Screening of new compounds for the management of erectile dysfunction

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Short Communiction Screening of new compounds for the mngement of erectile dysfunction Sumnt Kumr Goswmi*,1, Mohmmed Nseeruddin Inmdr 1 1 Deprtment of Phrmcology, Al-Ameen College of Phrmcy,

Inhiitors of phosphodiesterse type 5 (PDE5) enzyme like in sildenfi l, vrdenfi l, tdlfi l etc. re in clinicl use for the mngement of erectile dysfunction.

1 Short Communiction Screening of new compounds for the mngement of erectile dysfunction Sumnt Kumr Goswmi*,1, Mohmmed Nseeruddin Inmdr 1 1 Deprtment of Phrmcology, Al-Ameen College of Phrmcy, Bengluru, Krntk, Indi ABSTRACT Ojective/Bckground: Erectile dysfunction, mle sexul dysfunction generlly develops due to impirment of relxtion of penile smooth muscles nd rteries. Inhiitors of phosphodiesterse type 5 (PDE5) enzyme like in sildenfi l, vrdenfi l, tdlfi l etc. re in clinicl use for the mngement of erectile dysfunction. Ongoing reserch focuses on development of potent medicine with fewer side effects. Ojective of this rticle is to discuss nd demonstrte procedures ville to screen new compounds for the mngement of erectile dysfunction. Methodology: Mny niml models (mice, rt, ct, rit, dog etc.) re ville to screen new chemicl compounds for incresing erectile function. In vitro (using isolted corpus cvernosum smooth muscle of penile tissue) nd in vivo (mesuring intrcvernosl pressure) studies my e performed for screening new compounds. In this rticle, we discuss out two such screening procedures using rt s n niml model. Results nd discussion: Compounds those relx isolted rt corpus cvernosum smooth muscles signifi cntly in vitro my increse penile erection in humns. Similrly, the compounds those increse intrcvernosl pressure y relxing erectile smooth muscle nd penile rteries in rt my increse erectile function in humns. Conclusion: Rt is suitle model for screening new compounds to mnge erectile dysfunction. Key words: Corpus cvernosum smooth muscle, intrcvernosl pressure, men rteril pressure INTRODUCTION Erectile dysfunction (ED) is mle sexul disorder tht minly ffects ged mles, smokers nd persons with conditions such s dietes, lood pressure, nd depression. It is defined s the inility of men to ttin nd mintin penile erection required for stisfctory sexul intercourse. Journl Sponsor Access this rticle online Wesite: DOI: /jyp Sexul function is regulted y centrl nervous system, nd it is lso controlled peripherlly. Penile erection is the end result of relxtion of penile smooth muscles nd rterioles, therefore, inility of penile smooth muscles to relx in the presence of sexul stimuli my led to ED. 1,2 Penis contins three smooth muscle structures i.e., one corpus spongiosum tht surrounds urethr nd two corpor cvernos (singulr-corpus cvernosum) whose relxtion is responsile for penile erection. New compound tht relxes isolted corpus cvernosum smooth muscles (CCSMs) significntly in vitro would e helpful therpeutic cndidte in the mngement of ED. Itlino et l., suggested using corpus cvernosum of rt for investigting erectile phrmcology s penile structure Address for correspondence: Dr. Sumnt Kumr Goswmi, Deprtment of Phrmcology, Al-Ameen College of Phrmcy, Bengluru , Krntk, Indi. E-mil: sumntg@gmil.com Journl of Young Phrmcists Vol 6 Issue 4 Oct-Dec

2 of men nd rt re similr. 1,3 Quinln et l. showed tht rt cn e used s model for the study of penile erection. Mjor pelvic gnglions (MPGs) lie on either side of the dorsolterl loes of the prostte nd utonomic nerves (from MPGs) those tht innervte penis re known s cvernous nerves (CN). Stimultion of CN or MPG cn increse intrcvernous pressure (ICP), mesure of penile erection. 4,5 New compound tht increses ICP significntly would e helpful in the mngement of ED. The new compound tht relxes CCSMs my decrese lood pressure y relxing lood vessels. Therefore, men rteril pressure (MAP) is lso mesured long with ICP. MATERIALS AND METHODS Effect of new compound on isolted rt corpus cvernosum Anesthetize mle Wistr rt weighing g with ketmine (60 mg/kg, i.p.) nd xylzine (10 mg/kg, i.p.) (ny other nesthesi cn e used). Deglove the prepuce foreskin covering penis followed y cutting dorsl penile nerves nd dorsl penile vein from the dorsl surfce, nd corpus spongiosum covering urethr from the ventrl surfce. Microsurgiclly remove tunic lugine tht surrounds corpus cvernosum nd cut long the tunic lugine tht holds two corpus cvernos. Cut oth the ends of shft (ody) of penis to get two corpus cvernos of size 3 mm 3 mm 15 mm. Wsh the tissues with Kres-Henseleit (KH) physiologicl slt solution of following composition (in mm): 118 NCl, 4.7 KCl, 1.2 KH 2 PO 4, 1.2 MgSO 4, 1.5 CCl 2, 25 NHCO 3, nd 11 glucose, mintined t 37 C nd erted with crogen gs (95% O 2 nd 5% CO 2 ). Tie one end of the tissue to steel hook t the ottom of orgn chmer contining KH solution mintined t required temperture, erted with crogen gs nd nother end to force trnsducer (Model no: MLT0201; ADInstruments, Austrli connected to PowerL/8SP dt cquisition system, Chrt softwre, version 7.0; ADInstruments, Austrli) using cotton/silk thred. Exert 500 mg of tension to the tissue nd equilirte for 1 h, replcing KH solution every 1 h. Contrct the tissue with 3 μm phenylephrine (su-mximl dose) nd once the tissue ttins mximum contrction, record the relxnt effect of either vehicle, new compound or sildenfil, the stndrd drug used in the mngement of ED (Figures 1 nd 2). Mesurement of ICP nd MAP Anesthetize the mle rt s descried erlier nd expose the penile tissue nd deglove the prepuce. Locte crus (sl portion of corpus cvernosum) nd plce the 24G needle connected to PE tuing (filled with 200 IU heprin sline) connected to the pressure trnsducer tht in turn should e connected to AD Instrument dt cquisition system (or ny other dt cquisition system). Push μl of heprin sline solution into the crus nd oserve the deflection in penis tht confirms tht needle is in the crus. Remove two testes from the scrotl sc nd out of the peritonel cvity. Lift the seminl vesicle (SV) nd plce CN/MPG on the dorsolterl loe of prostrte t the se of SV on the ipolr steel electrode (wire dimeter 1 mm or less; distnce etween two poles: 3 mm) (Figure 3). Shve the hir of the neck nd mke smll cut on the skin of the neck. Locte the crotid rtery supplying to rin on either side of the trche long with the vgus nerve. Detch the vgus nerve from crotid rtery tking utmost cre not to dmge/cut vgus nerve. Cnnulte crotid rtery for plcing Millrs mikrotip ctheter (SPR-320, pressure trnsducer mrketed y AD Instrument, Austrli; use 3F size for rt, 2F size for mice; PE tues connected to ny other pressure trnsducer lso cn e used) 6 (Figure 4). Stimulte CN/MPG with 5 ms pulses of 5V t frequency of 12 Hz for 30 s nd record the chnge in ICP nd MAP in rts treted with vehicle, new compound or sildenfil (Figure 5). Voltge, frequency nd durtion of stimultion cn e optimized for your own experiment. Note: After, needle is inserted to crus of corpus cvernosum, ICP increses up to mmhg. After c Figure 1: Antomy of rt penile tissue nd isoltion of corpus cvernosum smooth muscle (CCSM). Deglove prepuce of the rt penile tissue () Locte nd cut two dorsl penile nerves nd one dorsl penile vein () Remove corpus spongiosum covering urethr (c) Locte two CCSMs on shft (ody) of the penis nd seprte y cutting cross the tunic lugine tht covers oth CCSM 52 Journl of Young Phrmcists Vol 6 Issue 4 Oct-Dec 2014

Goswmi nd Inmdr: Rt model of erectile function stle seline of ICP is mintined, CN/MPG cn e stimulted to see the effect of tretment on ICP. After the stimultion ceses, ICP return to norml.

Mximum ICP recorded re under the curve, nd ICP/MAP cn e used for sttisticl clcultion.

Figure 2: Screening of new compound using isolted corpus cvernosum smooth muscle (CCSM) in vitro.

3 Goswmi nd Inmdr: Rt model of erectile function stle seline of ICP is mintined, CN/MPG cn e stimulted to see the effect of tretment on ICP. After the stimultion ceses, ICP return to norml. The sme procedure cn e repeted 3 times for n niml in the intervl of 10 min ech for checking reproduciility of the dt collected. Mximum ICP recorded re under the curve, nd ICP/MAP cn e used for sttisticl clcultion. RESULTS Relxtion of isolted corpus cvernosum smooth muscle nd increse in intrcvernosl pressure provide informtion out erectile function. Figure 2: Screening of new compound using isolted corpus cvernosum smooth muscle (CCSM) in vitro. () CCSM is mounted in orgn th () Recording the effect of vehicle, new compound nd sildenfil using 4 chnnel orgn th nd AD Instrument dt cquisition system Figure 3: Mesurement of (intrcvernous pressure) BN: Butterfly needle, P: Penis, T: Testis, B: Bldder, Pr: Prostrte, SV: Seminl vesicle, E: Electrode c d e f g h i Figure 4: Mesurement of men rteril pressure (MAP). () Anesthetize rt nd ws nd shve the hir on the neck () Mke n incision into the skin of neck nd seprte the muscles (c) Locte the crotid rtery long with vgus nerve nd seprte the rtery (d) Tie the crotid rtery with thred (silk thred, 3-0 size is etter) nd lock it y ulldog clip, 1.5 cm prt (e) Tie nother thred loosely (f) Lift the crotid rtery y n rtery forceps nd cut it slightly with n iris scissor (g) Open prtilly collpsed rtery y curved iris forceps nd insert Millrs mikro-tip pressure ctheter (3F size) in to the rtery (h) Secure the inserted ctheter (with thred tied [round the ctheter] over rtery) nd remove ulldog clip (i) mesure MAP using AD Instrument dt cquisition system Journl of Young Phrmcists Vol 6 Issue 4 Oct-Dec

Tle 1: Optimiztion of experiment Nture of troule Isolted tissue is not responding to phrmcologic tretment ICP does not increse fter nerve stimultion Action to e tken Prtilly remove tunic lugine

4 Tle 1: Optimiztion of experiment Nture of troule Isolted tissue is not responding to phrmcologic tretment ICP does not increse fter nerve stimultion Action to e tken Prtilly remove tunic lugine Prepre fresh solution of phrmcologicl gents If KH solution is getting precipitted, prepre fresh solution Mount new tissues Check if PE tuing is locked y clotting of lood in it. If it is due to clotting of lood then remove needle (with PE tuing) from crus nd push pproximtely μl of heprine sline solution to remove the clot nd gin put the needle in crus Check whether nerve/mpg is on electrode or not Check whether you hve selected suitle stimulting prmeter or not Check whether stimultor nd electrodes re working or not Check whether tip of the needle is in crus or not Check integrity of CN/MPG Check the concentrtion of heprine in solution nd use the solution within 6-12 h Use n electrode of less dimeter ecuse more is the dimeter more will the mount of het generted Wit for out 10 min efore stimulting nerve/mpg gin CN is getting urnt ICP dt is not reproducile fter susequent stimultion Animl is wking up during Use supplementl dose of nesthesi to increse the durtion of nesthesi ICP nd MAP mesurement ICP: Intrcvernous pressure, MAP: Men rteril pressure, CN: Cvernous nerves, MPG: Mjor pelvic gnglion, KH: Kres-Henseleit crdiovsculr function. The ehvior of nimls lso my e ffected y presence of study personnel, so it is suggested to use video cmer for recording sexul ehvior nd then nlyze individul prmeters of sexul ehvior fterwrds. The positive point of sexul ehvior study is tht the effect of tretment cn e studied in reproduction. For pst decde or so, most of the reserchers hve relied more on the ICP nd MAP studies thn the sexul ehvior studies simply ecuse the collected dt re ccurte, tkes less time to cquire the dt (Tle 1). CONCLUSION Figure 5: Mesurement of intrcvernous pressure (ICP) nd men rteril pressure (MAP). First, second nd third chnnels on computer screen re displying MAP (deep green line), ICP (pink line) nd stimultion (lue line) respectively DISCUSSION Mesurement of ICP nd MAP is gold stndrd for determining the effect of new compound on erectile function. Sexul ehvior study is n lterntive method for screening new compound for their effect on sexul function of mle rts in the presence of femle rts. However, the study is more time tking s we hve to trin nimls efore the ctul study nd require femle rts lso. Femle rts need to e dministered estrogen (or derivtive) nd progesterone to ring them to estrous (het) phse so tht they llow mle rts to copulte with them. To void pregnncy of femle rts they my e ovriectomised tht need 6-10 dys for the wound to hel. 7 The study lso does not provide ny dt on the effect of tretment on Rt is suitle model to screen new compounds for the mngement of erectile dysfunction. ACKNOWLEDGEMENT This study ws conducted t Al-Ameen College of Phrmcy with the help of finncil grnt (R&D/KR-03/ NMPB) from Ntionl Medicinl Plnts Bord, New Delhi, Indi. We re thnkful to Mr. Rohitsh of University of Rhode Islnd, USA for reviewing mnuscript nd providing suggestion to improve it. REFERENCES 1. Andersson KE. Mechnisms of penile erection nd sis for phrmcologicl tretment of erectile dysfunction. Phrmcol Rev. 2011;63: Guy AT, Sprk RF, Bnsl S, Cunninghm GR, Goodmn NF, Nnkin HR, et l. Americn Assocition of Clinicl Endocrinologists medicl guidelines for clinicl prctice for the evlution nd tretment of mle sexul dysfunction: A couple s prolem updte. Endocr Prct. 2003;9: Itlino G, Clrò A, Pgno F. A simplified in vitro preprtion of the corpus cvernosum s tool for investigting erectile phrmcology in the rt. Phrmcol Res. 1994;30: Journl of Young Phrmcists Vol 6 Issue 4 Oct-Dec 2014

5 4. Quinln DM, Nelson RJ, Prtin AW, Mostwin JL, Wlsh PC. The rt s model for the study of penile erection. J Urol. 1989;141: Tochrus C, Smitsiri Y, Jeenpongs R. Bute super Rox. enhnces penile erection in rts. Phytother Res. 2006;20: Rjsekrn M, White S, Bquir A, Wilkes N. Rho-kinse inhiition improves erectile function in ging mle Brown-Norwy rts. J Androl. 2005; 26: Agmo A. Mle rt sexul ehvior. Brin Res Brin Res Protoc. 1997;1: Journl of Young Phrmcists Vol 6 Issue 4 Oct-Dec

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