using optical imaging with live mice. We also effectively ablated tumors by the illumination with far-red light to the mice, presumably through the combined effects of photodynamic therapy (PDT) and released chemotherapy drug, without any sign of acute systemic toxicity

Visible Light Controlled Release of Anticancer Drug through Double Activation of Prodrug

We designed and synthesized a novel double activatable prodrug system (drug–linker–deactivated photosensitizer), containing a photocleavable aminoacrylate-linker and a deactivated photosensitizer, to achieve the spatiotemporally controlled release of parent drugs using visible light. Three prodrugs of CA-4, SN-38, and coumarin were prepared to demonstrate the activation of deactivated photosensitizer by cellular esterase and the release of parent drugs by visible light (540 nm) via photounclick chemistry. Among these prodrugs, nontoxic coumarin prodrug was used to quantify the release of parent drug in live cells. About 99% coumarin was released from the coumarin prodrug after 24 h of incubation with MCF-7 cells followed by irradiation with low intensity visible light (8 mW/cm²) for 30 min. Less toxic prodrugs of CA-4 and SN-38 killed cancer cells as effectively as free drugs after the double activation

Folate-PEG Conjugates of a Far-Red Light-Activatable Paclitaxel Prodrug to Improve Selectivity toward Folate Receptor-Positive Cancer Cells

We recently demonstrated the far-red light-activatable prodrug of paclitaxel (PTX), Pc-(L-PTX)₂. Upon illumination with a 690 nm laser, Pc-(L-PTX)₂ showed combinational cell killing from rapid photodynamic therapy damage by singlet oxygen, followed by sustained chemotherapy effects from locally released PTX. However, its high lipophilicity (log <i>D</i>_7.4 > 3.1) caused aggregation in aqueous solutions and has nonselectivity toward cancer cells. To solve these important problems, we prepared folic acid (FA)-conjugated and photoactivatable prodrugs of PTX with a polyethylene glycol (PEG) spacer of various chain lengths: FA-PEG_<i>n</i>-Pc-L-PTX [<i>n</i> = 0 (0k, <b>5</b>), ∼23 (1k, <b>7a</b>), ∼45 (2k, <b>7b</b>), ∼80 (3.5k, <b>7c</b>), or ∼114 (5k, <b>7d</b>)]. The PEGylated prodrugs <b>7a–d</b> had a much improved hydrophilicity compared with the non-PEGylated prodrug, Pc-(L-PTX)₂. As the PEG length increased, the hydrophilicity of the prodrug increased (log <i>D</i>_7.4 values: 1.28, 0.09, −0.24, and −0.59 for 1k, 2k, 3.5k, and 5k PEG prodrugs, respectively). Fluorescence spectral data suggested that the PEGylated prodrugs had good solubility in the culture medium at lower concentrations (<1–2 μM), but showed fluorescence quenching due to limited solubility at higher concentrations (>2 μM). Dynamic light scattering indicated that all of the prodrugs formed nanosized particles in both phosphate-buffered saline and culture medium at a concentration of 5 μM. The PEG length affected both nonspecific and folate receptor (FR)-mediated uptake of the prodrugs. The enhanced cellular uptake was observed for the prodrugs with medium-sized PEGs (1k, 2k, or 3.5k) in FR-positive SKOV-3 cells, but not for the prodrugs with no PEG or with the longest PEG (5k), which suggests the optimal range of PEG length around 1k–3.5k for effective uptake of our prodrug system. Consistent with the cellular uptake pattern, medium-sized PEGylated prodrugs showed more potent phototoxic activity (IC_50s, ∼130 nM) than prodrugs with no PEG or the longest PEG (IC₅₀, ∼400 nM). In conclusion, we have developed far-red light-activatable prodrugs with improved water solubility and FR-targeting properties compared with the nontargeted prodrug

Site-Specific and Far-Red-Light-Activatable Prodrug of Combretastatin A‑4 Using Photo-Unclick Chemistry

Although tissue-penetrable light (red and NIR) has great potential for spatiotemporally controlled release of therapeutic agents, it has been hampered because of the lack of chemistry translating the photonic energy to the cleavage of a chemical bond. Recently, we discovered that an aminoacrylate group could be cleaved to release parent drugs after oxidation by SO and have called this “photo-unclick chemistry”. We demonstrate its application to far-red-light-activated prodrugs. A prodrug of combretastatin A-4 (CA4) was prepared, CMP–L–CA4, where CMP is dithiaporphyrin, a photosensitizer, and L is an aminoacrylate linker. Upon irradiation with 690 nm diode laser, the aminoacrylate linker of the prodrug was cleaved, rapidly releasing CA4 (>80% in 10 min) in CDCl₃. In tissue culture, it showed about a 6-fold increase in its IC₅₀ in MCF-7 after irradiation, most likely because of the released CA4. Most significantly, CMP–L–CA4 had better antitumor efficacy in vivo than its noncleavable (NC) analog, CMP–NCL–CA4. This is the first demonstration of the in vivo efficacy of the novel low-energy-light-activatable prodrug using the photo-unclick chemistry