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Visible Light Controlled Combination Strategy to Treat Non-muscle Invasive Bladder Cancer.

Record Type:	Language materials, manuscript : Monograph/item
Title/Author:	Visible Light Controlled Combination Strategy to Treat Non-muscle Invasive Bladder Cancer./
Author:	Rahman, Kazi Md. Mahabubur.
Description:	1 online resource (254 pages)
Notes:	Source: Dissertations Abstracts International, Volume: 85-03, Section: A.
Contained By:	Dissertations Abstracts International85-03A.
Subject:	Pharmaceutical sciences. -
Online resource:	click for full text (PQDT)
ISBN:	9798380349123

Visible Light Controlled Combination Strategy to Treat Non-muscle Invasive Bladder Cancer.
Rahman, Kazi Md. Mahabubur.

Visible Light Controlled Combination Strategy to Treat Non-muscle Invasive Bladder Cancer. - 1 online resource (254 pages)

Source: Dissertations Abstracts International, Volume: 85-03, Section: A.

Thesis (Ph.D.)--State University of New York at Buffalo, 2023.

Includes bibliographical references

In the USA, bladder cancer (BC) is the fourth most common cancer among men and the ninth most prevalent among women, claiming more than 17,000 lives annually. Of the 82,000 new bladder cancer cases diagnosed each year, more than 70% (> 57,000 patients) are diagnosed with non-muscle invasive bladder cancer (NMIBC). During this stage, the tumor is superficial or has only spread to the inner lining of the bladder without involving the surrounding muscle layer. It is possible to save a patient's bladder or life if a tumor is controlled at this stage in order to avoid its progression to muscle-invasive disease. Transurethral resection of the bladder tumor and subsequent instillation of BCG (Bacille Calmette-Guerin) or chemotherapy are standard treatments for NMIBC. In BC, both chemotherapy and BCG are associated with significant side effects, including bladder contraction, bleeding, and recurrences within the first five years. Because bladder cancer has a high recurrence rate, regular cystoscopies are crucial, making it one of the most expensive cancers to treat. Therefore, there is a great need for more effective treatment with fewer adverse effects.Photodynamic therapy (PDT) uses a photosensitizer drug and visible light to kill cancer cells by inducing necrosis and apoptosis through the generation of singlet oxygen species. PDT is already approved for several types of cancer and is in clinical trials for bladder cancer (PDT in BC is discussed in Chapter 2). Hexaminolevulinate (HAL) was approved by the FDA for the diagnosis of BC due to its ability to produce fluorescent protoporphyrin IX (PpIX) in cancer cells when it is administered to the bladder. Because it is produced preferentially in tumors, PpIX can be a good photosensitizer for BC. PpIX-PDT was successfully used in the clinic for BC without serious complications, but it fell short in efficacy. The purpose of this study was to improve PpIX-PDT efficacy by combining it with prodrugs that can be activated by the singlet oxygen generated by PpIX-PDT. Upon HAL instillation, PpIX is produced in the mitochondria of cancer cells and eventually diffuses out into the cytoplasm or the extracellular space, so both mitochondria-targeted and non-targeted prodrugs were developed and evaluated. Due to the strict bladder barrier, we have developed prodrugs with a range of physicochemical properties (molecular size and aqueous solubility). Prodrugs were selected from either clinically approved or in-clinical-trial drugs (e.g., paclitaxel (PTX), Combrestatin A4 (CA4), SN-38, and mitomycin C (MMC)). Our prodrugs have all been tested in vitro and some have been tested in vivo in an orthotopic rat bladder tumor model. The in vitro studies were conducted using AY-27, a rat bladder cancer cell line, while the in vivo studies were performed using the same cell line in a rat orthotopic bladder NMIBC model.Because PpIX is available outside of the mitochondria, non-targeted prodrugs have been developed to improve prodrug diffusion by linking the prodrug to small protecting groups such as methyl (Mt), morpholine (MP), and piperidine (PP). PP-L-PTX (piperidine-linked paclitaxel) and MP-L-PTX (morpholine-linked paclitaxel) were synthesized by linking paclitaxel with piperidine and morpholine, respectively. MP-L-CA4 (morpholine-linked CA4) and MP-L-SN-38 (morpholine-linked SN-38) were developed by linking morpholine to Combrestatin A4 (CA4) and SN-38, respectively. Mt-L-MMC (methyl-linked mitomycin C) was developed by linking methyl groups to mitomycin C. A 532 nm laser was used to test the photo and dark toxicity of all the prodrugs in 2D culture using AY-27 cells. To a concentration of 1 µM, none of the prodrugs showed dark toxicity. A combination of prodrugs and PpIX-PDT (0.5 mM HAL-generated PpIX) resulted in 95% cell killing, while PpIX-PDT alone generated only about 30% cell killing. Phototoxicity of the prodrugs was also evaluated in the 3D spheroids model and up to about 95% cell killing was observed with 5 µM concentration of prodrugs combined with 0.5 mM HAL. As a whole, MP-L-CA4, MP-L-SN-38, and Mt-L-MMC exhibited significantly greater efficacy than PP-L-PTX and MP-L-PTX. A similar effect was also observed by imaging live and dead spheroids with Calcein AM and propidium iodide, respectively.The efficacy of the prodrug and the PpIX-PDT combination was determined in vivo in an orthotopic NMIBC model in rats (Chapter 3). To develop an NMIBC tumor, 1 million AY-27 cells were instilled in the rat bladder after acid-base conditioning. The rats were treated with PpIX-PDT and PpIX-PDT + prodrug combination on day 7 after tumor instillation. In vivo studies used one mitochondria-targeted prodrug (Rh-L-PTX, rhodamine-linked paclitaxel, previously developed) and one non-targeted prodrug (Mt-L-MMC). A light dose-determining study with PpIX-PDT alone helped to determine that 50 mW for 20 minutes of illumination with a green 532 nm laser would be appropriate in a combination study. PpIX-PDT + prodrug combinations could achieve significantly better antitumor efficacy compared to PpIX-PDT alone. Dark toxicity and phototoxicity efficacy were confirmed using quantitative tumor data analysis (e.g., comparing tumor-covered area, tumor volume, etc.). Based on the histological evaluation, no significant bladder toxicity was associated with the PpIX-PDT and combination group bladders. Despite the significant efficacy of combination therapy, we wanted to further improve drug penetration into the bladder. We, therefore, developed a mitochondria-targeted prodrug using a much smaller parent drug than paclitaxel. In comparison to Rh-L-PTX, SN-38 was linked with rhodamine, which decreased its aqueous solubility and molecular size (1482 vs 985 and 6.3 vs 3.2 in gram/mole and ClogP, respectively). We examined Rh-L-SN-38 for its selectivity for cancer cells in vitro and in vivo (Chapter 4). In vitro, selectivity was compared between BdEC (human bladder epithelial cell line) and RT112 (human NMIBC cell line). According to fluorescence microscopy, Rh-L-SN-38 targets cancer cells more than normal cells, with maximum fluorescence intensity of 60 versus 40 in RT112 vs BdEC after 2 hr. A 4-fold increase in intracellular concentration was found in cancer cells compared with healthy epithelial cells, with intracellular concentrations of 12.6 mM and 3.3 mM in RT112 and BdEC cells, respectively, after 2 hr of prodrug incubation. The in vivo selectivity of the drug was assessed using an orthotopic rat bladder tumor model developed using the AY-27 cell line. It was found that both Rh-L-PTX and Rh-L-SN-38 could achieve a significantly higher drug diffusion rate (19 - 34 times) in the tumor as compared to the non-tumor area. Additionally, PpIX was found to form ~3 times more in the tumor area than in the non-tumor area. Overall, these results show that NMIBC can be treated with high selectivity when PpIX-PDT and targeted prodrugs are combined.To prevent bladder muscle damage from PDT (e.g., Photofrin PDT damaged the bladder when used with 630 nm), we previously used a green laser (532 nm) with PpIX. However, 532 nm displays some limitations in bladder tissue penetration (<1 mm), and light intensity can be reduced by the instillation medium. These could be avoided by using a 635 nm laser (tissue penetration depth ~ 1 cm) and its intensity is less affected by the instillation media. With HAL instillation, PpIX is mainly produced (~ 3 times more) in the tumor tissue and can also be excited with 635 nm light. Hence, we examined how green (532 nm) and red (635 nm) lasers differ in their efficacy and safety. AY-27 cells were used to evaluate in vitro efficacy, and the rat orthotopic NMIBC model to evaluate in vivo efficacy. Using 635 nm and 532 nm lasers, PpIX-PDT IC50 was found to be 18 and 22 mW/cm2, respectively. For quantitative analysis of the in vivo effectiveness of the treatment, we have developed a simple tool to quickly detect bladder tumors using fluorescence imaging using a portable LED light and emission filter attached to a smartphone. We performed an in vivo dose-dependent efficacy comparison by illuminating both lasers for 20 minutes at 50, 70, and 100 mW total power. The red laser was found to be more effective than the green laser in PpIX-PDT in vitro. The PpIX-PDT + prodrug combination has also been evaluated in vivo using a red laser at 50 mW for 20 minutes.

Electronic reproduction.
Ann Arbor, Mich. :
ProQuest,
2024

Mode of access: World Wide Web

ISBN: 9798380349123Subjects--Topical Terms:

1180569
Pharmaceutical sciences.
Subjects--Index Terms:

Bladder cancerIndex Terms--Genre/Form:

554714
Electronic books.

Visible Light Controlled Combination Strategy to Treat Non-muscle Invasive Bladder Cancer.
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520 $a In the USA, bladder cancer (BC) is the fourth most common cancer among men and the ninth most prevalent among women, claiming more than 17,000 lives annually. Of the 82,000 new bladder cancer cases diagnosed each year, more than 70% (> 57,000 patients) are diagnosed with non-muscle invasive bladder cancer (NMIBC). During this stage, the tumor is superficial or has only spread to the inner lining of the bladder without involving the surrounding muscle layer. It is possible to save a patient's bladder or life if a tumor is controlled at this stage in order to avoid its progression to muscle-invasive disease. Transurethral resection of the bladder tumor and subsequent instillation of BCG (Bacille Calmette-Guerin) or chemotherapy are standard treatments for NMIBC. In BC, both chemotherapy and BCG are associated with significant side effects, including bladder contraction, bleeding, and recurrences within the first five years. Because bladder cancer has a high recurrence rate, regular cystoscopies are crucial, making it one of the most expensive cancers to treat. Therefore, there is a great need for more effective treatment with fewer adverse effects.Photodynamic therapy (PDT) uses a photosensitizer drug and visible light to kill cancer cells by inducing necrosis and apoptosis through the generation of singlet oxygen species. PDT is already approved for several types of cancer and is in clinical trials for bladder cancer (PDT in BC is discussed in Chapter 2). Hexaminolevulinate (HAL) was approved by the FDA for the diagnosis of BC due to its ability to produce fluorescent protoporphyrin IX (PpIX) in cancer cells when it is administered to the bladder. Because it is produced preferentially in tumors, PpIX can be a good photosensitizer for BC. PpIX-PDT was successfully used in the clinic for BC without serious complications, but it fell short in efficacy. The purpose of this study was to improve PpIX-PDT efficacy by combining it with prodrugs that can be activated by the singlet oxygen generated by PpIX-PDT. Upon HAL instillation, PpIX is produced in the mitochondria of cancer cells and eventually diffuses out into the cytoplasm or the extracellular space, so both mitochondria-targeted and non-targeted prodrugs were developed and evaluated. Due to the strict bladder barrier, we have developed prodrugs with a range of physicochemical properties (molecular size and aqueous solubility). Prodrugs were selected from either clinically approved or in-clinical-trial drugs (e.g., paclitaxel (PTX), Combrestatin A4 (CA4), SN-38, and mitomycin C (MMC)). Our prodrugs have all been tested in vitro and some have been tested in vivo in an orthotopic rat bladder tumor model. The in vitro studies were conducted using AY-27, a rat bladder cancer cell line, while the in vivo studies were performed using the same cell line in a rat orthotopic bladder NMIBC model.Because PpIX is available outside of the mitochondria, non-targeted prodrugs have been developed to improve prodrug diffusion by linking the prodrug to small protecting groups such as methyl (Mt), morpholine (MP), and piperidine (PP). PP-L-PTX (piperidine-linked paclitaxel) and MP-L-PTX (morpholine-linked paclitaxel) were synthesized by linking paclitaxel with piperidine and morpholine, respectively. MP-L-CA4 (morpholine-linked CA4) and MP-L-SN-38 (morpholine-linked SN-38) were developed by linking morpholine to Combrestatin A4 (CA4) and SN-38, respectively. Mt-L-MMC (methyl-linked mitomycin C) was developed by linking methyl groups to mitomycin C. A 532 nm laser was used to test the photo and dark toxicity of all the prodrugs in 2D culture using AY-27 cells. To a concentration of 1 µM, none of the prodrugs showed dark toxicity. A combination of prodrugs and PpIX-PDT (0.5 mM HAL-generated PpIX) resulted in 95% cell killing, while PpIX-PDT alone generated only about 30% cell killing. Phototoxicity of the prodrugs was also evaluated in the 3D spheroids model and up to about 95% cell killing was observed with 5 µM concentration of prodrugs combined with 0.5 mM HAL. As a whole, MP-L-CA4, MP-L-SN-38, and Mt-L-MMC exhibited significantly greater efficacy than PP-L-PTX and MP-L-PTX. A similar effect was also observed by imaging live and dead spheroids with Calcein AM and propidium iodide, respectively.The efficacy of the prodrug and the PpIX-PDT combination was determined in vivo in an orthotopic NMIBC model in rats (Chapter 3). To develop an NMIBC tumor, 1 million AY-27 cells were instilled in the rat bladder after acid-base conditioning. The rats were treated with PpIX-PDT and PpIX-PDT + prodrug combination on day 7 after tumor instillation. In vivo studies used one mitochondria-targeted prodrug (Rh-L-PTX, rhodamine-linked paclitaxel, previously developed) and one non-targeted prodrug (Mt-L-MMC). A light dose-determining study with PpIX-PDT alone helped to determine that 50 mW for 20 minutes of illumination with a green 532 nm laser would be appropriate in a combination study. PpIX-PDT + prodrug combinations could achieve significantly better antitumor efficacy compared to PpIX-PDT alone. Dark toxicity and phototoxicity efficacy were confirmed using quantitative tumor data analysis (e.g., comparing tumor-covered area, tumor volume, etc.). Based on the histological evaluation, no significant bladder toxicity was associated with the PpIX-PDT and combination group bladders. Despite the significant efficacy of combination therapy, we wanted to further improve drug penetration into the bladder. We, therefore, developed a mitochondria-targeted prodrug using a much smaller parent drug than paclitaxel. In comparison to Rh-L-PTX, SN-38 was linked with rhodamine, which decreased its aqueous solubility and molecular size (1482 vs 985 and 6.3 vs 3.2 in gram/mole and ClogP, respectively). We examined Rh-L-SN-38 for its selectivity for cancer cells in vitro and in vivo (Chapter 4). In vitro, selectivity was compared between BdEC (human bladder epithelial cell line) and RT112 (human NMIBC cell line). According to fluorescence microscopy, Rh-L-SN-38 targets cancer cells more than normal cells, with maximum fluorescence intensity of 60 versus 40 in RT112 vs BdEC after 2 hr. A 4-fold increase in intracellular concentration was found in cancer cells compared with healthy epithelial cells, with intracellular concentrations of 12.6 mM and 3.3 mM in RT112 and BdEC cells, respectively, after 2 hr of prodrug incubation. The in vivo selectivity of the drug was assessed using an orthotopic rat bladder tumor model developed using the AY-27 cell line. It was found that both Rh-L-PTX and Rh-L-SN-38 could achieve a significantly higher drug diffusion rate (19 - 34 times) in the tumor as compared to the non-tumor area. Additionally, PpIX was found to form ~3 times more in the tumor area than in the non-tumor area. Overall, these results show that NMIBC can be treated with high selectivity when PpIX-PDT and targeted prodrugs are combined.To prevent bladder muscle damage from PDT (e.g., Photofrin PDT damaged the bladder when used with 630 nm), we previously used a green laser (532 nm) with PpIX. However, 532 nm displays some limitations in bladder tissue penetration (<1 mm), and light intensity can be reduced by the instillation medium. These could be avoided by using a 635 nm laser (tissue penetration depth ~ 1 cm) and its intensity is less affected by the instillation media. With HAL instillation, PpIX is mainly produced (~ 3 times more) in the tumor tissue and can also be excited with 635 nm light. Hence, we examined how green (532 nm) and red (635 nm) lasers differ in their efficacy and safety. AY-27 cells were used to evaluate in vitro efficacy, and the rat orthotopic NMIBC model to evaluate in vivo efficacy. Using 635 nm and 532 nm lasers, PpIX-PDT IC50 was found to be 18 and 22 mW/cm2, respectively. For quantitative analysis of the in vivo effectiveness of the treatment, we have developed a simple tool to quickly detect bladder tumors using fluorescence imaging using a portable LED light and emission filter attached to a smartphone. We performed an in vivo dose-dependent efficacy comparison by illuminating both lasers for 20 minutes at 50, 70, and 100 mW total power. The red laser was found to be more effective than the green laser in PpIX-PDT in vitro. The PpIX-PDT + prodrug combination has also been evaluated in vivo using a red laser at 50 mW for 20 minutes.
520 $a Rh-L-SN-38 and Mt-L-MMC have been used as targeted and non-targeted prodrugs in combination with PpIX-PDT. A combination of Mt-L-MMC and PpIX-PDT could generate significantly better efficacy than PpIX-PDT alone in terms of tumor volume, tumor-covered bladder area, fraction of bladder covered by tumor, and bladder weight per unit area. Lastly, we evaluated the toxicity of PpIX-PDT alone and in combination with prodrugs. The histology study of the rat bladder found no significant injury or toxicity. Overall, 635 nm lasers with PpIX-PDT combined with Mt-L-MMC could be a promising option for further development in clinical trials.In general, we have developed both targeted and non-targeted prodrugs of anticancer drugs to improve PpIX-PDT's efficacy. In both in vitro and in vivo settings, both classes of prodrugs achieved significantly better efficacy when combined with PpIX-PDT compared to PpIX-PDT alone. There was no significant dark or phototoxicity associated with the combination treatment. Therefore, the PpIX-PDT + prodrug combination could be a promising treatment strategy for NMIBC.
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