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Historical background of photodynamic therapy
Treatment of skin disease in the presence of light has been used since 1400 BC. Earlier Egyptian, Indian and Chinese civilisations used light in the treatment of psoriasis, rickets and skin cancer. This was later known as phototherapy. Phototherapy uses either ultraviolet (UV) or visible light, and could be performed with or without a photosensitizer (PS). Whenever a PS is not in-use, phototherapy is commonly applied for the treatment of dermatological problems such as eczema, neonatal jaundice and in the treatment of vitamin D deficiency. Photochemotherapy is another concept of treatment which is done in the presence of a photosensitizer, usually the psoralens. It is commonly employed in the treatment of psoriasis, atopic dermatitis, alopecia areata and many other kinds of skin problems [3].
Photodynamic therapy is actually a type of photochemotherapy in which light, photosensitizer and molecular oxygen are required for treatment.. In the beginning of the twentieth century, treating diseases with light started to gain interest, and the first oncological application of chemical and light in cancer treatment was realized in 1903, during which eosin and light were used to treat skin cancer.
Ten years after, in 1913 Frederic Meyer-Betz conducted the first PDT test on himself by injecting 200 mg of haematoporphyrin into his body and subsequently exposing himself to sun light. He experienced swelling and photoreaction especially in the light-exposed areas of his body, such as hands and face. Since then, many discoveries were made (Figure 12) throughout the years, and in 1975, Dougherty et al. successfully treated skin cancer by usinghaematoporphyrin derivative (HpD) in 98 out of 113 patients in his study. The studies that followed afterwards showed that this technique is effective in treating early-stage cancers but failed to produce better outcome due to the problem of targeting and specificity [49].
Porphyrin: Historical Background and Current Advancement
The word porphyrin originates from the Greek word porphura, which means purple. It is widely occurs in nature and is represented by chlorophyll and heme, which are also known as metalloporphyrins due to the presence of metal in the centre of their porphyrin rings (Figure 20). Ferrous (II) is known to be present in the centre of heme ring, and it was only in this form that the heme has the capability to bind oxygen for transportation in the blood. Chlorophyll on the other hand has magnesium as the central ring metal and the light capturing ability of plant for photosynthesis is actually magnesium dependant. Indeed, porphyrin-related molecules have contributed in multiple ways to the sustainability of living organisms including the respiratory and photosynthesis processes which are two of the most important processes in life.
ADLER AND LONGO METHOD (1964)
In the Adler method which was developed nearly 30 years later, the reaction of aldehyde and pyrrole were conducted in refluxing propionic acid at 141˚C for γ0 minutes in an open vessel [80] and the porphyrin crystals were isolated at the end of the reaction by cooling, with about 20% yield (Figure 23). This method has the advantage of being easy, milder and is capable of producing numerous different meso-substituted porphyrins [92]. It allows the utilization of a greater variety of aldehydes and hence different symmetrical meso-tetraarylporphyrins could be produced [95], besides being able to increase into bigger synthesis scales [80, 96].
THE MACDONALD [2 + 2] PORPHYRIN SYNTHESIS METHOD
This method usually utilizes dipyrromethane as an intermediate and this is the most common pathway to synthesize porphyrin. The dipyrromethane molecules could be classified as either symmetrical or unsymmetrical, depending on the starting molecule prior to the condensation reaction. As an example in Figure 24, the condensation on two molecules of bromoethylpyrrole in hot methanol produces a symmetrical substituted dipyrromethane.
Catalytic hydrogenation of the benzyl ester group produces carboxylic acid and this is followed by formylation of the carboxylic group with Vilsmeier reagent to form the final diformyldipyrromethane. The formyl group (-CHO) is the source of bridging carbons in the MacDonald [2+2] porphyrin formation [97].
The solubilisation of FA and PS-FA
Solubilising FA was proven to be a challenge especially at lower pH medium. This is in accordance to available literature which describes similar occurrence. As an example, Younis et al. reported the solubility study of FA in different pH buffer media at 37˚C [160]. They described that the solubility of FA is very minimum at acidic pH (1 mg/L at pH 3) and increases as the pH increases (18 mg/L at pH 10). It is hence understood that the difficulties in solubilising FA at low pH media could be expected and due to this problem, perhaps the stability study of FA in such medium (pH 4) was compromised.
PS-FA has a different solubility profile as compared to FA alone. Due to the presence of a PS, it was already projected that the conjugate could only be solubilised in DMSO and at a lower concentration as compared to FA. Its solubility in methanol and the tested buffer medium is also very poor. After conducting a solubility study with the conjugate, it was decided that the conjugate will be solubilised first in DMSO to help its dissolution and subsequently the respected medium will be added. Figure 45 described the steps taken in solubilising PS-FA conjugate.
The evaluation of Folic acid stability
Different techniques including UV/Visible spectrophotometer and HPLC with UV/visible and/or fluorescence detector were reported in the literature as useful in detecting FA and its degradation products. With the aim of obtaining the most reliable results from the experiments conducted, it was decided that HPLC analysis would be the most suitable technique with both UV/visible and fluorescence detection.
A method reported by Araujo et al. (2012) was chosen as a reference [161] and as outlined in Table 5 it is being adapted accordingly. Based on this method, a mixture of methanol and water was used as the eluting solvent, and formic acid was added at a concentration of 0.1%. The addition of formic acid is beneficial in improving the resolution of peak during elution.
Although technically trifluoroacetic acid or TFA is more efficient for this purpose, TFA is a much stronger acid and is more destructive as compared to formic acid. Our research team has also discovered preliminarily that TFA could cause degradation of FA or complexes containing FA in the HPLC chain during analysis.
Peptides and Solid-phase peptide synthesis
Peptides are small proteins but with no tertiary, three-dimensional structures as is the characteristic of proteins. Medium size peptides could have secondary structures. The utilization of peptide-based therapeutic agents is gaining much attention in many different fields of pharmaceutics, owing to the improvement in its production techniques, the ability to reduce its metabolic breakdown and also the feasibility of different administration routes.
Although there are some problems that could be associated with peptides in vivo, they have more advantages as compared to proteins and antibodies. Indeed, they have less immunogenicity and higher chances for tissue penetration due to their smaller size, better stability profile and lower manufacturing cost [170]. Although some restrictions do exist, the application of peptides has nevertheless advances, with a lot of protein- or peptide-based drug products already available in the clinics. More than 60 peptide-based products are approved currently by FDA, around 140 drugs are currently in clinical trials and more than 500 peptide molecules are in pre-clinical stage [165]. The introduction of modern peptide synthesis technique in the last few decades has contributed to the increasing application of peptides in therapeutics. Solid-phase peptide synthesis (SPPS) is a significant breakthrough and more researchers have opted for this technique due to its ease of usage, feasible and reliable production of peptide. During the initial years, solid phase protocol was only devoted to peptide synthesis. The organic chemists considered this method as not very reliable because the intermediaries were neither isolated, nor characterised. The biologists and pharmacologists, who were in great need of numerous new molecules for the design of new drugs, encouraged the chemists to reconsider their position. In fact, the implementation of robotic high-throughput screening which enables the testing of thousands of compounds per day was the reason why there is a high demand of fast compounds production. Methods which promise shorter time or lower production cost were then eagerly taken up. This is clearly the case with combinatorial chemistry and multiple parallel syntheses technique such as the solid-phase synthesis which is well suited to perform reactions in parallel, because it readily enables the automated performance of multistep synthetic sequences. Solid phase synthesis technique has enabled the synthesis of up to 50 amino acid-length peptides and the synthesis is usually conducted in an opposite direction (C- to N-terminus) than the native peptide synthesis in cells (Figure 54) [166].
Table of contents :
Chapter I: General Overview
[Cancer, Photodynamic Therapy and Drug Targeting]
1.0 General Introduction
1.1 Cancer and Its Therapeutics
1.1.1 General Overview
1.1.2 Development of Cancer: Cells, Genes and Mutations
1.1.3 Angiogenesis: Background review
1.1.4 Angiogenesis: The process
1.1.5 Neuropilin-1 receptor and cancer
1.1.6 Folate receptor and cancer
1.1.7 Treatment approach for cancer
A. Surgery
B. Chemotherapy
C. Radiotherapy
D. Immunotherapy
E. Hormone therapy
F. Bone marrow transplant
1.1.7 Current Anti-angiogenesis Treatment
1.2 Photodynamic Therapy
1.2.1 Historical background of photodynamic therapy
1.2.2 Overview of PDT
1.2.3 Components of Photodynamic Therapy
A. Light
B. Oxygen
C. Photosensitizer
1.2.4 Classification of photosensitizers
1.2.5 Porphyrin: Historical Background and Current Advancement
A. Structure of porphyrin
B. Synthesis of Porphyrin
C. Photophysical characteristics
1.2.6 Photodynamic action in vivo
1.3 Targeted Cancer Therapy: A Strategy in Improving the Delivery of Photosensitizer
1.3.1 Improving PS Delivery for PDT
1.3.2 Targeting with peptide
A. DKPPR peptide
B. Target application of DKPPR and KDKPPR in PDT
1.3.3 Targeting with folic acid
1.4 Objectives of Study
Chapter II: The Evaluation of Folic Acid Stability: As Free Molecule and as Conjugate for PDT Application
2.1 Introduction
2.1.1 Folic acid
2.1.2 Folic acid in cancer therapy
2.1.4 Conjugation with a Model Photosensitizer
2.1.5 Experimental approach: Design of Experiment
2.1.6 Statistical analysis
2.2 Results and Discussion
2.2.1 The solubilisation of FA and PS-FA
2.2.2 The evaluation of Folic acid stability
2.2.3 The evaluation of PS-FA conjugate stability
2.2.4 Statistical analysis
2.3 Conclusion and Future Perspective
Chapter III: Modification of KDKPPR Peptide to Investigate the Effect on its Binding on NRP Receptot
3.1 Introduction
3.1.1 Peptides
3.1.2 Peptides and Solid-phase peptide synthesis
3.1.3 Peptides and drug development
3.1.4 KDKPPR peptide
3.1.5 Peptide modifications performed in this study
A. Alanine-scanning
B. Replacement of K2 lysine with arginine (KDRPPR)
C. Retro, inverso and retro-inverso peptides
3.1.6 ELISA competitive binding assay
3.2 Results and Discussion
3.2.1 NMR analysis
3.2.2 ELISA competitive binding assay
3.3 Conclusion and Future Perspective
Chapter IV: Synthesis of Porphyrin (P1COOH) and DKPPR Peptide Platform through Click Chemistry
4.1 Introduction
4.1.1 Click chemistry
4.1.2 The advantages and disadvantages of click chemistry
4.1.3 Porphyrin-based click chemistry
4.1.4 Microwave-assisted click chemistry
4.1.5 Peptides in click chemistry
4.1.6 Synthesis plan
4.2 Results and Discussion
4.2.1 The synthesis of platform building blocks
4.2.2 Building porphyrin blocks through click chemistry
4.2.3 Photophysical properties
A. Absorption
B. Fluorescence
C. Singlet oxygen
4.2.4 Building DKPPR blocks through click chemistry
4.2.5 Building the Porphyrin-DKPPR platform through click chemistry
4.2.6 ELISA Competitive Assay
4.3 Conclusion and Future Perspective
Conclusion
References
