Mr. L. Jayashankar

Photosensitizer : A drug or chemical compound used in photodynamic therapy (treating cancer). When absorbed by cancer cells and exposed to light of specific wavelengths, the drug becomes active and kills the cancer cells ^1,2 .

Photodynamic therapy

Photo-therapy is the term used to describe treatments which use light to achieve their effects. Examples include the treatment of seasonal affective disorder (SAD) in winter by the controlled use of artificial light, and blue light exposure which is used to treat new-born babies with neonatal jaundice. In the case of the latter, blue light reacts with bilirubin, the yellow pigment responsible for producing the skin discolouration, and turns it into a more soluble form which is easier to excrete from the body.

Photodynamic therapy (also called PDT, photo radiation therapy, phototherapy, or photochemotherapy) is a revolutionary treatment aimed at detecting cancers and treating them without surgery or chemotherapy.  It is based on the discovery that certain chemicals known as photosensitizing agents can kill one-celled organism when the organisms are exposed to a particular type of light. PDT is the destruction of cancer cells through the use of a fixed-frequency laser light in combination with a photosensitizing agent like Porphyrins, Phthalocyanins, etc. Depending on the part of the body being treated, the drug is either injected into the bloodstream or applied to the skin. Apart from cancer, PDT is also promised for its efficiency in other complications associated with Cardiovascular (e.g., alternative to angioplasty), Chronic skin diseases [e.g. Psoriasis (in development)], Autoimmune (e.g. Rheumatoid arthritis), Macular degeneration, Antibacterial (wound healing, oral cavity), Endometriosis, Precancerous conditions.

Studies have shown that PDT can be as effective as surgery or radiation therapy in treating certain kinds of cancers and precancerous conditions, and may have some advantages

• It is less invasive than surgery.
• It can be targeted very precisely.
• Unlike radiation, it can be repeated several times at the same site if necessary.
• It may result in less scarring.

However, PDT has its limits. It can only treat areas where light can reach, so it is mainly used to treat areas on or just under the skin, or in the lining of internal organs. While the drugs may travel throughout the body, the treatment only works at the area exposed to light, so PDT can’t be used to treat extensive cancers. The current drugs available also leave people very sensitive to light for a time, requiring special precautions ^1,2,17,18 .

New light on Medicine                                  

This light-induced toxicity may also help to explain a rare blood disease “Porphyria ” which evinces porphyrins is the photosensitive agents. Porphyria is actually a collection of diseases in which pigments called porphyrins accumulate in the skin, bones, and teeth that turn caustic on exposure to light ³ .Many porphyrins are benign in the dark but are transferred by sunlight into caustic, flesh-eating toxins. While struggling to find a cure for Porphyria, scientist came to realize that porphyrins could be not just a problem but also a tool for medicine. If a porphyrin is injected into cancerous tumor, it can be activated by light to destroy the tissue.

Consequences of Porphyrins

During the mid-nineteenth century, scientists began to unlock the mystery as to why certain biological substances such as chlorophyll (chlorin) and heme display unique colours. Close structural similarity between heme and chlorin was demonstrated by Verdeil in 1844, followed by Hoppe-Seyer in 1880 ⁴ . The final steps of structural elucidation were initiated by Willstatter and culminated in the work of Hans Fischer in the early twelfth century. These scientists demonstrated that the addition or removal of hydrogen atoms from this class of compounds facilitated the transformation of one porphyrin-type to another with distinctive pigment changes. 

Porphyrins the Bioinorganic component of an important class of cyclic tetra pyrrole pigments are essential constituents of a number of important biological systems. The Porphyrin-type nucleuses along with metal ions are found in cytochromes, peroxides and catalases. Other biologically important porphyrins that occur in nature and in the human body are hemin, an iron porphyrin- the prosthetic group of hemoglobin and myoglobin; chlorophyll- magnesium porphyrin- like compound involved in plant photosynthesis; and Vitamin B­­­12 - cobalt porphyrin-like compound commonly known as cobalamine. As a result of their vital role in life processes, metallo-porphyrins have always warranted chemist’s attention ⁴ .

Porphyrins are a component of hemoglobin, which in turn is a component of red blood cells. Hemoglobin is what carries oxygen in the blood. When porphyrins are not used as a component of hemoglobin, they can absorb energy from photons (particles of light) and transfer this energy to surrounding oxygen molecules. Toxic oxygen species such as singlet oxygen and free radicals are thus formed ^1,3,5,6,7 . Singlet oxygen, the predominant cytotoxic agent produced during PDT is a highly reactive form of oxygen that is produced by inverting the spin of one of the outermost electrons. These chemicals are very reactive and can damage proteins, lipids, nucleic acids and other cellular components.

These porphyrins belong to a class of compounds that form vital constituents of several important and diverse biological functions. All forms of life depend on the ability of porphyrins to undergo oxidation-reduction and electron transfer reactions. This process in chlorophyll’s and iron-porphyrin heme containing cytochromes converts light to chemical energy ^6,8 . In addition to their biological implications, the cyclic tetra dentate framework of the four central nitrogen atoms makes porphyrins unique chelating agents, almost every metal on the periodic table is capable of forming a metalloporphyrin complex ^{1,8,9,10,11,12,13} . More than eighty different natural and synthetic metalloporphyrins are known. The fact that porphyrins can be used in combination with almost any metal produces a Vast ranges of electronic, spectral are structural properties, and this has caught the interest of many inorganic, organic, physical and biochemists.

Properties of Porphyrin

Porphyrins are “a large class of deeply coloured red or purple, fluorescent crystalline pigments, with natural or synthetic origin, having in common a substituted aromatic macrocyclic ring consisting of four pyrrole-type residues, linked together by four methine bridging groups”. All porphyrin-like compounds have a strong absorption band around 400nm called band. Unfortunately this band is not useful for PDT since blue light does not penetrate very deeply into tissue; thus the weaker satellite absorption bands (Q bands) between 600nm and 800nm are used for treatment. Porphyrin exhibits weak absorption maxima around 630nm while chlorins and bacterochlorins have strong absorption maxima around 650nm and 710nm respectively ^1,13 .

Porphyrins evinces as an ideal photosensitizers since they are non-toxic, selectively retained in tumor tissue in high concentrations, water soluble to a certain level, cleared in a reasonable time from the body and rapidly from the skin which avoid  photosensitive reaction. Porphyrins have got competent amphiphilicity. Amphiphilicity is analogous to zwitterionicity. Margaron etal and others have reported that amphiphilic photosensitizers are generally more photodynamically active than symmetrically hydrophobic or hydrophilic molecules ^1,14,18 .

Photocatalytic process of Porphyrins in PDT

Several unique properties of these complexes have been reported in the literatures . Porphyrins have applications in a variety of novel chemical and medicinal procedures including the use of water-soluble porphyrins in PDT as photosensitizers for detection and treatment of cancer and also an effective modality against antibiotic resistant bacteria and cell free viruses ^14,15 .           

Mechanism of Porphyrin sensitization

The reason that singlet oxygen is generated in the cells is because of simple photophysics. The below figure (Fig.1) shows a simplified Jablonski diagram (with vibrational levels omitted). Provided that the porphyrin possesses an absorption maximum at a wavelength corresponding with that of the incident laser light, shining light on a highly colored porphyrin causes excitation to the singlet excited state ( ¹ P*). The singlet excited porphyrin can decay back to the ground state with release of energy in the form of fluorescence - enabling identification of tumor tissue. If the singlet state lifetime is suitable (and this is true for many porphyrins) it is possible for the singlet to be converted into the triplet excited state ( ³ P*) which is able to transfer energy to another triplet. One of the very few molecules with a triplet ground state is dioxygen, which is found in most cells. Energy transfer therefore takes place to afford highly toxic singlet oxygen ( ¹ O ₂ ) from ground state dioxygen ( ³ O ₂ ), provided the energy of the ³ P* molecule is higher than that of the product ¹ O ₂ ¹ .

Fig. 1. Photophysics of PDT sensitization (vibrational levels omitted). (Simplified Jablonski diagram)

Type-I and Type-II photoreactions in the PDT.

Here the ¹ P is the photosensitizer in a singlet ground state, ³ P* is a photosensitizer in a triplet excited state, S is a substrate molecule, P ^{- is reduced} photosensitizer molecule, S ⁺ is an oxidized substrate molecule,O ₂ is molecular oxygen (triplet ground state), O ₂ ^– is the superoxide anion O ₂ is the superoxide radical, P ⁺ is the

oxidized photosensitizers, ³ O ₂ is triplet ground-state oxygen , ¹ O ₂ in a singlet excited   state, and S(O) is an oxygen adduct of a substrate ¹ .

In PDT, the photosensitizing agent is injected into the bloodstream and absorbed by cells all over the body. The agent remains in cancer cells for longer times than it does in normal cells. When the treated cancer cells are exposed to laser light (usually 16minutes), the photosensitizing agent absorbs the light and produces an active form of oxygen that destroys the treated cancer cells. Porphyrin have been determined to be ligands for the mitochondrial peripheral benzodiazepine receptor (PBR) and correlation  between the photodynamic activities of several porphyrin photosensitizers and their binding affinities for the PBR were shown by verma et al ^1,15 .

The light sources

The activating light is most often generated by lasers or in some cases by arc lamps or fluorescent light sources. Lasers are most frequently used as the source. The laser light used in PDT can be directed through a fiber optic to deliver the proper amount of light. Most of the light photons at wavelengths between 630 and 800 nanometers (nm) travel 23 centimeters through the surface tissue and muscle between input and exit at the photon detector ^1,5 .

Porphyrins a sophisticated and an effective weapon

Twenty-three centimeters is 9 inches. Logically, therefore, if we illuminate the whole body, front and back, light in the range of 630 to 800nm can reach almost any part of the human body.  Ongoing research includes pioneering treatments for bladder cancer, brain cancer, breast metastases, skin cancers, gynecological malignancies, colorectal cancers, thoracic malignancies, oral cancer, neck cancers, head cancers, coronary artery  disease, for macular degeneration and pathogenic myopia, AIDS, Autoimmune disease, transplantation rejection, leukemia and for common adult blindness. So simply saying it is a sophisticated and an effective weapon for the treatment of the various ailments ^1,3 .

Porphryins and FDA

In December 1995, the U.S.Food and Drug Administration (FDA) approved a photosensitizing agent called porfimer sodium, or photofrin, to relieve symptoms of esophageal cancer that is causing an obstruction and for esophageal cancer that cannot be satisfactorily treated with laser alone, In January 1998, the FDA approved porfimer sodium for the treatment of early non-small cell lung cancer in patients for whom the usual treatments for lung cancer are not appropriate. Recently the FDA has recommended that BPDMA, given the trade name Visudyne (injection), be approved for use in Visudyne therapy, which is essentially PDT to destroy the neo-vasculature on the retina ¹ . The National cancer institute and other institutions are supporting clinical trials to evaluate the use of PDT for the several types of cancers. Tin etiopurpurin (purlytin) is photosensitizer being investigated for use in PDT a chlorin

The most commonly used and studied photosensitizer to date is photofrin, the only commercially available photosensitizer. At the time of photofrin review in this innovative PDT it has been used on nearly 10000 patients in the United States , Canada , Netherlands , Japan , France , and Italy (and is pending approval for use in eight other countries).

Many new compounds have been synthesized in an attempt to create a better photosensitizer than photofrin. Often, researchers examine new chromophores week absorption maxima at wavelengths longer than 630nm, citing that photofrin really weak absorption band at 630nm does not allow optimal penetration. Researchers are also looking at different laser types, Photosensitizers that can be applied to the skin to treat superficial skin cancers, and new photosensitizing agents that may increases the potency of PDT against cancers that are located further below the skin or inside an organ ¹ .

Antibacterial and antiviral activity of porphyrin photosensitization

The development of photodynamic therapy (PDT) has also provided an effective modality against antibiotic-resistant bacteria and cell free viruses. The antibacterial activity of porphyrin induced photodynamic therapy shows unique properties.

Porphyrins possess a high binding-affinity to cellular components, membranes, proteins and DNA. Living cells as well as dead cells are stained rapidly by different porphyrins. Appropriate illumination generates an emission of red fluorescence and generates toxic oxygen species. Cancer cells stemming from solid tumors cells and bacterial infected tissues show preferential retention of porphyrins. In vivo administration of various sensitizers to tumor bearing animals and humans resulted in retention of the porphyrins in the tumors, while the normal surrounding tissues had a low comparable porphyrin contents. Photodynamic therapy of solid tumors was found highly efficient in eradication of the inflicted tissues and the damage, -initiating necrosis or apoptosis of Photodynamic therapy, occurred within a very small time frame.

Photodynamic interactions were described to take place wherever sensitizer, light and oxygen are simultaneously present. Inflammatory tissue was described to manifest similarities in porphyrin retention and therefore bacterial and viral infected tissues may become targets for photodynamic treatment. It is independent of the antibiotic sensitivity spectrum of the treated pathogen and it has an efficient and non-recovering anti-microbial killing effect upon illumination of Gram positive bacteria. Bacterial PDT is affected by the use of various sensitizers, as a general rule non-charged or positively charged molecules are effective in photoinactivation of Staphylococcus sp. In order to photosensitive Gram (-) bacteria such as  Pseudomonas aeruginosa, it is necessary to introduce a small peptide polymyxin-B nona-peptide (PBNP) which stimulate the translocation of  porphyrin through the outer membrane of these bacteria and makes PDT possible. Gram negative cell killing by the use of PBNP and DP broadens the antibacterial spectrum of photodynamic inactivation and opens new horizons for this modality as a wide spectrum drug when antibiotic resistance is the main concern ^14,15 .

Porphyrin against AIDS

The rapid spread of human immunodeficiency virus (HIV), the Causative agent of acquired immunodeficiency syndrome (AIDS), throughout the world has promoted an intense search for antiretroviral therapeutics.  An analysis of nonpeptide compounds with useful pharmacological properties has led to test the ability of porphyrins to inhibit HIV protease (HIVPR) ¹⁶ .

Impersonation of Phthalocyanines

Among these sensitizers, phthalocyanine (c) derivatives have shown great promise. Phthalocyanins are porphyrin-like second-generation sensitizers for photodynamic therapy (PDT) of cancer. Their intense absorption in the far red and long-lived excited triplet state is among the important attributes that make them ideally suited for PDT. In addition, by selecting appropriate metal ligands and peripheral substituents, Pc derivatives that are powerful sensitizers for PDT cause only minimal damage to the cells thus, making them potentially useful for the PDT ^1,2 .

Other Porphyrin derivatives

Extensively porphyrins, chlorins, and bactereochlorins are the most useful photosensitizers for photodynamic therapy. Apart from these, certain other photosensitizers proves its photodynamic activeness against infrared range light. Such compounds include, Phthalocyanins, texaphyrins belonging to the class of Porphyrinoides. Several benzoporphyrin derivatives (BPDs) were synthesized from

Proporphyrin ^1,2,14,15 .

The Success of Porphyrin in the Photo Dynamic Therapy motivated Chemists and Biologist to work deep into the Porphyrin related entities. Since porphyrin evinces its wide applications, future work in porphyrin is decisive for the further development of better photosensitizer.

References

1. IAN J. MACDONALD* and J. DOUGHERTY, (2001). Basic principles of photodynamic therapy, Journal of Porphyrins and Phthalocyanines, 5, pp 105-129

2. EUGENY A. LUKYANETS, (1999). Phthalocyanines as photosensitizers in the Photodynamic therapy of cancer, Journal of Porphyrins and Phthalocyanines, 3, pp 424-432

3. Susan Aldridg, Ph.D., medical journalist, (2003), PHOTODYNAMIC THERAPY ON SKIN CANCER</Home/gid1=3265>www.cancer.gov, ww.mcw.edu/whelan/html/2

4. Leslie D. Dobbins, Natarajan Ravi, Ph.D., an Albert N. Thompson, Jr, Ph.D (1998) Mossbauer spectroscopic charecterization of a water soluble porphyrin, Department of chemistry, Spelman college georgia institute of technology,United States

5. Lars-Oliver Klotz, Corinne Pellieux, Karlis Briviba, Christel Pierlot, Jean- Marie Aubry and Helmut Sies, (1999) Mitogen-activated protein Kinase (p38-, JNK-, ERK-) activation pattern induced by extracellular and intracellular singlet oxygen and UVA, European Journal of Biochemistry, 260, pp 917-922

6. Sham M Sondhi, Shefali Rajvanshi and Monika Johar, (2002) Synthesis and anticancer activity evaluation of some hemin and hematoporphyrin derivatives, Indian journal of chemistry, 41B, pp 388-393

7. Seema Gupta, B S Dwarakanath, K Muralidhar and Viney Jain, (2003) Role of apoptosis in photodynamic sensitivity of human tumor cell lines, Indian journal experimental biology, 41, pp 33-40

8. Sham M Sondhi, Nidhi Singhal and Rajeshwar P Verma, (2001). Synthesis of hemin and porphyrin derivatives and their evaluation for anticancer activity, Indian journal of chemistry, 40B, pp 113-119

9. Mario Nappa and Joan S. Valentine, (1978) The influence of axial ligands on metalloporphyrin visible absorption spectra. Complexes of Tetraphenyl porphyrinatoinc, Journal of American Chemical Society, 78, pp 5075-5080

10. J. -M. Barbe, C. Ratti, P. Richard, C. Lecomte, R. Gerardin, and R. Guilard, (1990). Tin(II) Porphyrins: Synthesis and spectroscopic properties of a series of divalent tin porphyrins, X-ray crystal structure of (2,3,7,8,12,13,17,18- octylethyl porphyrinato)tin(II), Inorganic chemistry, 29, pp 4126-4130

11. Dennis P. Arnold and John P. Bartley, (1994) Tin Porphyrins. 6. Tin-119 Chemical Shifts and Line Widhts of Tin(iv) Complexes of tetraphenyl- Tetra- p -tolyl and Octaethylporphyrin, Inorganic chemistry, 33,pp 1486-1490

12. Ines Scalise and Edgardo N. Durantini (2002) ,Synthesis and photodynamic activity of metallo 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrins, sixth international electronic conference on synthetic organic chemistry,(ECSOC-6), www.mdpi.org/ecsoc-6,1-30sep2002

13. Kurstan L. Cunningham, Kristina M. McNett, Rodney A. Pierce, Keith A. Davis, Holden H. Harris, David M. Falck, and David R. McMillin (1997) EPR Spectra, Luminense Data, and Radiationless Decay Processes of copper(II) Porphyrins, Inorganic chemictry, 36, pp 608-613

14. Elena Reddi, Mkara Ceccon, Giuliana Valduga, Giulio Jori, Jerry C. Bommer, Fausto Elisei, Loredana Latterini, and Ugo Mazzucato, (2002) Photophysical properties and Antibacterial Activity of Meso-substituted Cationic Porphyrins, Photochemistry and Photobiology, 75, 5, pp 462-470

15. Zvi Malik, Hava Ladan, Yeshayau Nitzan and Zehava Smetana , Life Sciences Department, Bar-llan University, Ramat-Gan 52900, Israel, (2004), Antimicrobial and anti Viral activity of porphyrin photosensitization,

16. Dianne L. DeCAmp, Lilia M. Babe, Rafael Salto Jeanne L. Lucich, Myoung-Seo Koo, Stephen B. Kahl, Charles S. Craik, (1992), Specific Inhibition of HIV – 1 Protease by Boronated porphyrins, Journal of medicinal chemistry, 35, pp 3426-3428.

17. http://www.cancer.org/docroot/ETO/content/ETO_1_3X_Photodynamic_Therapy.asp

18. http://www.thenakedscientists.com/HTML/articles/article/davinacolumn1.htm/

About Authors:

Mr. L. Jayashankar ^a *, Dr. R. Vijayaraghavan ^b

* a Department of Chemistry, Pharmaceutical Chemistry unit, Vellore Institute of Technology, Vellore-632 014, India.

b Professor, Department of Chemistry, Pharmaceutical Chemistry unit, Vellore Institute of Technology, Vellore-632 014, India

* For Correspondence: L. Jayashankar, c/o. Dr. R. Vijayaraghavan b , Department of Chemistry, Pharmaceutical Chemistry unit, Vellore Institute of Technology, Vellore-632 014, India. E-mail: l.jayashankar@gmail.com, Phone: 9810043362