Photochemical internalisation (PCI) with focus on bladder cancer treatment

Det ble vist at HAL-PDT påvirket proteinuttrykk og induserte proteinmodifikasjoner. Det tyder på at PDT utløser en kompleks cellulær respons som involverer flere biologiske prosesser, slik som transport, stressrespons, DNA-reparasjon, overlevelse og celledød. Vi modellerte fire humane GAT-er ved molekylmodellering (ICM) og dokket GABA, ALA og MAL molekyler til bindingssetet i hver modell, og elektrostatiske potensialer (ESPs) i porene for translokasjon i modellene ble beregnet. Resultatene tyder på at ALA kan være substrat for alle GAT-ene, mens MAL er substrat for GAT-2, GAT-3 og BGT-1. I tillegg kan forskjeller i ESP mellom transportørene bli utnyttet i utviklingen av selektive inhibitorer, for eksempel for å redusere smerte i PDT ved bruk av ALA eller MAL. For å forbedre effekten av bleomycin som cytostatika ved blærekreft ble PCI-bleomycin studiet i tre cellelinjer (T24, AY-27, A431), ved hjelp av en ny amfifil fotosensitizer tetrafenyl klorin disulfonat (TPCS2a). Våre resultater viser at PCI øker bleomycin-effekten, og at denne effekten blir ytterligere forsterket av et nytt peptid-basert medikament ATX-101, som kan svekke DNA- reparasjon. Vi viser også at PCI øker DNA-skadenivået, noe som tyder på at den observerte effekten skyldes økt cellulær internalisering av bleomycin. For å etablere en protokoll for PCI-bleomycin i dyreforsøk med TPCS2a fotosensitizer ble kinetikk og lokaliseringen av TPCS2a studert i en etablert dyremodell for blærekreft i rotte, ved bruk av histologi og ex vivo fluorescensmålinger. Dette viser at TPCS2a har et overfladisk distribusjonsmønster i tumorvevet i rotteblæren, og at det er nesten fullstendig eliminert fra tumorvevet etter 72 timer. En optimal protokoll for TPCS2a ble utviklet for dyremodellen.

In the study (ex vivo) using an orthotopic rat bladder tumor model, we observed a superficial distribution pattern of the photosensitizer TPCS2a in the mucosal layer of the bladder wall. We found that TPCS2a was almost cleared from the tumors after 72 hours. The highest retention of TPCS2a was shown at 24 hours after intravesical instillation with a concentration of 3 mg/ml. Based on these results, an optimal PCI protocol was determined for further in vivo study in this tumor model, including a 24-hour TPCS2a-to-light time interval and a dose of 3 mg/ml TPCS2a.

This project was a preclinical study of the treatment effects focused on bladder cancer, using a novel drug deliver tool, photochemical internalization (PCI) which was mediated by a photosensitizer disulfonated tetraphenyl chlorin (TPCS2a), to enhance cellular uptake of the chemotherapeutic agent bleomycin.

Our work has provided a practical PCI protocol in an orthotopic rat bladder tumor model, which will be utilized for the study of PCI-enhanced therapeutic effects on non-muscle invasive bladder cancer, using a potent chemotherapeutic agent and an optimal light dose.

The strategy of a cocktail of PCI-bleomycin and the peptide drug ATX-101, under condition where individual drug levels have no or low toxicity, reaches a promising therapeutic effect in all three cell lines tested (bladder cell lines AY-27 and T24, and skin cell line A431). Combining PCI-chemotherapy treatments with inhibitors of DNA repair therefore seems to be a promising therapeutic strategy for increased efficacy “on site”.

Kinetic and photodynamic characteristics of TPCS2a in several cell lines have been studied. Protocols for these experiments and TPCS2a-PCI have been developed by the candidate.

A selected chemotherapeutic agent was used in combination with PCI technique in rat and human bladder carcinoma cell lines. Therapeutic effects of single and combination drug doses have been examined in four cell lines. Results have shown that PCI can enhance therapeutic effect of the selected agent in bladder cancer cells.

The location and penetration depth of TPCS2a in the rat bladders have been examined with fluorescence microscope using frozen sections from earlier animal exeriments. This study was cooperated with pathologist Anna Bofin (LBK, NTNU) and pathologist Qian Peng (The Norwegian Radium Hospital, UiO).

Moreover, a study on homology molecular modeling of GABA transporters and analysis of uptake of PDT prodrugs have been finished.This study pioneers in structure-based characterization of 5-Aminolevulinic Acid (ALA) and Methyl ALA transports via the four GABA transporters using the homology modeling approach. Although ALA-based PDT has been used successfully for the treatment of a variety of skin cancers, pain is a limiting factor. ALA-based PDT in combination with selective inhibitors of the GABA transporters may be an attractive approach to develop pain-reduce strategy and improve the PDT efficacy in the future. This study was cooperated with professor Ingebrigt Sylte, UiT.

A manuscript of this study has been submitted.

Authors: Baglo, Yan; Gabrielsen, Mari; Sylte, Ingebrigt; Gederaas, Odrun Arna.

Title: Homology Modeling of Human gamma-Butyric Acid Transporters and the Binding of Prodrugs 5-Aminolevulinic Acid and Methyl Aminolevulinic Acid used in Photodynamic Therapy.

Journal: PLOS ONE, submitted 20th Dec 2012. Case 01563752; PL#0N3_AR; ref:_00DU0Ifis._500U06AE0w:ref

1. Analysis of stress and DNA damage responses induced by HAL-PDT, in vitro study

Study of mechanisms of PDT resistance is important in order to improve the therapeutic efficacy e.g. by combining PDT with chemotherapy or gene-therapy. The purpose of this study was to monitor expression and activation of several proteins associated with cellular survival and DNA repair in response to HAL-PDT in human bladder cancer cells. The results showed that cellular apoptosis and survival pathways were activated after HAL-PDT, and combining HAL-PDT with inhibition of stress protein could improve the efficacy in the treatment of bladder cancer, as well as combining TPCS2a-PCI with inhibition of DNA repair. More investigations will be done in 2012. This study is supervised by Professor Marit Otterlei, IKM.

2. Homology molecular modeling of GABA transporters and analysis of uptake of ALA

Structural knowledge is essential for the understanding of how drugs interfere with cellular communication and regulation. Membrane transporter proteins are not easy to study by X-ray crystallography and NMR. Therefore, the homology modeling is a valuable approach for obtaining structural information when the experimental structure of the transporter of interest is not available. In order to clarify more severe pain reactions observed during ALA-based PDT in comparison with ALA esters, a homology molecular model of GABA transporter has been constructed to gain insight into pro-drug ALA uptake mechanism. This transporter was shown to be responsible of the uptake of ALA into LM3 mammary adenocarcionoma cells (Rodriguez et al. 2006, Br J Pharmacol. Apr; 147(7):825-33). This study is cooperated with Dr. Mari Gabrielsen, UiT and supervised by Professor Ingebrigt Sylte, UiT. Biological experiments will be done at IKM in 2012.

3. Determination of optimal concentration and time of light treatment of photosensitizer TPCS2a, in vivo study

Using a rat bladder cancer model, the optimal concentration of TPCS2a and the optimal time of light treatment after drug administration were investigated. The manuscript writing is ongoing. This study was cooperated and supervised by scientist researcher Odrun Gederaas.

4. Determination of optimal light dose of combining PCI and chemotherapy

Using the rat cancer model, the treatment efficacy of different light doses combining with chemotherapy were investigated based on study 3 described above. This study was cooperated and supervised by Odrun Gederaas, and histological study was cooperated with Anna Bofin, LBK.