Photosensitizing effects of xenobiotics are of increasing concern in public health
since modern lifestyle often associates sunlight exposure with the presence of chemical
substances in the skin. An important number of chemicals like perfumes, sunscreen
components, or therapeutic agents have been reported as photosensitizers.
In this context, a considerable effort has been made to design a model system for
photosafety assessment. Indeed, screening for phototoxicity is necessary at the
early phase of drug discovery process, even before introducing drugs and chemicals
into clinical therapy, to prevent undesired photoreactions in humans. In the case
of new pharmaceuticals, their phototoxic potential has to be tested when they absorb
in the regions corresponding to the solar spectrum, that is, for wavelengths
>290 nm. So, there is an obvious need for a screening strategy based on in vitro
experiments. The goal of the present thesis was the photochemical study of different
photoactive drugs to investigate the key molecular aspects responsible for their
photosensitivity side effects.
In a first stage, rosuvastatin was considered in chapter 3 as representative
compound of the statin family. This lipid-lowering drug, also known as “superstatin”,
contains a 2-vinylbiphenyl-like moiety and has been previously described
to decompose under solar irradiation, yielding stable dihydrophenanthrene analogues.
During photophysical characterization of rosuvastatin, only a long-lived
transient at ca. 550 nm was observed and assigned to the primary photocyclization
intermediate. Thus, the absence of detectable triplet-triplet absorption and
the low yield of fluorescence ruled out the role of the parent drug as an efficient
sensitizer. In this context, the attention was placed on the rosuvastatin main photoproduct
(ppRSV). Indeed, the photobehavior of this dihydrophenanthrene-like
compound presented the essential components needed for an efficient biomolecule
photosensitizer i.e. (i) a high intersystem crossing quantum yield (ΦISC =0.8), (ii)
a triplet excited state energy of ca. 67 kcal mol−1
, and (iii) a quantum yield of singlet oxygen formation (Φ∆) of 0.3. Furthermore, laser flash photolysis studies
revealed a triplet-triplet energy transfer from the triplet excited state of ppRSV
to thymidine, leading to the formation of cyclobutane thymidine dimers, an important
type of DNA lesion. Finally, tryptophan was used as a probe to investigate the
Type I and/or Type II character of ppRSV-mediated oxidation. In this way, both
an electron transfer process giving rise to the tryptophanyl radical and a singlet
oxygen mediated oxidation were observed. On the basis of the obtained results,
rosuvastatin, through its major photoproduct ppRSV, should be considered as a
potential sensitizer.
Then, itraconazole (ITZ), a broad-spectrum antifungal agent, was chosen as
main character of chapter 4. Its photochemical properties were investigated in connection
with its reported skin photosensitivity disorders. Steady state photolysis,
fluorescence and phosphorescence experiments were performed to understand ITZ
photoreactivity in biological media. The drug is unstable under UVB irradiation,
suffering a primary dehalogenation of the 2,4-dichlorophenyl moiety that occurs
mainly at the ortho-position. In poorly H-donating solvents, as acetonitrile, the
major photoproduct arises from intramolecular attack of the initially generated
aryl radical to the triazole ring. In addition, reduced compounds resulting from
homolytic cleavage of the C-Cl bond in ortho or para positions and subsequent Habstraction
from the medium are obtained to a lesser extent. In good H-donating
solvents, such as ethanol, the main photoproducts are formed by reductive dehalogenation.
Furthermore, irradiation of a model dyad containing a tryptophan unit
and the reactive 2,4-dichlorophenyl moiety of itraconazole leads to formation of
a new covalent link between these two substructures revealing that homolysis of
the C-Cl bond of ITZ can result in alkylation of reactive amino acid residues of
proteins, leading to formation of covalent photoadducts. Therefore, it has been established
that the key process in the photosensitization by itraconazole is cleavage
of the carbon-halogen bond, which leads to aryl radicals and chlorine atoms. These
highly reactive species might be responsible for extensive free radical-mediated biological
damage, including lipid peroxidation or photobinding to proteins.
In chapter 5, photobehavior of imatinib (IMT) was addressed. This is a
promising tyrosine kinase inhibitor used in the treatment of some types of human
cancer, which constitutes a successful example of rational drug design based on the
optimization of the chemical structure to reach an improved pharmacological activity.
Cutaneous reactions, such as increased photosensitivity or pseudoporphyria,
are among the most common nonhematological IMT side effects; however, the
molecular bases of these clinical observations have not been unveiled yet. Thus,
to gain insight into the IMT photosensitizing properties, its photobehavior was
studied together with that of its potentially photoactive anilino-pyrimidine and
pyridyl-pyrimidine fragments. In this context, steady-state and time resolved fluorescence,
as well as laser flash photolysis experiments were run, and the DNA
photosensitization potential was investigated by means of single strand breaks
detection using agarose gel electrophoresis. The obtained results revealed that the drug itself and its anilino-pyrimidine fragment are not DNA-photosensitizers.
By contrast, the pyridyl-pyrimidine substructure displayed a marked photogenotoxic
potential, which was associated with the generation of a long-lived triplet
excited state. Interestingly, this reactive species was efficiently quenched by benzanilide,
another molecular fragment of IMT. Clearly, integration of the photoactive
pyridyl-pyrimidine moiety in a more complex structure strongly modifies its
photobehavior, which in this case is fortunate as it leads to an improved toxicological
profile. Thus, on the bases of the experimental results, direct in vivo
photosensitization by IMT seems unlikely. Instead, the reported photosensitivity
disorders could be related to indirect processes, such as the previously suggested
impairment of melanogenesis or the accumulation of endogenous porphyrins.
Finally, a possible source of errors in the TEMPO/EPR method for singlet
oxygen detection was analyzed. For many biological and biomedical studies, it is essential
to detect the production of 1O2 and to quantify its production yield. Among
the available methods, detection of the characteristic 1270 nm phosphorescence of
singlet oxygen by time-resolved near infrared (TRNIR) emission constitutes the
most direct and unambiguous approach. An alternative indirect method is electron
paramagnetic resonance (EPR) in combination with trapping. This is based on
the detection of the TEMPO free radical formed after oxidation of TEMP (2,2,6,6-
tetramethylpiperidine) by singlet oxygen. Although the TEMPO/EPR method has
been largely employed, it can produce misleading data. This was demonstrated by
the present study, where the quantum yields of singlet oxygen formation obtained
by TRNIR emission and by the TEMPO/EPR method were compared for a set of
well-known photosensitizers. The results revealed that the TEMPO/EPR method
leads to significant overestimation of singlet oxygen yield when the singlet or triplet
excited state of the photosensitizers were efficiently quenched by TEMP, acting as
electron donor. In such case, generation of the TEMP+•
radical cation, followed by
deprotonation and reaction with molecular oxygen gives rise to a EPR detectable
TEMPO signal that is not associated with singlet oxygen production. This knowledge
is essential for an appropriate and error-free application of the TEMPO/EPR
method in chemical, biological and medical studies. / Nardi, G. (2014). MECHANISTIC STUDIES ON THE PHOTOTOXICITY OF ROSUVASTATIN, ITRACONAZOLE AND IMATINIB [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/48535
| Identifer | oai:union.ndltd.org:upv.es/oai:riunet.upv.es:10251/48535 |
| Date | 31 March 2015 |
| Creators | Nardi, Giacomo |
| Contributors | Lhiaubet, Virginie Lyria, Miranda Alonso, Miguel Ángel, Universitat Politècnica de València. Departamento de Química - Departament de Química |
| Publisher | Universitat Politècnica de València |
| Source Sets | Universitat Politècnica de València |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/doctoralThesis, info:eu-repo/semantics/acceptedVersion |
| Rights | http://rightsstatements.org/vocab/InC/1.0/, info:eu-repo/semantics/openAccess |
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