Introduction

Sildenafil, the active agent in Viagra™, is a highly selective inhibitor of the cGMP-binding cGMP-specific phosphodiesterase-5 (PDE5). The action of sildenafil to block cGMP hydrolysis by PDE5 in vascular smooth muscle of the penile corpora cavernosa accounts for its efficacy in treatment of male erectile dysfunction.^1,2,3 The widespread use of this therapy has generated great interest in understanding the action of sildenafil in this and other biological systems. PDE5 occurs in many tissues other than penile corpora cavernosa including platelets and the smooth muscle of the systemic vasculature, the airways, and the gastrointestinal tract. To date, most research has focused on the effect of sildenafil on vascular events in penile corpora cavernosa, but there is increased interest in potential efficacy of sildenafil in modulating cyclic nucleotide signaling in other tissues.^{4,5,6,7,8,9,10,11} In addition, biochemical characteristics of the interaction of sildenafil with PDE5 and perhaps other proteins are still poorly understood. Investigation of these basic processes is compromised by the fact that purified sildenafil is not commercially available. Herein, we describe a simple and inexpensive procedure for isolating sildenafil from Viagra™ tablets that can be readily obtained commercially. This protocol can be easily adjusted to purify either large or small amounts of sildenafil, and it utilizes materials and techniques available in most laboratories. It is also likely that other PDE5 inhibitors can be purified using a similar protocol because it exploits hydrophobic interactions between these compounds and the Sephadex G-25 resin using low ionic strengths.

Methods and results

Scheme for purification of sildenafil from Viagra™ tablets

Sildenafil, CA Index name (1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methyl-piperazine), was extracted from commercially available Viagra^® tablets. Two 50 mg tablets of Viagra™ (Pfizer) were placed in 120 ml deionized water at room temperature (20°C). Mixture was manually agitated for 15 min and then centrifuged for 20 min at 27 000 g at 4°C to remove particulate material. Supernatant (110 ml) was carefully decanted and applied to a 285-ml Sephadex G-25 (superfine) column (4.1 17.5 cm²) equilibrated in deionized H₂O at 20°C. Column was washed with five column volumes of deionized water (1.5 l). Under these conditions, sildenafil (Figure 1) was retained at least five column volumes beyond the total column volume due to hydrophobic interactions between sildenafil and the resin at low ionic strength. Since most small molecules elute from Sephadex G-25 within one column volume, this extensive washing minimized the likelihood that other small molecules were still present.

Figure 1.

Profile of sildenafil elution from Sephadex G-25. Sildenafil extracted from two 50 mg tablets of Viagra™ was applied to a Sephadex G-25 column (4.1 17.5 cm²) equilibrated in deionized water at room temperature. Following a wash with five column volumes of water, the sildenafil was eluted with 1% formic acid. The appearance of sildenafil in the column fractions was monitored by increased UV absorbance at 289 nm. Molecular structure of sildenafil is shown in the inset.

Full figure and legend (24K)

Water above the resin bed was removed, and 1% formic acid was applied to elute the sildenafil. Sildenafil eluted as a sharp peak (Figure 1). Its appearance was monitored by increased absorbance at 289 nm. The sample was lyophilized, resuspended in deionized water, and relyophilized. The fluffy white powder in the lyophilization flask was difficult to collect. Care was exercised in bleeding air into the flask following lyophilization in order to prevent significant loss of crystals by dispersion. The sample was either stored in crystalline form, in water, or in solution of 0.1% formic acid. When dissolved in water, the solution was slightly hazy, but this could be largely clarified by addition of small amount of formic acid. Sildenafil in solution was stored at -20°C. The recovery was 40–80%. When the purified sildenafil is rechromatographed on an analytical Sephadex G-25 column equilibrated in 1% formic acid as shown in Figure 2, the compound elutes as a sharp peak, but even at this ionic strength, it is still retained beyond the total column volume (1.2 column volumes) as indicated by the elution position of [³H]H₂O.

Figure 2.

Comparison of elution position of sildenafil and [³H]H₂O from Sephadex G-25 in 1% formic acid. Rechromatography of purified sildenafil and [³H]H₂O on analytical Sephadex G-25 (0.9 12.5 cm²) equilibrated in 1% formic acid. Fractions were 0.5 ml each.

Full figure and legend (23K)

The Sephadex column can be reused several times if stored properly. Immediately following elution of the sildenafil, the column bed is washed with water to remove the formic acid and then washed with several volumes of 0.2% sodium azide. After three to four uses, the resin must be replaced because it no longer retains the sildenafil.

Characterization of sildenafil purified using Sephadex G25

The preparation of sildenafil purified on Sephadex G-25 was characterized in several ways. The absorbance spectrum shown in Figure 3 indicates that maximum absorbance occurs over a relatively broad absorbance range (279–289 nm) with a trough at 264 nm. Molar extinction coefficient (at 289 nm) of 13 800 at pH 5.2 was determined using a sample of sildenafil provided by Pfizer. Inhibitory potency of sildenafil purified using Sephadex G-25 for human lung PDE5 (Figure 4) indicated that the IC₅₀ was 4 nM. This value agrees well with values determined in this lab and with literature values for potency of sildenafil.^7,8,9 Physical properties of this preparation were similar to those of a purified sample from Pfizer. Molecular weight of sildenafil (474 g/mol) was confirmed by positive ion nanospray and MALDI mass spectrometry. No significant levels of impurities were detected. Analysis by nanospray mass spectroscopy revealed a major peak at 475.37 and a minor peak at 476.37. By collision-induced dissociation fingerprinting, five major fragments of different masses in addition to the original mass of 475.33 were observed. The masses of these fragments were also found in the sample from Pfizer.

Figure 3.

Absorbance profile of sildenafil purified using Sephadex G-25. Ultraviolet absorbance spectrum for sildenafil (0.08 M) purified from the Sephade G-25 was determined over wavelengths ranging from 250 to 320 nm.

Full figure and legend (15K)

Figure 4.

Inhibition potency of sildenafil that had been purified from Viagra tablets. Sildenafil fractions in Figure 1 were lyophilized and resuspended in 0.1% formic acid as described in the text. The IC₅₀ for the preparation was determined using a partially purified human lung PDE5. PDE activity was determined by a modified assay as described earlier using 0.4 M [³H]cGMP as substrate.^10,11

Full figure and legend (13K)

Preparation of radiolabeled sildenafil

Sildenafil (6.7 mg) purified according to this procedure was also used to prepare sildenafil radiolabeled with tritium. Sildenafil powder was sent to Amersham Pharmacia Biotech (Piscataway, NJ, USA) for tritium labeling. Resulting tritiated sildenafil stock solution was 6 Ci/mmol and 34 M. Tritium label is expected on the methyl and propyl groups of the pyrazole ring. The tritiated sildenafil bound specifically to both partially purified and purified bovine lung PDE5, and it showed the same IC₅₀ for PDE5 inhibition as was observed for unlabeled sildenafil (data not shown). The K_D of 4.8 nM (in the presence of 10 M cGMP) agreed well with the IC₅₀.

Other PDE5 inhibitors

It is likely that in the future other PDE5 inhibitors can be purified from commercial sources using this protocol as a guide. We have shown that purified tadalafil (Cialis™) is also retained well beyond the total volume of the Sephadex G-25 column equilibrated in water, and it can be eluted with 1% formic acid. Unlike sildenafil, tadalafil does not elute immediately upon application to the resin, and requires a larger volume of the 1% formic acid. However, when tadalafil is chromatographed on Sephadex G-25 in 1% formic acid, it elutes at 4 column volumes (as measured by the elution position of tritiated water).

Concluding remarks

The procedure described herein provides an easy and inexpensive protocol for purification of sildenafil from commercial sources for use in biochemical studies of PDE5 function, for in vitro tissue studies of cGMP signaling pathways, and for studies in experimental systems where infusion of the drug is indicated. A readily available source of this compound will expand the potential and the feasibility for investigation of the mode of action of sildenafil and other PDE5 inhibitors.

References

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Acknowledgements

We thank Lisa Manier and the Vanderbilt University Mass Spectrometry Research Center, the E Bronson Ingram Cancer Center, and the Diabetes Center of Vanderbilt University School of Medicine. We also thank Pfizer for providing purified sildenafil to be used as a standard. This work was supported by NIH DK58277 and DK40029.