Moses Bio

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/cm(2)) for 30 min. Less toxic prodrugs of CA-4 and SN-38 killed cancer cells as effectively as free drugs after the double activation.

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 in vitro. 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.

Far-Red Light-Activatable Prodrug of Paclitaxel for the Combined Effects of Photodynamic Therapy and Site-Specific Paclitaxel Chemotherapy

Paclitaxel (PTX) is one of the most useful chemotherapeutic agents approved for several cancers, including ovarian, breast, pancreatic, and nonsmall cell lung cancer. However, it causes systemic side effects when administered parenterally. Photodynamic therapy (PDT) is a new strategy for treating local cancers using light and photosensitizer. Unfortunately, PDT is often followed by recurrence due to incomplete ablation of tumors. To overcome these problems, we prepared the far-red light-activatable prodrug of PTX by conjugating photosensitizer via singlet oxygen-cleavable aminoacrylate linker. Tubulin polymerization enhancement and cytotoxicity of prodrugs were dramatically reduced. However, once illuminated with far-red light, the prodrug effectively killed SKOV-3 ovarian cancer cells through the combined effects of PDT and locally released PTX. Ours is the first PTX prodrug that can be activated by singlet oxygen using tissue penetrable and clinically useful far-red light, which kills the cancer cells through the combined effects of PDT and site-specific PTX chemotherapy.

Synthesis and Singlet Oxygen Reactivity of 1,2‐Diaryloxyethenes and Selected Sulfur and Nitrogen Analogs

1,2‐Diaryloxyethene has recently been proposed as a linker in singlet oxygen‐mediated drug release. Even though 1,2‐diaryloxyethenes look very simple, their synthesis was not an easy task. Previous methods are limited to symmetric molecules, lengthy step and low yield. We report on a facile synthetic method not only for 1,2‐diaryloxyethenes but also their sulfur and nitrogen analogs in yields ranging from 40 to 90% with more than 90% purity at the vinylation reaction.

Photodynamic therapy via FRET following bioorthogonal click reaction in cancer cells

Longer wavelength light (650-800nm) is desired to treat large tumors in photodynamic therapy (PDT). However, shorter wavelength light is needed in PDT for thin tumors, not to cause undesirable local side effects. We proposed a strategy for stepwise optical imaging and PDT using a bioorthogonal click chemistry and fluorescence resonance energy transfer (FRET). We prepared azidyl rhodamine (Rh-N3, clickable FD) and cyclooctynyl phthalocyanine [Pc-(DIBAC), clickable PS], with which, here, we demonstrate that the non-catalytic click chemistry is rapid and efficient in cancer cells and FRET from a fluorescence dye (FD) to a photosensitizer (PS) is sufficient to generate enough singlet oxygen killing cancer cells by using shorter wavelength light.

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 D7.4 > 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-PEGn-Pc-L-PTX [n = 0 (0k, 5), ∼23 (1k, 7a), ∼45 (2k, 7b), ∼80 (3.5k, 7c), or ∼114 (5k, 7d)]. The PEGylated prodrugs 7a–d 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 D7.4 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 (IC50s, ∼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.

Abstract 1361: Progress in light activatable prodrug for the combinational treatment of PDT and site-specific chemotherapy: paclitaxel prodrugs

We developed the photoactivatable prodrug of Paclitaxel (PTX) for the combinational treatment of chemo and photodynamic therapy (PDT). PTX causes dose-limiting side effects as other anticancer drugs when administered systemically. On the other hand, PDT suffers from incomplete ablation and subsequent reoccurrence in part due to the short half-life and poor diffusion rate of singlet oxygen. We prepared a conjugate of PTX with phthalocyanine via a singlet oxygen cleavable linker, as a unique prodrug of PTX, to overcome the problems of PDT and systemic chemotherapy. The PTX prodrug was evaluated for tubulin polymerization, the release rate of PTX from prodrug upon illumination at 690 nm, stability in complete media, and the combination effect in killing ovarian cancer cells in vitro (SKOV-3). The PTX prodrug did not enhance the tubulin polymerization unlike PTX. While it was stable in the media under dark, it rapidly released PTX upon illumination with far-red light: > 90% release in 30 min. The prodrug showed much lower dark toxicity compared to PTX. When illuminated with 690 nm at 5.6 mW/cm2, the prodrug showed very potent phototoxicity: IC50 = 3.9 nM, through the combinational effect of PDT and PTX. In conclusion, we found that the PTX prodrug have desired properties as light-activatable prodrug expressing the combinational effect of PDT and local PTX chemotherapy. Animal study is underway to evaluate the antitumor effect of the PTX prodrug. Citation Format: Pritam Thapa, Mengjie Li, Moses Bio, Pallavi Rajaputra, Yajing Sun, Sukyung Woo, Youngjae You. Progress in light activatable prodrug for the combinational treatment of PDT and site-specific chemotherapy: paclitaxel prodrugs. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1361.

Abstract 1671: Local release of combretastatin A-4 from NIR-light activatable prodrugs overcomes areal and temporal limitations of photodynamic therapy

A unique prodrug strategy for treating localized cancers, in which NIR light-illuminated prodrug effectively ablates tumors through the combined effects of photodynamic therapy (i.e., singlet oxygen [SO]) and locally released anticancer drugs has been proposed. Due to short distance of action ( Citation Format: Pallavi Rajaputra, Moses Bio, Gregory Nkepang, Pritam Thapa, Sukyung Woo, Youngjae You. Local release of combretastatin A-4 from NIR-light activatable prodrugs overcomes areal and temporal limitations of photodynamic therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1671.

Abstract 4394: Selective accumulation and specific tumor damage by folate receptor-targeted CA-4 prodrug as part of a combination of photodynamic therapy and site-specific chemotherapy

A non-invasive or minimally invasive tumor ablation regimen is an attractive tool with which to control tumors. This approach is complementary to primary treatment options without causing systemic side effects or severe physical burdens from the treatment itself. Clinically approved photodynamic therapy (PDT) is one such regimen. In PDT, photosensitizers are activated by visible and near IR light to destroy tumors. However, the therapeutic efficacy of PDT is limited due to the spatial and temporal restrictions of the effector of PDT, singlet oxygen. We have been developing a new prodrug strategy that can overcome these limits by using a unique combination of PDT and site-specific chemotherapy. The prodrug is composed of photosensitizer and anticancer drug via a singlet oxygen-cleavable linker. Upon illumination, the prodrugs cause PDT damage and simultaneously release anticancer drugs only at the illuminated target area. In our previous publications, we reported 1) the design and synthesis of prodrugs of combretastatin A-4, 2) visible/ near IR light-controlled release of the drug from the prodrug, 3) bystander effects from the released anticancer drug, and 4) the superior antitumor effect of the prodrug compared to a simple combination of PDT and chemotherapy. Recently, a more advanced CA-4 prodrug was developed to achieve selective delivery to tumors via folate receptors (FRs) overexpressed in numerous cancer cell lines. The targeted prodrug showed FR-mediated uptake to FR-expressing cancer cells (colon 26 cells) in vitro and to FR-expressing tumors in vivo. Notably, the targeted CA-4 prodrug effectively ablated the tumors without causing collateral damage to the surrounding skin. Citation Format: Nkepang Gregory, Moses Bio, Pallavi Rajaptura, Samuel G. Awuah, Youngjae You. Selective accumulation and specific tumor damage by folate receptor-targeted CA-4 prodrug as part of a combination of photodynamic therapy and site-specific chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4394. doi:10.1158/1538-7445.AM2015-4394