Hydroxy urea attenuates Sildenaphil citrate (Viagra) induced
heamolysis in vitro : atomic force microscopic study.
Radhaballabh Bhar1, Tapan Guha2#, Upasana Das Adhikari3, Heena Kohad2 and Vedachal Ayier 1.Department of Instrumentation Science, Jadavpur University, Kolkata 700032, India 2.Modern Dental College and Research Centre, Airport Road, Gandhinagar , Indore, India 3.University of Calcutta,92 APC Road, Kolkata 700009, India 4.UGGC-DAE CSR, University Campus, Khandwa Road,Indore,452017,India # Corresponding author (email Keywords : Viagra, Sickle cell, Hydroxy Urea ABSTRACT : It has been recently reported by us that PDE 5 (Phosphodiesterase 5) inhibitor Sildenaphil citrate(Viagra), at high concentration, is haemolytic in vitro with the aid of Atomic Force Microscopy. Hydroxy carbamide( Hydroxy urea) is an antineoplastic drug and used for treating Sickle celled diseases and AIDS. We have observed that Hydroxy Urea is not haemolytic in vitro. We report here that Hydroxy Urea attenuates Sildenaphil citrate induced haemolysis in vitro. INTRODUCTION : Sildenaphil Citrate (Viagra) is used routinely in treating Erectile Dysfunction(ED) and Pulmonary Arterial Hypertension(PAH). It inhibits Phosphodiesterase 5 competitively because it is a structural analog of cyclic Guanosyl Monophosphate(cGMP). It has been reported by us recently that Sildenaphil Citrate is haemolytic in vitro at very high concentration at room temperature (1). The haemolytic action is expressed after a certain time period. There are published reports that Sildenaphil Citrate increases force of Right Atrial Contraction `. It has been also reported that Vaginal application of Viagra increases Endometrial thickness in women with thin endometrium causing miscarriage, it also decreases Natural Killer cells. Viagra increases sperm motility and penile rigidity. It is also used in treatment of high altitude sickness. Viagra is used by adult men ,not suffering from ED, to improve sexual performance, it is contradicted in patients suffering from Cardiac problems. Hydroxy Urea is used in myelo proliferative Disorders like Polycythemia vera and painful attacks in Sickle celled diseases. It also has anti retroviral properties like AIDS. In Sickle celled diseases it augments synthesis of Fetal haemoglobin by an unknown mechanism but it is thought that it increases NO(nitric oxide) level causing activation of Guanyl cyclase with resultant rise in cyclic GMP , as a result gammaglogin synthesis is increased, resulting into augmentation of Fetal Haemoglobin synthesis. NO level is increased in male reproductive organ by sexual stimulation and augments activation of Guanylate cyclase resulting into cGMP in penis giving rise to vasodilation and relaxation of smooth muscles causing erection. PDE 5 degrades cGMP. Sildenaphil citrate being a selective inhibitor of PDE5, increases cGMP levels hence results into sustained erection. Both Sildenaphil citrate (Viagra ) and Hydroxy urea increases cGMP level through NO pathway. Sildenaphil citrate causes haemolysis in vitro at room temperature and high concentrations, observed and reported by us recently. This inspired us to study the effect of Hydroxy urea on Red Blood Cell to determine if it is haemolytic. We studied the effect of Sildenaphil citrate, effect of Hydroxy urea and combined effect of the two drugs on human red bold cells in vitro. MATERIALS AND METHODS: Whole human blood was collected and diluted immediately in normal saline to make a normal Red Blood Cell( RBC) suspension in Saline(PCV is 1%) Hydroxy urea was dissolved in normal saline to make a solution of concentration 5mg/ml. Similarly a solution of Sildenafil citrate of concentration 5mg/ml was prepared. . 1. Normal RBC suspension and Hydroxy urea solution were mixed in 1:1 ratio. 2. Normal RBC suspension and Hydroxy urea with Sildenaphil citrate solutions were mixed in All these samples were incubated at room temperature for 90 minutes and then centrifuged for 1 Supernatants, after centrifugation, were observed and discarded and pellets were re suspended in fresh normal saline by shaking the tubes and films were drawn on glass slides. The slides were air dried and cut into small pieces for imaging under Atomic Force Microscope (AFM). The AFM instrument make was Digital Instruments, Santa Barbara, CA, Nanoscope –E series Scanning Probe Microscope with standard top view in contact mode. The AFM images were captured in real time. RESULTS AND DISCUSSION: After centrifugation of the tubes supernatant appeared red in colour in Sildenaphil citrate treated sample indicating extensive haemolysis. Hydroxy urea treated sample did not show haemolysis at all and the sample containing both Sildenaphil citrate and Hydroxy urea was different from the other samples , which indicated partial haemolysis of negligible amount (it showed little red tinge). The control supernatant was colourless without any sign of haemolysis. AFM images of films on glass slides revealed normal cytoarchitecture of RBCs in control sample, namely, pits or holes surrounded by blebs reported by Guha et al in 2002 (2)(Fig.1).
Cytoarchitecture of Hydrohy urea treated RBCs appeared more or less similar to normal cells
indicated by presence of surface pits and globular structures(Fig.2). Sildenafil citrate treated
haemolysed RBC surfaces indicated disappearance of pits and blebs (Fig.3) and in Hydroxy urea
with Sildenafil citrate treated cells membrane appeared unevenly granular and disorganised (Fig.4).

[1] Kohad,H. et al, EMSI golden jubilee conference Proceedings(2011), 325-327.
[2] Guha, T., Bhattacharyya, K., Bhar, R., Ganesan, V., Sen, A. And Brahmchary, R.L., Current
Science, 83 (6),(2002),693-694.
Figure 1. Normal RBC suspension and Hydroxy urea solution were mixed in 1:1 ratio
Figure 2. Normal RBC suspension and Hydroxy urea with Sildenaphil citrate solutions were mixed in 1:1:1

Source: http://www.emc2012.org.uk/documents/Abstracts/Abstracts/EMC2012_0253.pdf

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