Viagra ( Sildenafil )
Tadalafil, commercially available as Cialis, is a treatment for erectile dysfunction and is a potent inhibitor of phosphodiesterase 5 (PDE5), localized in the brain .
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9.4 Pharmacokinetic interaction between tadalafil and bosentan in healthy male
Tadalafil , an oral PDE5 inhibitor, is being investigated as a treatment for pulmonary arterial hypertension. Bosentan is an oral endothelin receptor antagonist widely used in the treatment of pulmonary arterial hypertension. Tadalafil is mainly metabolized by cytochrome P450 (CYP) 3A4, and as bosentan induces CYP2C9 and CYP3A4, a pharmacokinetic interaction is possible between these agents. This open-label, randomized study investigated whether any pharmacokinetic interaction exists between tadalafil and bosentan. Healthy adult men ( n = 15; 19–52 years of age) received 10 consecutive days of tadalafil 40 mg once daily, bosentan 125 mg twice daily, and a combination of both in a 3-period crossover design. Following 10 days of multiple-dose coadministration of bosentan and tadalafil, compared with tadalafil alone, tadalafil geometric mean ratios (90% confidence interval [CI]) for AUCtau and Cmax were 0.59 (0.55, 0.62) and 0.73 (0.68, 0.79), respectively, with no observed change in tmax. Following coadministration of bosentan with tadalafil, bosentan ratios (90% CI) for AUCtau and Cmax were 1.13 (1.02, 1.24) and 1.20 (1.05, 1.36), respectively. Tadalafil alone and combined with bosentan was generally well tolerated. In conclusion, after 10 days of coadministration, bosentan decreased tadalafil exposure by 41.5% with minimal and clinically irrelevant differences (< 20%) in bosentan exposure  .
Discovery of small molecules for the treatment of Alzheimer’s disease
Praveen P.N. Rao , . Arash Shakeri , in Small Molecule Drug Discovery , 2020
11.2.12 Tadalafil derivatives
Tadalafil , commercially available as Cialis, is a treatment for erectile dysfunction and is a potent inhibitor of phosphodiesterase 5 (PDE5), localized in the brain  . PDEs are essential in hydrolysis of cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) which are closely associated with neurotransmitter release, neuroplasticity, and neuroprotection  . PDE inhibitors have been able to effectively restore cognitive deficits in animal models. In this regard, Li and coworkers through drug repurposing approach designed and developed a set of novel tadalafil derivatives as dual AChE and PDE5 inhibitors  . According to their SAR studies, the N-methyl group of piperazine-2,5-dione was replaceable without significant loss of PDE5 inhibitory activity. A library of 40 derivatives were synthesized and compound 14a ( Fig. 11.15 ) was identified as the lead compound. In the first step of synthesis Pictet-Spengler reaction was carried out where the primary amine of l -tryptophan methyl ester hydrochloride and the aldehyde functional group of piperonal undergo condensation followed by cyclization to get the intermediate possessing the tadalafil core. In the next step, it was reacted with 2-chloroacetyl chloride, followed by coupling with 2-(1-benzylpiperidin-4-yl)ethane-1-amine to afford 14a ( Fig. 11.15 ).
Figure 11.15 . Synthesis and activity of AChE/PDE5 inhibitor MTDL 14a. (i) Piperonal, i-PrOH, reflux, 24 h; (ii) 2-chloroacetyl chloride, Et3N, DCM, −10°C to rt, 6 h; (iii) 2-(1-benzylpiperidin-4-yl)ethan-1-amine, Et3N, MeOH, reflux, overnight.
Compound 14a exhibited excellent AChE inhibition (IC50 = 0.032 μM), comparable to the potency of donepezil. It was significantly less potent for BuChE (IC50 = 3.88 μM) exhibiting strong selectivity toward AChE  . Using immobilized metal ion affinity-based fluorescence polarization (IMAP-FP) assay to evaluate PDE5 inhibitory activity, 14a exhibited moderate inhibition (IC50 = 1.53 μM). It also showed good BBB permeation in PAMPA assay, and in the in vivo cognitive behavior assay, it was able to decrease the latency and the number of errors in the water maze experiment in scopolamine-induced cognitive deficit mouse model. Molecular docking studies of 14a in crystal structures of hAChE and hPDE5A exhibited favorable interactions.
In their pursuit of second-generation AChE/PDE5 inhibitors, Li and coworkers further optimized 14a  . A small library of 19 compounds were synthesized where derivative 14b ( Fig. 11.15 ) was identified as the most potent compound with improved water solubility. The ChE inhibitory activity of 14b was evaluated at 40 μM and compared with known inhibitors huperzine A and donepezil. Compound 14b exhibited 2.1-fold increase in inhibitory activity toward AChE (IC50 = 0.015 μM) in comparison to 14a. However, the BuChE inhibition decreased by 1.95-fold (IC50 = 7.61 μM) with the a 507 selectivity index toward AChE. In the PDE5 inhibitory evaluation, 14b showed 2.1-fold decrease in the inhibition (IC50 = 3.23 μM) compared to 14a and exhibited no activity toward other isoforms of PDEs. Compound 14b was BBB permeable and showed excellent safety profile in its corresponding citrate form at a dosage of 200 mg/kg, oral route in mice model  . The researchers were successful in improving the water solubility of 14a by modifying its structure to 14b ( Fig. 11.15 ) as a citrate salt which showed good water solubility to enhance its oral absorption. These studies highlight the importance of drug repurposing concept in developing novel treatments in the field and the potential of dual AChE/PDE5 inhibitors as possible candidate in treatment of AD.
5.2.2 Comparison of VAR with other PDE-5 inhibitors from the pharmacokinetic perspective
Even though VAR shares the same mechanism of action and selectivity for PDE-5 with sildenafil and tadalafil , there are noted pharmacokinetic disparities that largely affect the efficacy profiles of the three drugs  . The three PDE-5 inhibitors are rapidly absorbed after oral administration and have a rapid onset of action. However, the absolute bioavailability for VAR and sildenafil is limited to only 15% and 40%, respectively, because of extensive presystemic metabolism in the gut wall and liver via CYP3A4 and/or CYP3A5 pathways. The absolute bioavailability of tadalafil has not been reported to date [64,72] . The half-life of tadalafil is 17.5 h, compared to 3.9 h for VAR, and 3.8 h for sildenafil  . Consequently, the duration of action of VAR and sildenafil is found to be about 8–12 h [6,75] , whereas that of tadalafil is about 36 h  . The Cmax of VAR (20.9 ng/mL) is significantly lower than that for sildenafil (450 ng/mL) and tadalafil (378 ng/mL), which might be expected based on its lower bioavailability  . VAR and sildenafil have broadly similar tmax of about 0.8 h which predicts a similar time of onset of action, whereas tadalafil has a tmax of about 2 h  . A high-fat meal has been found to have no significant effect on the rate and extent of tadalafil absorption, but delayed the absorption of VAR and sildenafil, possibly affecting the onset of action [72,78] . Consequently, sildenafil and VAR package inserts state that efficacy may be delayed following a high-fat meal, independently from the type of formulation, while tadalafil can be administered independently of food intake  .
Counterfeit Drugs Studied by NMR
PDE-5 Inhibitors and Others
The PDE-5 inhibitors for the treatment of erectile dysfunctions are the most counterfeited drugs worldwide. Besides sildenafil, vardenafil, and tadalafil of low quality, more than 60 structural analogs are on the market, which have never been tested in clinical trials and, therefore, not approved. Thus, they are potentially highly dangerous for the users. Due to the similarity in structure that makes the identification by HPLC difficult, NMR spectroscopy plays a major role, for either the identification of known PDE-5 inhibitors or structure elucidation of new PDE-5 inhibitors, mostly after purification. Since the market of potency enhancers is still growing and the synthesis is easy to perform, because the starting materials are readily available, the number of new analogous PDE-5 inhibitors will further increase in the future.
The situation is even worse, because many of the PDE-5 inhibitors are ingredients in innumerable dietary supplements and herbal formulations, often not declared on the label. Here, the identification of the only active ingredient is challenging, because the supplements and formulation are complex mixtures of countless components. Even the hyphenation of HPLC and mass spectrometric techniques is sometimes not able to provide enough information about known analogous PDE-5 inhibitor. However, NMR spectroscopy can serve as a first-line method for the detection of counterfeit dietary supplements. This holds also true for other drugs that improve sexual performance, such as yohimbine, phentolamine, testosterone, and other androsterone derivatives.
A similar example has recently been reported by Wiest et al., who found the active ingredients acetaminophen, sulfamethoxazole, indomethacin, and trimethoprim in a Vietnamese herbal medicine by means of NMR spectroscopy as a leading technique accompanied by LC-ESI-MS and IR spectroscopy, the latter necessary for identification of inorganic phosphate.
Strategy and Drug Research
The emergence of PDE5 as a validated drug target based on the commercial success of sildenafil (4), vardenafil (5 ), and tadalafil ( 6) has stimulated substantial interest in this particular PDE in the last 10 years. More details on these and many other PDE5 inhibitors can be found in Section 2.23.6 .
The regulatory domain contains allosteric binding sites for cGMP, and cGMP binding at these sites increases the rate of cGMP hydrolysis. 101,102 These sites are kinetically distinct, and occupation of both is required for activation of the enzyme by phosphorylation at Ser92 by either PKA or protein kinase G (PKG). 103–105 It has been demonstrated that there appears to be a positive correlation between the level of phosphorylation and the catalytic activity of PDE5 in cultured vascular smooth muscle cells. 106 The affinity of these allosteric sites for cGMP is comparatively high (Km as low as 200 nM), and the high selectivity of the site for cGMP has been attributed to the ionization state of Asp289. 107
Phosphorylation of the isolated PDE5 regulatory domain causes a conformational change in this portion of the protein, and this conformational change contributes to the increased binding affinity of the cyclic nucleotide (about 10-fold). 108 Addition of cGMP has been shown to enhance the affinity of tritiated tadalafil and vardenafil. This interesting observation suggests the possibility that PDE5 inhibitors may potentiate their own binding to the enzyme as cyclic nucleotide levels increase as a result of inhibition. 109 The physiological relevance of this information was supported by experiments using rabbit corpus cavernosum tissue, where binding of cGMP to PDE5 and PKG was found to greatly exceed the free concentration of the cyclic nucleotide in the tissue. 110
The regulatory subunit of PDE5 also contains two GAF domains. These regions are contributors to cGMP binding, monomer dimerization, and enzyme regulation. 111 Structural analysis of the portions of the regulatory domain that contribute to each of these features was carried out by Corbin and Francis. 112 It was shown that the GAF a modules can homodimerize with high affinity (KD b modules have even higher affinity (∼20 pM). Dimer stability is controlled by the amino acid sequence linking the two regions. GAF a has higher affinity for cGMP (KD b and the amino acids immediately flanking it are critical for cGMP-mediated stimulation of Ser102 phosphorylation by PKA and PKG. As noted above, PDE5 phosphorylation is an important activating step for enzymatic activity.
Section 2.23.3 describes some of the work that has been carried out to identify key residues for cGMP binding and catalysis. As noted, a divalent cation, particularly zinc, is important for both of these functions. Point mutations in the enzyme indicate that two key histidine residues, His607 and His643, are critical for metal cation binding. Substitution of these amino acids with alanine resulted in a substantial drop in enzyme activity, and a decrease in binding by Mg 2+ or Mn 2+ . It was interesting to note that mutation of several other highly conserved residues within the putative metal binding motif present in PDE5 (e.g., Asn604, His675, or aspartic acids at positions 644, 714, or 754) did not have a substantial influence on catalysis. 113
PDE5 is widely known as a key regulator of vascular tone. In this role, it controls blood flow in the corpus cavernosum tissue of the penis, where it is the primary cGMP-hydrolyzing activity present. Inhibition of enzymatic activity reduces the outflow of blood, leading to engorgement and, ultimately, an erection following sexual stimulation. Recent work using knockout mice lacking in endothelial nitric oxide synthase illustrated that PDE5 expression and activity were downregulated, resulting in priaprism in the knockout mice. 114 Increased PDE5 activity in the inner medullary collecting duct in the kidney has been demonstrated in pregnant rats. This leads to excessive sodium retention, and resistance to the effects of ANP. ANP signals via cGMP, and resistance to the diuretic effects of the peptide hormone are directly related to enzymatic activity. 115 In pulmonary endothelial cells, PDE5 has been recently shown to regulate growth and apoptosis. This process is regulated in part by ANP, as administration of the peptide to pulmonary microvasculature endothelial cells in culture leads to an increase in cGMP concentration, and results in growth inhibition and apoptosis. 116
DOSY NMR for Drug Analysis
3.3 DOSY 1 H NMR analysis of formulations of genuine Cialis® and a Chinese imitation
Nowadays, drugs for the treatment of erectile dysfunction that inhibit the phosphodiesterase type 5 enzyme (PDE-5), sildenafil citrate (Viagra®), tadalafil (Cialis®) or vardenafil hydrochloride (Levitra®, see Figure 2 ) are among the most counterfeited or imitated. Imitations come generally from Asia (India and China most often) which do not recognize the European and American patent laws so that products manufactured legally in such countries are illegal in Europe, USA and other countries. The original Cialis® tablet from Eli Lilly Laboratories and one copy commercialized in China were analyzed. The DOSY 1 H NMR spectra of their solutions are presented in Figure 5 . It is obvious that the two formulations do not contain the same active ingredient. Their common excipients are the lubricant magnesium stearate (Mg st, Δ), the tablet binder hypromellose (#) and lactose (□) as a diluent. Moreover, the brand formulation is composed of another cellulose derivative with a higher molecular weight known to be hydroxypropylcellulose (▀). Signals of sodium lauryl sulphate (laur; ▴), a wetting agent, and triacetin (♦), a hydrophilic plasticizer, are also present.
Figure 5 . 2D DOSY 1 H NMR spectra recorded at 500 MHz of Cialis formulations (solvent CD3CN/D2O, 80/20). (a) the brand formulation from Eli Lilly (Cialis®); (b) Chinese Cialis. T, tadalafil; V, vardenafil; H, homosildenafil; ▪, hydroxypropylcellulose; #, hypromellose; □, lactose; Mg st (Δ), magnesium stearate; Laur (▴), sodium lauryl sulphate; ♦, triacetin; ¦, diethylphtalate; PEG, polyethylene glycol; ?, unknown. A deeper section of some signals is shown in the boxes. For the recording and processing conditions, see legend of Figure 3 .
The intensity of the peaks located in the aromatic region of the 1 H NMR spectrum demonstrates that the Chinese fake formulation of Cialis contains a mixture of two active pharmaceutical ingredients. These two compounds were purified by chromatography and then identified by NMR and LC–MS–MS (liquid chromatography–mass spectrometry–mass spectrometry) as vardenafil (V) and homosildenafil (H) ( Figure 5 ). Their amounts were 34.3 ± 0.2 and 7.2 ± 0.1 mg per tablet, respectively.
Similar to tadalafil, vardenafil is a PDE-5 inhibitor, and indications and contraindications for this medicine are close to those of other PDE-5 inhibitors. However, the structural differences between tadalafil and vardenafil are reflected in dissimilarities of the clinical pharmacokinetics and pharmacodynamics. 30 The brand formulation of vardenafil (Levitra®) is available in 2.5, 5, 10, and 20 mg doses and the normal starting dose is 10 mg. The level of vardenafil found in the Chinese formulation might induce an overdosage in patients. In the case of vardenafil, there was no evidence of a dose-dependent improvement in efficacy beyond the 10 mg dose but problems of drug–drug interactions may occur. For example, vardenafil is contraindicated with nitrates as it potentiates their hypotensive effect. Also vardenafil (10 or 20 mg) when concomitantly administered with α-blockers (such as terazosin and tamsulosin) and with nifedidine to healthy volunteers resulted in some subjects experiencing hypotension. 30 The presence of homosildenafil (see Figure 2 ) has already been described in functional food marketed for penile erectile dysfunction 31 and in dietary supplements 32 but toxicological data are not known or not available. Thus, due to possible side effects or drug–drug interactions, a significant risk is faced by consumers who purchase drugs marketed for erectile dysfunction via the Internet.