Transcript Melanin Binding Characteristics of α

Melanin Binding Characteristics of α-1 Adrenergic Receptor Antagonists
Jeffrey S.
1
Gaynes ,
Cedomir
1
Micic
, Jeffrey A.
1
Borgia
Bruce I.
2
Gaynes ,
1Department
of Biochemistry, Rush University Medical Center, Chicago, IL
2Department of Ophthalmology, Loyola University Medical Center, Chicago, IL
Introduction
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Intraoperative floppy iris syndrome (IFIS) describes an entity
encountered during surgical removal of a cataract that is
characterized by a flaccid iris and inhibition of dilation of the pupil
required for successful cataract surgery.
The occurrence of IFIS during cataract surgery is particularly
troubling as it dramatically increases the likelihood of surgical
complications during the cataract procedure and is often
recalcitrant to any form of treatment.
Patients may experience more pain, a longer recovery period and
less improvement in visual acuity than a patient with an
uncomplicated cataract removal in which IFIS is absent.
In many cases IFIS is related to use of various systemic drugs,
notably, those medications used to treat benign prostatic
hypertrophy such as Flomax (tamsulosin) in men.
Interestingly, IFIS has been noted to occur up to a year after
discontinuation of Flomax
Purpose
The purpose of this investigation is to elucidate the melanin binding
affinity of Flomax/tamsulosin and other similar α-1 receptor antagonists.
If such affinity exists, this such affinity may imply a cause for IFIS
occurrence well after discontinuation of Flomax.
Data Analysis
Methods
Synthetic Experiment:
Synthetic melanin was synthesized by combining L-DOPA with polyphenol oxidase (tyrosinase) followed by incubation
and centrifugation for a total yield of 10.0mg of substance. 1.0mg/mL solutions of drug (Flomax/tamsulosin and
prazosin) were incubated with 3.0mg of synthetic melanin overnight with constant agitation. After 24 hours of
incubation the melanin samples are washed by resuspending in phosphate buffer three times. After the samples are
washed 100µl of acetonitrile (Sigma-Aldrich, St. Louis, MO) are added and the samples are centrifuged. The eluent is
then collected from each sample and analyzed via liquid chromatography – tandem mass spectrometry (LC-MS/MS).
Tissue Experiment:
Retinal Pigmented Epithelium – Choroid (RPE) and iridial tissue is excised from four bovine eyes obtained from a local
abattoir. In duplicate, 1.0mg/mL solutions of drug (Flomax/tamsulosin, chloroquine, and doxazosin) is are incubated
for 24 hours with each type of tissue with constant agitation. Following incubation samples are washed by
resuspending in phosphate buffer three times. After the samples are washed 100µl of acetonitrile (Sigma-Aldrich, St.
Louis, MO) is added to each and the samples are centrifuged. The eluent is then collected from each sample and
analyzed via liquid chromatography – tandem mass spectrometry (LC-MS/MS).
Liquid Chromatography – Tandem Mass Spectrometry (LC-MS/MS):
Concentration of drug that bound to melanin (synthetic and tissue) is determined by means of LC-MS/MS.
The instrument used is a Waters Voyageur HPLC linked to a Thermo TSQuantum Triple Quad Mass Spectrometer. A
two solvent, fifteen minute method with a 7 minute linear gradient (5-95% MeOH +0.1% formic acid) was used along
with a Waters XBridge C18, 2x100 mm, 3.5µm particle size column with internal standards 1000µg/mL  100ng/mL.
• There was no statistically significant difference in the amount of
tamsulosin or chloroquine bound to RPE tissue. In contrast,
doxazosin was found to demonstrate statistically significantly
reduced RPE binding compared to tamsulosin and chloroquine
alike.
• In the iris, chloroquine was again found to demonstrate the
highest binding level, followed by tamsulosin and doxazosin.
Although tamsulosin was found to bind to iris tissue in amounts
exceeding doxazosin, the absolute levels was lower than
compared with RPE. Doxazosin demonstrated the lowest level of
binding for both RPE and iris respectively.
• For all statistically analyses, α = 0.05 (one way ANOVA with
Bonferroni sub testing).
Conclusion
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It is thought that due to the presence of a highly characteristic secondary
amine group in both the anti-malarial drug chloroquine (a compound
with a high melanin binding affinity) and Flomax/tamsulosin that is not
found in any other α-1 receptor antagonist, Flomax will demonstrate a
substantial melanin binding affinity.
Figure 1 – Structure of Flomax
Drug Binding Data
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Results of this study suggest that tamsulosin demonstrates
significant binding potential to both RPE and iris tissue alike.
The amphilic nature of chloroquine is mimicked by tamsulosin
which is widely used for treatment of benign prostatic
hypertrophy.
It is posited that due to melanin binding, tamsulosin is adsorbed
into iris tissue and tightly bound to iridial melanin resulting in
prolongation of its normal pharmacologic activity as an α1
receptor antagonists.
Although IFIS is seen with other α-1 antagonists such as
doxazosin, the incidence of IFIS with α -1 antagonists aside from
tamsulosin is reduced.
In summary, despite limitation, the results of this study support
the hypothesis that tamsulosin demonstrated binding affinity to
ocular melanin. Further study is required to elucidate the
relationship between tamsulosin melanin binding and the
occurrence of IFIS.
Figure 2 – Structure of Chloroquine
References
• Chang, D., & Campbell, J. (2005, April). Intraoperative Floppy Iris
Syndrome Associated with Tamsulosin. Journal of Cataract and
Refractive Surgery 31(4), 664-673.
• Gaynes, BI. (2007, March 26). Tamsulosin (Flomax) and Cataract
Surgery [Letter to the editor]. Investigative Ophthalmology and
Visual Science, 47(9).
Figure 3 – Drug Bound to Synthetic Melanin
Figure 4 – Drug Bound to RPE Tissue
Figure 5 – Drug Bound to Iris Tissue
*Denotes statistically significant difference from chlroquine (p<0.05)
Acknowledgments
• This work was supported in part by the Department of Veterans
Affairs, Richard A. Perritt Charitable Foundation, The Illinois
Society for the Prevention of Blindness and Niles North High
School.