Sildenafil Citrate, a Selective Phosphodiesterase Type 5 Inhibitor:

Sildenafil Citrate, a Selective Phosphodiesterase Type 5 Inhibitor: Research and Clinical Implications in Erectile Dysfunction Robert B. Moreland, Irwin Goldstein, Noel N. Kim and Abdulmaged Traish

Under normal physiological conditions, following sexual stimulation, release of nitric oxide (NO) from penile non-adrenergic, non-cholinergic nerves and the endothelium activates guanylyl cyclase and induces intracellular cGMP synthesis in erectile tissue trabecular smooth muscle cells. Increased cGMP levels reduce intracellular Ca 2+ concentrations, inhibiting smooth muscle contractility and thereby initiating the erectile response. Phosphodiesterase type 5 (PDE type 5) is the predominant enzyme responsible for cGMP hydrolysis in trabecular smooth muscle. Activation of PDE type 5 terminates NO-induced, cGMP-mediated smooth muscle relaxation, resulting ultimately in restoration of basal smooth muscle contractility and penile flaccidity. Sildenafil citrate is a potent PDE type 5 reversible and selective inhibitor that blocks cGMP hydrolysis effectively (Ki ~3 nM). Under conditions of excessive adrenergic tone or impaired neurovascular status, following sexual stimulation, sildenafil acts to enhance NO-mediated smooth muscle relaxation, resulting in improved penile erection in men with erectile dysfunction. In this review, we summarize the current state of knowledge of the physiology of penile erection and the pharmacology, metabolism and clinical experience with sildenafil citrate in the management of erectile dysfunction. Erectile dysfunction (ED), the persistent inability to achieve or maintain an erection for satisfactory sexual performance, is a common and important medical problem1–3. Based on a random community-based sample of men 40 to 70 years of age, erectile dysfunction was self-reported in over half of the respondents4. Ten percent claimed complete dysfunction, while 25% and 17% noted moderate and minimal dysfunction, respectively. The prevalence of erectile dysfunction increases with age, doubling between the ages of 40 R.B. Moreland, I. Goldstein and N.N. Kim are at the Department of Urology and A. Traish is at the Departments of Biochemistry and Urology, Boston University School of Medicine, Boston, MA 02118, USA. All correspondence should be addressed to A. Traish.

TEM Vol. 10, No. 3, 1999

and 70 years, for moderate erectile dysfunction, and tripling over the same three decade time period for complete dysfunction. Although age is a significant correlate, other factors were found to predict complete erectile dysfunction. These included treated diabetes mellitus, heart disease and hypertension, medications for diabetes and cardiovascular disease, and diminished values of high-density lipoproteins. Men with treated heart disease and hypertension, who either began or continued cigarette smoking, significantly increased their risk of complete erectile dysfunction4. Considerable advances have been made in the clinical management of erectile dysfunction over the past several decades1,5–7. Historically the

domain of mental health professionals and endocrinologists, contemporary treatment interventions for erectile dysfunction have involved the urologist and have evolved from penile implantation surgery in the 1970s, intracorporal injection of vasoactive agents in the 1980s and transurethral insertion of prostaglandin E1 in 1997. Research on the biochemical and physiological mechanisms regulating erectile tissue trabecular smooth muscle contractility has led to important advances in the pharmacological management of erectile dysfunction. Basic and clinical research has led to newer pharmacological strategies, such as sildenafil citrate, which are safe, effective, reliable, non-invasive, easy to administer and appropriate for a broad range of patients with erectile dysfunction3. Such advances have produced far-reaching changes in the process of care and clinical practice patterns in the management of those afflicted with erectile dysfunction. The primary care physician is playing an increasingly important role in sexual health care medical issues. •

Role of Smooth Muscle Contractility in Erectile Function

The state of relaxation or contraction of the arteriolar and trabecular smooth muscle determines penile erection or flaccidity1,2,8,9. Several neurotransmitters are implicated in erectile function. The exact nature of the neurotransmitters that initiate and/or terminate penile erection remains under investigation. The non-adrenergic, non-cholinergic neurotransmitter nitric oxide (NO) plays a crucial role in attenuating smooth muscle contraction and inducing smooth muscle relaxation and penile erection (Fig. 1). Activation of neurogenic and endothelial NO synthases results in synthesis of NO. Released NO diffuses into smooth muscle cells and binds to the heme component of soluble guanylyl cyclase, stimulating cyclic guanosine monophosphate (cGMP) synthesis. Binding of cGMP to cGMP-dependent protein kinases (PKGs) or cGMPdependent ion channels results in reduction of intracellular Ca21, via Ca21

1043-2760/99/$ – see front matter © 1999 Elsevier Science. All rights reserved. PII: S1043-2760(98)00127-1

97

Acetylcholine Norepinephrine αAR?

MR

αAR?

MR?

Endothelium ↑ Ca2+, IP3

↑ Ca2+, IP3

L-Arg

Arach. acid

L-NNA/LNAME

O2

Prostaglandin G/H synthase

Endothelial NOS

PGE2 Nitric oxide (NO)

PGE2 NANC nerve

Norepinephrine

NO EP R

−

+

MR

α1AR PLC

AC

GC ↑ cGMP

Adrenergic nerve

α2AR

↑ cAMP

↓ cAMP

ACh

↑ IP3

Sarcoplasmic reticulum PDE 5 ↓ Ca2+

↑ Ca2+ Cholinergic nerve

5'GMP

Relaxation

Contraction

Sildenafil

Smooth muscle Figure 1. A conceptual framework depicting mechanisms involved in regulating trabecular smooth muscle tone. The nitric oxide (NO) pathway is highlighted. NO synthesis in the non-adrenergic, non-cholinergic (NANC) nerve is not depicted, but occurs in a fashion similar to the endothelium, with the exception of a different enzyme isoform, neuronal NO synthase, mediating conversion of L-arginine and O2 into NO. Putative a-adrenergic receptor (AR) on the endothelium may counteract the activity of a-AR on smooth muscle through physiological antagonism by release of NO or prostanoids. This functional antagonism, together with synergistic release of NO from NANC nerves leads to increased intracellular cGMP and cAMP, reducing intracellular Ca21 and mediating relaxation. The activity of phosphodiesterases (PDEs) in the smooth muscle contributes to maintaining cyclic nucleotide levels and the overall balance between contraction and relaxation. Sildenafil acts directly upon PDE type 5 by inhibiting its activity to increase the stability of cGMP. AC, adenylate cyclase; ACh, acetylcholine; Arach. acid, arachidonic acid; EP R, PGE receptor; GC, guanylate cyclase; IBMX, 3-isobutyl-1-methyl-xanthine; L-Arg, L-arginine; IP3, inositol trisphosphate; MR, muscarinic receptor; L-NAME, NG-nitro-L-arginine methyl ester; L-NNA, NG-nitro-L-arginine; NO, nitric oxide; NOS, NO synthase; PGE2, prostaglandin E2; PLC, phospholipase C.

sequestration and extrusion, and activation of myosin light chain phosphatases9–12, causing diminution of smooth muscle contractility and enhancing penile erection.

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Several vasoactive agents, including NO, vasoactive intestinal polypeptide (VIP), prostaglandin E1 (PGE1), forskolin, phosphodiesterase inhibitors and a-adrenergic receptor antagonists

initiate and/or enhance corpus cavernosum smooth muscle relaxation5–7,12. Each of these agents affects smooth muscle contractility via a specific and distinct mechanism, which ultimately TEM Vol. 10, No. 3, 1999

Table 1. Selectivity of sildenafil and zaprinast for various PDE isozymes

Isozyme

Name

PDE 1

Calmodulin/ Ca2+ sensitive cGMP stimulated cGMP inhibited cAMP specific (rolipram sensitive) cGMP binding

PDE 2 PDE 3 PDE 4 PDE 5 PDE 6 PDE 7 PDE 8 PDE 9

Photoreceptor cAMP specific (rolipram insensitive) cAMP specific (IBMX insensitive) cGMP specific (sildenafil insensitive)

Substrate

Ki (nM) Sildenafil

Ki (nM) Zaprinast

cAMP, cGMP cAMP, cGMP cAMP

281 >30 000 16 200

6650 >100 000 >100 000

cAMP cGMP

7680 3.5a,b

cGMP

33

77 400 285a 856b 385

cAMP

ND

ND

cAMP

ND

ND

cGMP

7000

29 000

Inhibition constants for zaprinast and sildenafil are taken from Refs 18, 19, 21. aHuman corpus cavernosum smooth muscle cells; bhuman platelets; ND, not determined (adapted from Ref. 13).

leads to changes in intracellular Ca21 and modulation of specific smooth muscle myosin light chain kinases (MLCKs) and myosin light chain phosphatases (MLCPs). These enzymes rapidly change the state of myosin phosphorylation and result in smooth muscle contraction or relaxation9–12. •

Role of NO and Phosphodiesterases in cGMP-mediated Signal Initiation and Termination in Trabecular Smooth Muscle Relaxation

Intracellular cGMP concentrations are regulated by the action of guanylyl cyclases and cGMP-specific phosphodiesterases (PDEs)13–16. Soluble guanylyl cyclase, when activated by NO, catalyzes the formation of cGMP from GTP, whereas cGMP-specific PDEs catalyze the hydrolysis of cGMP to GMP. Termination of signal transduction by hydrolysis of cGMP depends on the specificity of PDE isozymes and the expression of the specific isozyme in the target tissues13–15. To date, nine PDE isozymes have been characterized, each with different cAMP or cGMP specificities (Table 1)13,14,17,18. PDE types 1, 2, 5, 6 and 9 metabolize cGMP with varying efficiencies. Of these isozymes, PDE types 5, 6 and 9 are specific for cGMP. Human erectile TEM Vol. 10, No. 3, 1999

corpus cavernosum smooth muscle expresses PDE types 2, 3, 4 and 5 (Refs 16, 19–21). PDE type 6 is expressed only in the retina13. The recently cloned PDE type 9 is highly expressed in kidney and spleen and to lesser extents in liver, lung, small intestine and brain17,18. It is currently unknown whether PDE type 9 is expressed in human corpus cavernosum. Kinetic analysis of PDE types 2 and 3 showed that higher substrate (cGMP) concentrations are required for activity, suggesting that these isozymes might not regulate physiological intracellular cGMP levels when present at low concentration14,16,20,21. However, PDE type 5 hydrolyzes cGMP at low substrate concentration (Km = 0.75–2 mM) and represents the main cGMP hydrolytic activity in human corpus cavernosum erectile tissue19–21. Because different tissues express specific PDE isozymes, regulation of signal termination is dependent on tissue type, cellular responses and cyclic nucleotide metabolism13–15. PDE type 5 was first identified as the main cGMP-binding protein in bovine lung22 and was later characterized as a cGMP-binding, cGMP-specific PDE (Refs 23–25). Bovine pulmonary PDE type 5 is expressed as a

6.9-kb mRNA, is comprised of 875 amino acids and forms a homodimer of 99.5-kDa subunits24. Human PDE type 5 has recently been cloned and is expressed in a wide variety of tissues as a 9-kb major transcript and an 8 kb minor transcript26. Human PDE type 5 is 95% identical to the bovine lung PDE type 5 and is comprised of 875 amino acids with subunit molecular mass of 100 kDa (Ref. 26). PDE type 5 contains two zinc atoms per monomer which bind with high affinity (Kd = ~0.5 mM) and are necessary for catalysis25. Each subunit contains one allosteric site for cGMP binding (Kd = ~1.3 and 3 mM) and the occupancy of these sites is necessary for PDE type 5 phosphorylation27,28. Phosphorylation has been shown to be important in the regulation of phosphodiesterase activity in PDE isoforms other than PDE type 5 (Ref. 29). PDE type 5 is phosphorylated by PKG (Ref. 30); however, the role of PDE type 5 phosphorylation in regulating PDE activity is still unclear27,28. The active site of PDE type 5 has been mapped by means of site-directed mutagenesis and characterization of the purified mutant enzymes27,28. Substitution of four amino acids alters cGMP selectivity of PDE type 5 towards 99

O N

HN H2N

Me

O

NH

N

Et O

O N

N

HN

N N

nPr

O

nPr O

HN

N N

NH

O −O-P=O

O

OH

O=S=O N

N Me cGMP

Sildenafil

Zaprinast

Figure 2. Chemical structures of cGMP and the phosphodiesterase type 5 selective inhibitors sildenafil and zaprinast.

cAMP more than 100-fold31. Within the active site, substitution of histidine 643 and aspartate 754 (common to all PDEs), histidines 603, 607 and 647, glutamate 672 and aspartate 714 produced large changes in kcat, while substitutions in tyrosine 602 and glutamate 775 dramatically increased the Km (wild-type, ~2 mM) without affecting the velocity of the enzyme32. Interestingly, the residues that were responsible for the binding of the competitive PDE type 5 selective inhibitor zaprinast were different from those defined in the active site32. Expression of PDE type 5 has been reported in tracheal, pulmonary and vascular smooth muscle13,14 as well as in platelets19. PDE type 5 activity isolated from total corpus cavernosum tissues may be derived in part from platelets contaminating the tissue 19,20. To ascertain whether human corpus cavernosum smooth muscle expresses PDE type 5 activity, we have used cultured human corpus cavernosum smooth muscle cells. Our results demonstrate that human trabecular smooth muscle cells express PDE type 5 as the major isozyme21. These observations were supported by recent cloning of human PDE type 5 and in situ hybridization in human corpus cavernosum26,33. Although the distribution of PDE type 5 activity in various tissues is less well characterized, PDE type 5 mRNA has been reported in corpus cavernosum, uterus, bladder,

100

prostate, colon, placenta, pancreas26, lung34 and Purkinje cells during neuronal development35. •

Role of Sildenafil Citrate in Regulation of Human Corpus Cavernosum Smooth Muscle Tone: Enhancement of Erectile Function

Inhibition of PDE type 5 activity in human corpus cavernosum would be expected to increase intracellular cGMP levels in response to stimuli that activate the NO–cGMP pathway. This increase in intracellular cGMP levels would further be expected to lead to enhanced trabecular smooth muscle relaxation1,3,8,9. Consequently, PDE type 5 inhibition is a potential target for pharmacotherapy of erectile dysfunction3,5,19,20. Sildenafil, (1-[4ethoxy-3-(6,7-dihydro-1-methyl-7-oxo3-propyl-1-H-pyrazolo [3,4-d]pyrimidin-5-yl) phenyl sulfonyl]-4-methylpiperazine), a selective, reversible, PDE type 5 inhibitor, has been approved by the Food and Drug Administration as an oral agent to treat male erectile dysfunction (Fig. 2)3,19,20,36,37. Sildenafil effectively inhibited the activity of PDE type 5 isolated from human corpus cavernosum tissue20, cultured human corpus cavernosum smooth muscle cells21 and human platelets19. Sildenafil exhibited high affinity for PDE type 5 and type 6 with inhibition constants (Ki) of ~3.5 and 33 nM, respectively19. The inhibition

constants for sildenafil and zaprinast for PDE isozymes are shown in Table 1. Based on these data, sildenafil citrate did not appreciably inhibit other PDE isozymes, except PDE type 6 (Ki = 33 nM). These observations may account for some of the transient visual side effects reported with the use of sildenafil by patients with erectile dysfunction3,40. Sildenafil enhances sodium nitroprusside- or transmural electrical stimulation-induced relaxation of precontracted corpus cavernosum muscle strips in organ baths, suggesting that sildenafil augments the activity of NOmediated relaxation12,19,38,39. Sildenafil citrate increases intracellular cGMP concentrations in cultured smooth muscle cells treated with sodium nitroprusside21 and in rabbit corpus cavernosum in vitro12,39. Inhibition of PDE type 5 activity by sildenafil correlated with the increased intracellular concentrations of cGMP and decreased phosphorylation of myosin light chain (40% to 28%) in rabbit corpus cavernosum in vitro12. Sildenafil citrate enhanced erectile function following pelvic nerve stimulation in anesthetized dogs as measured by increased intracavernosal pressure41. These responses were attenuated by Lnitroarginine, suggesting involvement of NO synthases and the generation of NO (Ref. 41). These studies indicate that sildenafil citrate is a potent, selective inhibitor of PDE type 5, and might explain its success in the treatment of male erectile dysfunction. •

Pharmaco-kinetics and Metabolism of Sildenafil

After oral administration of sildenafil citrate, maximal plasma concentrations are reached within 60–120 min20. Approximately 96% of sildenafil and its metabolite are bound to plasma proteins. The reversible binding of sildenafil and its active metabolite determines the free active drug available to enter the smooth muscle and inhibit its target enzyme. Sildenafil is metabolized in the liver by cytochrome P450 and is converted into an active metabolite with characteristics similar to the parent compound. The half-life of sildenafil and its active metabolite is ~4 h (Refs 20, 42). TEM Vol. 10, No. 3, 1999

•

Clinical Experience with Sildenafil

The results of several clinical trials with oral sildenafil citrate in the treatment of men with erectile dysfunction have been reported recently3,20,36,37. In one such double-blind, placebocontrolled, multi-institutional trial, sildenafil (25 mg, 50 mg and 100 mg) or placebo were administered to 532 men with erectile dysfunction in a 24 week dose-response study. Increasing the dose of sildenalfil was found to be associated with significant increases in scores for achieving and maintaining erections. At 100 mg of sildenafil, the mean score for achieving erections was 100% higher after treatment than at baseline. After the 24 weeks of treatment, improved erections were noted in 56%, 77% and 84% of the men taking 25 mg, 50 mg and 100 mg sildenafil, respectively, compared with 25% in those taking placebo3. In another double-blind, placebocontrolled, multi-institutional trial, sildenafil (25 mg, 50 mg and 100 mg) or placebo were administered to 329 different men with erectile dysfunction in a 12 week dose-escalation study20. In the last four weeks of this study, 69% of all attempts at sexual intercourse were successful in men receiving sildenafil compared with 22% in men receiving placebo. For those receiving sildenafil, the mean number of successful intercourse attempts per month was 5.9 compared with 1.5 for those taking placebo. The mean scores for erectile function, orgasmic function, intercourse satisfaction and overall satisfaction were significantly higher for men taking sildenafil compared with those taking placebo. No changes in libido were noted for men taking sildenafil compared with placebo. The efficacy of sildenafil citrate in men with severe erectile dysfunction was examined in a meta-analysis of data from 3361 patients who were followed in ten double-blind, placebocontrolled, fixed dose or flexible dose studies of sildenafil (50–100 mg)36. Severe erectile dysfunction was defined by having erectile dysfunction for at least six months and having a baseline score of 0 or 1 out of 5 for the ability TEM Vol. 10, No. 3, 1999

to initiate or maintain erections. A total of 2085 patients met the criteria for severe erectile dysfunction, of whom 66% were classified as having organic, 12% psychogenic and 22% mixed erectile dysfunction. After sildenafil treatment, the percentages of men able to score 4 or 5 out of 5 for the ability to initiate or maintain erections were 46% and 48%, respectively, compared with 8% and 8%, respectively, for placebo. The efficacy of sildenafil citrate on aging men was examined in a similar meta-analysis of 2982 men (2240 men 65 and under, and 742 men over 65) from eight double-blind, placebocontrolled, fixed dose or flexible dose studies of sildenafil (25–100 mg)37. It was found that patients over 65 years of age had similar responses to sildenafil as patients less than 65 years old in terms of their ability to obtain or maintain an erection for satisfactory sexual intercourse. In clinical trials, sildenafil sideeffects included headache (32% at the 100 mg dose), flushing (21% at the 100 mg dose), dyspepsia (17% at the 100 mg dose), rhinitis (12% at the 100 mg dose) and visual disturbances (10% at the 100 mg dose)3,40. Of note, sildenafil treatment was not examined in patients with retinitis pigmentosa, active peptic ulcer disease, poorly controlled diabetes mellitus, stroke or myocardial infarction within six months, or a history of alcohol or substance abuse 3 as such individuals were excluded from entering these studies. The safety of sildenafil was further assessed in a review of a series of double-blind, placebo-controlled studies and openlabel sildenafil treatment extension studies3,40. Data from a total of 4274 patients (2722 sildenafil, 1552 placebo; age 19–87 years) were available for analysis. The most commonly reported, adverse side-effects of sildenafil were headache (16% sildenafil, 4% placebo), facial flushing (10% sildenafil, 1% placebo), dyspepsia (7% sildenafil, 2% placebo), nasal congestion (4% sildenafil, 2% placebo), urinary tract infection (3% sildenafil, 2% placebo), abnormal vision (3% sildenafil, 0% placebo), diarrhea (3%

sildenafil, 1% placebo), dizziness (2% sildenafil, 1% placebo) and rash (2% sildenafil, 1% placebo). These symptoms were predominantly transient and mild or moderate in nature. To date, no cases of priapism have been reported. Based on available data in double-blind studies, the rate of discontinuation of treatment because of adverse events was similar for sildenafil (2.5%) and placebo (2.3%). In open-label, extension studies with sildenafil, the rate of discontinuation of treatment secondary to adverse events was 2%. The most common reason for treatment discontinuation was headache. Transient visual disturbances (mild to moderate color tinge to vision, changes in perception of color, hue or brightness) have been reported3,40 and may be related in part to sildenafil citrate inhibition of PDE type 6 (Ref. 19) in the retina13. Although increases in cGMP have been reported to be associated with programmed cell death of retinal rods and cones in long-term inhibition of PDE type 6 (Ref. 43), no chronic visual sequelae have been reported in clinical trials with sildenafil citrate. Of note, the incidence and nature of visual adverse events were similar in diabetic and non-diabetic patients40. One of the main contraindications of sildenafil use is treatment with nitrates (nitroglycerine, amyl nitrate, etc.) for ischemic coronary artery disease3,40. Combinations of nitrates and sildenafil have resulted in severe systemic systolic hypotension and have proved fatal. Although concern exists for sildenafiltreated men with coronary artery disease and erectile dysfunction, there is no indication from the current clinical trial data that administration of sildenafil increases the risk of myocardial infarction40. •

Sildenafil and Erectile Dysfunction: Future Implications for Basic and Clinical Research

The clinical availability of an effective, convenient, safe and acceptable oral medication, such as sildenafil, for treatment of men with erectile dysfunction poses multiple research and clinical questions. Recent studies have 101

shown widespread expression of PDE type 5 mRNA in various tissues, such as the lung, kidney, pancreas, colon, stomach, bladder and prostate26. Thus, because sildenafil is administered systemically, it remains to be determined whether this drug has potential adverse side-effects on the metabolism or physiological functions in these tissues. Furthermore, the possible compensatory upregulation of PDE type 5 in response to sildenafil is unknown. Upregulation of PDE type 5 may lead to sildenafil tolerance, requiring higher systemic doses of the drug, and leading to more frequent adverse sideeffects. Thus, long-term (5–10 years) use of sildenafil might produce sideeffects that are not detectable in shortterm clinical studies. High concentrations of cGMP have been shown to induce apoptotic cell death of retinal rods and cones43. In addition, sildenafil inhibits retinal PDE type 6 activity (Ki = 33 nM). A PDE type 5-specific pharmacotherapeutic agent for treatment of erectile dysfunction would be more desirable. High concentrations of cGMP have also been shown to prevent vascular smooth muscle cell proliferation by PKG-dependent mechanisms44. In view of the widespread expression of PDE type 5 mRNA in many tissues, the effects of sildenafil on cell growth, and programmed cell death need to be investigated. This raises the question of what are the long-term effects of sildenafil use on cell growth, cell death, tissue physiological function and tissue properties. Although sildenafil is efficacious in patients with mild, moderate or severe erectile dysfunction3,36, it remains to be determined whether sildenafil can improve erectile dysfunction in patients with erectile tissue fibrosis. Such fibrotic tissue states are often accompanied by an impaired corporal veno-occlusive mechanism, and have been reported in patients with diabetes, cardiovascular and/or peripheral vascular disease45. Thus, whether sildenafil is an effective agent in the treatment of men with erectile dysfunction of various etiologies remains unknown.

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Will Sildenafil Be an Effective Prophylactic Agent in Minimal and Moderate Erectile Dysfunction? A functional veno-occlusive mechanism is crucial for erectile function, and is dependent on tissue structure and the ratio of trabecular smooth muscle to connective tissue in the corpus cavernosum1,45,46. Changes in penile blood oxygenation during erection47 may be necessary for maintaining a functional balance of connective tissue and smooth muscle45,46. Nocturnal penile tumescence (NPT), which occurs coincident with rapid eye movement sleep, may be a mechanism for physiological oxygenation of the corpus cavernosum to maintain tissue structure for veno-occlusive function45,46. Arterial insufficiency, vascular risk factors, radical prostatectomy, aging, and/or loss of NPT are associated with an increased incidence of erectile dysfunction45,46. Potentially, one application of sildenafil is in the preservation and maintenance of NPT with age. This premise may be the basis for use of sildenafil as a prophylactic agent. Will there Be Available Pharmacologic Strategies to Enhance Erectile Function in Sildenafil Treatment Failures? The results of clinical trials with sildenafil show ~50–70% success rates3,20,36,37,40. Thus, one would assume that sildenafil alone is ineffective in ~30–50% of patients with erectile dysfunction. Based on clinical experience with other pharmacotherapeutic agents for erectile dysfunction, combination and multiagent therapy have proved more effective in men who do not respond to single agent treatment. Potentially, men with erectile dysfunction who do not respond to sildenafil treatment may benefit from a combination with other pharmacological agents. Will Sildenafil Be Useful for Treatment of Female Sexual Dysfunction, Specifically Female Sexual Arousal Disorder? The approval of a safe and effective oral agent to increase blood flow to erectile tissue in men with erectile dysfunction has raised the possibility of a similar

pharmacological effect on vaginal and clitoral tissue in selected females with sexual dysfunction48. At the present time, however, only limited information is available on the physiological, biochemical and pharmacological mechanisms of vaginal vasocongestion and clitoral engorgement48–50. Recently, PDE type 5 has been reported in human clitoral corpus cavernosum smooth muscle cells and this activity has been shown to be inhibited by sildenafil (Ki = 7.2 nM) and zaprinast (Ki = 400 nM)51. While research into female sexual function and clitoral and vaginal engorgement disorders is ongoing, the potential use of sildenafil in treatment of female sexual dysfunction must await further basic and clinical studies on female sexual function to define more precisely the mode of action of this pharmacological agent. This review summarizes the current state of knowledge of the usefulness of sildenafil in the treatment of male erectile dysfunction; however, many questions remain unanswered regarding the long-term impact of sildenafil use in treatment of erectile dysfunction. The increased public awareness of male and female sexual function will advance new areas of research into understanding the complex physiological mechanisms of sexual function and promote the development of new systemic agents for the treatment of sexual dysfunction. •

Acknowledgements

We are grateful to Ms Jerie McGrathCerqua for her administrative assistance. This work was supported by NIH grants DK 39080, DK 40025 and DK47950. References 1 Saenz de Tejada, I. (1992) In the physiology of erection, signposts to impotence. Contemp. Urol. 7, 52–68 2 Andersson, K-E. and Wagner, G. (1995) Physiology of erection. Physiol. Rev. 75, 191–236 3 Goldstein, I., Lue, T.F., Padma-Nathan, H., Rosen, R.C., Steers, W.D. and Wicker, P.A. (1998) Oral sildenafil in the treatment of erectile dysfunction. Sildenafil study group. New Engl. J. Med. 338, 1397–1404 4 Feldman, H.A., Goldstein, I., Hatzichristou, D.G., Krane, R.J. and McKinlay, J.B.

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32 Turko, I.V., Francis, S.H. and Corbin, J.D. (1998) Potential roles of conserved amino acids in the catalytic domain of the cGMP-binding c-GMP-specific phosphodiesterase. J. Biol. Chem. 273, 6460–6466 33 Rulten, S.L. et al. (1998) Isolation of a human phosphodiesterase 5 cDNA and in situ analysis of its expression in human corpus cavernosum. J. Urol. 159, 92 (abstr. 351) 34 Sanchez, L.S., de la Monte, S.M., Filippov, G., Jones, R.C., Zapol, W.M. and Bloch, K.D. (1998) Cyclic-GMP-binding, cyclicGMP-specific phosphodiesterase (PDE5) gene expression is regulated during rat pulmonary development. Pediatr. Res. 43, 163–168 35 Kotera, J. et al. (1997) Expression of rat cGMP-binding cGMP-specific phosphodiesterase mRNA in Purkinje cell layers during postnatal neuronal development. Eur. J. Biochem. 249, 434–442 36 Steers, W.D. (1998) Meta-analysis of the efficacy of sildenafil (Viagra™) in the treatment of severe erectile dysfunction. J. Urol. 159, 238 (abstr. 910) 37 Wagner, G. (1998) Analysis of the efficacy of sildenafil (Viagra™) in the treatment of male erectile dysfunction in elderly patients. J. Urol. 159, 239 (abstr. 912) 38 Stief, C.G., Uckert, S., Becker, A.J., Truss, M.C. and Jonas, U. (1998) The effect of the specific phosphodiesterase (PDE) inhibitors on human and rabbit cavernous tissue in vitro and in vivo. J. Urol. 159, 1390–1395 39 Jeremy, J.Y., Ballard, S.A., Naylor, A.M., Miller, M.A. and Angelini, G.D. (1997) Effects of sildenafil. A type 5 cGMP phosphodiesterase inhibitor and papverine on cyclic GMP and cyclic AMP levels in rabbit corpus cavernosum in vitro. Br. J. Urol. 79, 958–963 40 Morales, A., Gingell, C., Collins, M., Wicker, P.A. and Osterloh, I.H. (1998) Clinical safety of oral sildenafil citrate (Viagra™) in the treatment of erectile dysfunction. Int. J. Impot. Res. 10, 69–74 41 Carter, A.J., Ballard, S.A. and Naylor, A.M. (1998) Effect of the selective phosphodiesterase type 5 inhibitor on erectile function on the anesthetized dog. J. Urol. 160, 242–246 42 Cooper, J.D., Muirhead, D.C., Taylor, J.E. and Baker, P.R. (1997) Development of an assay for simultaneous determination of sildenafil (Viagra™) and its metabolite (UK-103,320) using automated sequential trace enrichment of dialysates and high performance liquid chromatography. J. Chromatogr. B Biomed. Appl. 701, 87–95 43 Fox, D.A., Campbell, M.L. and Blocker, Y.S. (1997) Functional alterations and apoptotic cell death in the retina following developmental or adult lead exposure. Neurotoxicology 18, 645–664 44 Boerth, N.J., Dey, N.B., Cornwell, T.L. and Lincoln, T.M. (1997) Cyclic GMP-

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Molecular Genetics of Type 1 Glycogen Storage Diseases Janice Yang Chou and Brian C. Mansfield

Glycogen storage disease type 1 (GSD-1), also known as von Gierke disease, is caused by a deficiency in the activity of the enzyme glucose-6-phosphatase (G6Pase). It is an autosomal recessive disorder characterized by hypoglycemia, hepatomegaly, kidney enlargement, growth retardation, lactic acidemia, hyperlipidemia and hyperuricemia. The disease presents with both clinical and biochemical heterogeneity consistent with the existence of two major subgroups, GSD-1a and GSD-1b, which have been confirmed at the molecular genetic level. GSD-1a, the most prevalent form, is caused by mutations in the G6Pase gene that abolish or greatly reduce enzymatic activity. The gene maps to chromosome 17q21 and encodes a microsomal transmembrane protein. Animal models of GSD-1a exist and are being exploited to delineate the disease more precisely. It has been proposed that GSD-1b is caused by a defect in the microsomal glucose-6-phosphate transporter. The gene responsible for GSD-1b has been mapped to chromosome 11q23 and a cDNA encoding a microsomal transmembrane protein has been identified. The function of this putative GSD-1b protein remains to be determined. These recent developments, along with newly characterized animal models of GSD-1a, are increasing our understanding of the interrelationship between the components of the G6Pase complex and type 1 glycogen storage diseases. Glycogen storage disease type 1 (GSD1) is a group of autosomal recessive disorders with a combined incidence of ~1 in 100 000 live births1. Patients J.Y. Chou and B.C. Mansfield are at the Heritable Disorders Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA. B.C. Mansfield is currently at Protein Development, Human Genome Sciences Inc., Rockville, MD 20852, USA.

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afflicted with GSD-1 are unable to maintain glucose homeostasis and present with hypoglycemia, hepatomegaly, kidney enlargement, growth retardation, hyperlipidemia, hyperuricemia and lactic acidemia, which are clearly related to defects in glucose metabolism. However, long-term problems can include complications less obviously related to glucose such as gout, hepatic adenomas with risk for

Published by Elsevier Science Ltd. PII: S1043-2760(98)00123-4

50 Park, K., Goldstein, I., Andry, C., Siroky, M.B., Krane, R.J. and Azadzoi, K.M. (1997) Vasculogenic female sexual dysfunction: the hemodynamic basis for vaginal engorgement insufficiency and clitoral erectile insufficiency. Int. J. Impot. Res. 9, 27–37 51 Park, K., Moreland, R.B., Atala, A., Goldstein, I. and Traish, A.M. (1998) Characterization of phosphodiesterase activity in human clitoral corpus cavernosum smooth muscle cells in culture. Biochem. Biophys. Res. Commun. 249, 612–617

malignancy, osteoporosis, platelet dysfunction, pulmonary hypertension and renal failure1,2. The GSD-1 abnormality is caused by a deficiency in the activity of the microsomal enzyme glucose-6phosphatase (G6Pase)1, which catalyzes the terminal steps in gluconeogenesis and glycogenolysis; the conversion of glucose-6-phosphate (G6P) to glucose and phosphate3 (Fig. 1). G6Pase is considered to be the key enzyme in glucose homeostasis. It is expressed primarily in the gluconeogenic organs, the liver and kidney, and to a lesser extent in the intestine and pancreas3. Before 1976, the prognosis for GSD-1 was guarded; however, over the past 20 years, dietary therapy, consisting of continuous nasogastric infusion of glucose or frequent oral administration of uncooked cornstarch, has greatly improved the prognosis1,2. As a result, many GSD-1 patients are now young adults. Whether the dietary therapy will eliminate or reduce the long-term complications of GSD-1 will be seen as this first generation of patients matures. Because the clinical aspects of GSD-1 have been reviewed extensively1,2, this review will focus primarily on the molecular genetics of GSD-1. •

GSD-1 is a Group of Autosomal Recessive Disorders

GSD-1 was first described by von Gierke in 1929. Cori and Cori4 showed that the disorder is caused by the absence of G6Pase activity, establishing for the first time that metabolic disorders could arise from enzyme deficiencies. With the increasing number of patients identified, it became apparent that a number of the patients TEM Vol. 10, No. 3, 1999