Customized photodynamic protocol with innovative porphyrins and redox modulators in premalignant cutaneous disorders - preclinical demonstration

PORPHYDERM

Project code: PN-III-P2-2.1-PED-2021-0360

Contract no: 637PED/2022

Programme: Proiect Experimental Demonstrativ (PED)

Domain: 1.3 - Biotehnology

Project duration: 28.06.2022 – 27.06.2024 (24 months)

Partner institutions

Proiectul este implementat de un consortiu multidisciplinar care are o lungă istorie de colaborare și expertiză în dezvoltarea preclinică de porfirine noi pentru PDT, precum și în medicina și terapia redox.

• Coordinator: Institutul Național de Cercetare - Dezvoltare în Domeniul Patologiei și Științelor Biomedicale „Victor Babeș”, project director: Dr. Gina MANDA
• Partner 1: Universitatea de Medicină și Farmacie “Carol Davila”, partner responsible: Prof. Rica BOSCENCU
• Partner 2: Biotehnos SA, partner responsible: Dr. Laura OLARIU

Budget

• From the national budget: 598.795 RON
• Own contribution: 53.895 RON
• Total value: 652.690 RON

ABSTRACT

The main objective is the development at preclinical level of a customized photodynamic therapy (PDT) protocol using smartly formulated porphyrinic photosensitizers and redox modulators for the treatment of premalignant cutaneous disorders such as actinic keratosis (AK). The project is in line with the call topic Bioeconomy - Biotehnology.

The medical application will be demonstrated in cells and mouse models by addressing both therapeutic efficacy and toxicological issues related to PDT on skin. We will start TLR 2 – technology/product concept formulated, with a portofolio of six patents and expertise in experimental PDT and in redox medicine. We will advance through TRL 3 – experimental proof of concept by performing in vitro and ex vivo studies using skin-relevant cells and animal skin explants. TRL 4 – technology/method validated in laboratory is expected to be reached by testing in animal models a customized procedure of keratolytic PDT in AK, using innovative photosensitizers and redox modulators. The activities will be organized in 5 work packages.

OBJECTIVES

Main objectives

Secondary objectives

STUDY DESIGN

PROJECTED RESULTS

1) At least 2 new porphyrinic compounds with improved properties as compared to commercial photosensitizers for the treatment of actinic keratosis (AK). Improvements are expected in terms of amphiphilicity, fluorescence for in vivo imaging, significant singlet oxygen yield for PDT and good stability / preservation of photodynamic properties in the topical formulation.

2) Technical specifications for all formulated porphyrinic compounds and phytochemicals, as well as for the selected PDT protocol and its outcome in AK mice.

3) Documentation for at least 3 patent requests, submitted to the Romanian Patent and Trademark Office.

4) Two porphyrinic compounds registered at the European Chemicals Agency.

5) At least 2 publications in journals with impact factor > 4, and at least 2 communications at relevant congresses in the field of dermatology, toxicology and redox biology (we take into consideration that patent-associated results should not be previously published).

6) A workshop organized at the end of the project for presenting results to stakeholders (academia, dermatologists and pharmaceutical SMEs).

7) New research services on preclinical testing of new PDT strategies in dermatology, to be implemented at: IVB (PDT on tumor cells and small laboratory animals), and at BTH (in vitro PDT on skin-relevant cells, technological process for formulation of porhyrins and redox-modulating phytochamicals), to be published in eERIS.

8) Detailed development and market strategy, taking also advantage of the COST Action CA20121 which creates the collaboration frame with pharmaceutical SMEs in redox medicine and pharmaceutics.

9) Dedicated web page and social media channels for promoting the knowledge generated in the project towards specialists, pharmaceutical industry and the general public.

SUMMARY OF STAGE I /2022

Synthesis and preliminary characterization of new porphyrin compounds

Recent statistics highlight cancer as the leading cause of death worldwide, predicting an increase in the number of cases compared to 2020 by 47% by 2040 [1,2]. For these reasons, the development of innovative strategies regarding the dissemination of preventive measures and the supply of active substances with antitumor potential are essential for fighting cancer worldwide. Non-melanoma lesions (actinic keratoses, squamous and basal cell carcinoma, cutaneous lymphoma, Kaposi's sarcoma, angiosarcoma) are among the most common forms of cancer. The development of a non-melanoma cancer is determined by genetic factors, history of HPV infection, history of chronic inflammatory skin conditions, exposure to toxic substances, sun, immunosuppression, skin phototypes I and II, PUVA as previous therapy [3, 4]. Actinic keratoses are described in the literature as "carcinoma in situ" (due to dysplastic keratinocytes similar to squamous cell carcinoma) or as a premalignant lesion [5]. The therapeutic approach in the case of actinic keratosis must aim at reducing the risk of malignancy and an early diagnosis of malignant formations that may appear in a field of actinic keratosis. Porphyrins, through their structural and spectral profile, have potential in the identification and therapy of malignant skin manifestations [6]. In addition, the major advantage of these heme-like structural types is the selectivity for tumor cells and their versatility, the possibility of structural modeling by attaching substituents with different degrees of polarity, to obtain an optimal hydrophilic/lipophilic ratio for cellular internalization.

In this context, the main objective of the project is to develop a protocol for photodynamic therapy (PDT) of actinic keratosis, using new porphyrinic derivatives. The activities within the project include the evaluation of the marker and antitumor agent potential of some porphyrinic compounds with asymmetric molecular architecture and appropriate spectral profile. A first stage of the project envisages obtaining new porphyrinic structures, through ecological synthesis techniques that respected the current requirements in the field of drug synthesis. It was aimed to obtain a set of data associated with the synthesis procedures and the parameters that define the structural profile of the new photosensitizers.

As part of Activity I.1 carried out by UMF, the following porphyrinic compounds were obtained:

• with asymmetric structures:

 5-(2,4-dihydroxyphenyl)-10,15,20-tris-(4-acetoxy-3-methoxyphenyl)porphyrin (P4.2)
 5-(2-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl)porphyrin (P 5.2)

• with symmetric structures (reference for asymmetric derivatives in in vitro studies)

 5,10,15,20-meso-tetrakis-(4-acetoxy-3-methoxyphenyl) porphyrin (P4.1 P2.1)
 5, 10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P 5.1)

The structural and spectral evaluation of the synthesized compounds was carried out in Activity I.1 by NMR, FTIR, UV-Vis and fluorescence analysis. Correlation of the obtained data confirmed the structures of the porphyrinic compounds synthesized at this stage. In conclusion, the preliminary studies led to obtaining, with good yields, 4 porphyrinic compounds by approaching a modern and ecological synthesis method.

In Activity I.2, 3 types of formulations for topical applications of porphyrinic compounds and modulators were analyzed, in the first stage the gel method was selected and detailed.

The in vitro study carried out in Activity I.3 showed that all porphyrinic compounds analyzed, evaluated in vitro on normal and tumor-specific skin cells (normal human keratinocytes HaCaT, human dermal fibroblasts HS27 and mouse melanoma cells B16F10) had good biocompatibility with normal and tumor skin cells. However, the following aspects are worth noting: 1) compound P5.2 can inhibit the multiplication of keratinocytes, which has therapeutic relevance in the case of actinic keratosis characterized by the hyper-proliferation of these cells, if temporary photosensitization of the skin is avoided; b) compounds P2.1 and P5.2, but not the porphyrinic derivative P4.2, increase LDH release by dermal fibroblasts, possibly due to undesirable effects that disrupt the membrane integrity of dermal fibroblasts.

All analyzed porphyrinic compounds incorporated into normal and tumor skin cells investigated, it is true with different intensities, thus proving fluorescent marker properties. The porphyrinic compounds P2.1 and P4.2 can be considered as candidates for fluorescence imaging in dermatology, and for PDT in dermatology oncology.

For the visibility of the project and its achievements, in Activity I.4 the web page of the project was created, which can be found at

https://www.ivb.ro/porphyderm

In Activity I.5, coordinated by UMF, the patent application with the title "Porphyrinic compound with fluorescent marker potential in dermato-oncology" was developed, in which all partners participated. The technical documentation of the patent application claims novel elements corresponding to the obtaining, spectral and biological evaluation of the compound 5-(2-hydroxy-3-methoxyphenyl)-10,15,20-tris-(4-carboxymethylphenyl) porphyrin (P5.2) . The patent application was registered at OSIM.

The objective and activities of phase I were 100% achieved.

SUMMARY OF STAGE II /2023

Intermediate preclinical study

STAGE II ACTIVITIES

Activity	Partener
Activitatea II.1 Synthesis and characterization of some porphyrinic compounds for preclinical studies	UMF (P1)
Activitatea II.2 Obtaining, formulating and characterizing some phytocompounds	BTH (P2)
Activitatea II.3 In vitro biological study	IVB (CO)
Activitatea II.4 Formulation of porphyrinic compounds and redox modulators for topical application	BTH (P2)
Activitatea II.5 Ex vivo biological study on skin explants	BTH (P2)
Activitatea II.6 Preliminary in vivo study in animal model	IVB (CO)
Activitatea II.7 Studiu preliminar in vivo in model animal	IVB (CO)
Activitatea II.8 Evaluation of skin homeostasis in an animal model of actinic keratosis with various treatments	BTH (P2)
Activitatea II.9 Dissemination of results: web page update, articles, communications, international partnership	IVB (CO) UMF (P1)

STAGE II RESULTS

Projected results	Achieved results
2 selected porphyrin compounds intermediate technical specifications	3 porphyrinic compounds with technical specifications: P2.1, P2.2 and P4.2
2 phytocompounds with their technical specifications	2 phytocompounds with technical specifications
1 final in vitro biological study report	Preliminary in vitro study report. The results are presented in the article sent for publication in an ISI journal.
1 interim study report on skin explants	The method of obtaining and testing skin explants for vertical diffusion studies of topical and transdermal preparations; • HPLC method for porphyrinic compounds; • Protocol for performing PDT on skin explants.
2 porphyrinic compounds and 2 phytocompounds formulated for topical application	Variants of gels for the topical formulation of photosensitizers and phytocompounds for PDT on the skin.
1 ex vivo PDT protocol on skin explants	Ex vivo PDT protocol on skin explants
2 animal models of actinic keratosis	2 characterized animal models (repeated exposure to UVB, transplantable tumors of human skin carcinoma cells with squamous cells in immunosuppressed nude mice); A third model is in progress (chemically induced carcinogenesis in FAV mice).
1 preliminary in vivo study report	Preliminary in vivo study report on C57BL/6 mice repeatedly exposed to increasing doses of UVB and treated in vivo with phytocompounds.
NRF2 target genes panel	Panel of target genes of NRF2, relevant for photodynamic therapy
1 protocol of combined PDT therapy and redox modulation in a preclinical model	1 protocol of combined PDT therapy and redox modulation in preclinical model
1 in vivo skin investigation procedure	In vivo skin investigation procedure
Updated web page	PORPHYDERM – National Institute of Pathology Victor Babeş – Bucharest (ivb.ro)
2 scientific communications	• 1 scientific communication at the Autumn Scientific Conference of AOSR 2023 with the title "Science for a healthy society", organized between 21-23.09.2023 OVIDIUS University of Constanța; • 1 oral presentation in the field of the transcription factor NRF2 in cancer at the "NRF2 in Noncommunicable Diseases: from Bench to Bedside" Course, organized within the COST Action CA20121 between 26-30.06.2023 at Smolenice Castle, Slovakia.
2 published scientific articles	• 1 article published in 2023 in Molecules magazine (ISI, impact factor 2023 4.927); 1 article submitted for publication in Pharmaceuticals magazine (ISI, impact factor 5.215).
2 patent applications submitted to OSIM	• 1 patent application was submitted to OSIM with no. of registration A/00775/28.11.2022 and with the title "Porphyrin compound with fluorescent marker potential in dermato-oncology" (submission reported in advance in stage I); • For one of the compounds tested during this stage, patent No. 132752 B1 was granted, published in RO-BOPI, 11 of 29.11.2023, with the title "Porphyrin derivative for theranostic use", authors: Rica Boscencu, Gina Manda, Radu Petre Socoteanu, Mihail Eugen Hinescu, Ionela Victoria Neagoe, Laura Olariu, Brandusa Dumitriu.
1 patent application in progress	1 patent application is being developed regarding the formulation of the photosensitizers selected in stages 1 and 2 of the project for topical application in PDT.
2 collaboration protocols with experts from abroad	3 collaboration protocols with experts from abroad