Humidity induced phase transformation of poloxamer 188 and its effect on physical stability of amorphous solid dispersion of AMG 579, a PDE10A inhibitor.

Poloxamer 188, a commonly used emulsifying and solubilizing agent, was found to be the cause of crystallization of an investigational drug, AMG 579, from its amorphous solid dispersion at accelerated storage conditions. Investigation of this physical stability issue included thorough characterization of poloxamer 188 at non-ambient conditions. At 40°C, poloxamer 188 becomes deliquescent above relative humidity of 75%. Upon returning to ambient conditions, the deliquescent poloxamer 188 loses water and re-solidifies. The reversible phase transformation of poloxamer 188 may cause physical and chemical stability issues and this risk should be assessed when selecting it as an excipient for formulation development.

Surface plasmon resonance biosensor assay for the analysis of small-molecule inhibitor binding to human and parasitic phosphodiesterases.

In the past decade, surface plasmon resonance (SPR) biosensor-based technology has been exploited more and more to characterize the interaction between drug targets and small-molecule modulators. Here, we report the successful application of SPR methodology for the analysis of small-molecule binding to two therapeutically relevant cAMP phosphodiesterases (PDEs), Trypanosoma brucei PDEB1 which is implicated in African sleeping sickness and human PDE4D which is implicated in a plethora of disease conditions including inflammatory pulmonary disorders such as asthma, chronic obstructive pulmonary disease and central nervous system (CNS) disorders. A protocol combining the use of directed capture using His-tagged PDE_CDs with covalent attachment to the SPR surface was developed. This methodology allows the determination of the binding kinetics of small-molecule PDE inhibitors and also allows testing their specificity for the two PDEs. The SPR-based assay could serve as a technology platform for the development of highly specific and high-affinity PDE inhibitors, accelerating drug discovery processes.

Fragment-assisted hit investigation involving integrated HTS and fragment screening: Application to the identification of phosphodiesterase 10A (PDE10A) inhibitors.

Fragment-based drug design (FBDD) relies on direct elaboration of fragment hits and typically requires high resolution structural information to guide optimization. In fragment-assisted drug discovery (FADD), fragments provide information to guide selection and design but do not serve as starting points for elaboration. We describe FADD and high-throughput screening (HTS) campaign strategies conducted in parallel against PDE10A where fragment hit co-crystallography was not available. The fragment screen led to prioritized fragment hits (IC50's ∼500µM), which were used to generate a hypothetical core scaffold. Application of this scaffold as a filter to HTS output afforded a 4µM hit, which, after preparation of a small number of analogs, was elaborated into a 16nM lead. This approach highlights the strength of FADD, as fragment methods were applied despite the absence of co-crystallographical information to efficiently identify a lead compound for further optimization.

Validated spectrofluorimetric method for determination of two phosphodiesterase inhibitors tadalafil and vardenafil in pharmaceutical preparations and spiked human plasma.

A valid, sensitive and rapid spectrofluorimetric method has been developed and validated for determination of both tadalafil (TAD) and vardenafil (VAR) either in their pure form, in their tablet dosage forms or spiked in human plasma. This method is based on measurement of the native fluorescence of both drugs in acetonitrile at λem 330 and 470 nm after excitation at 280 and 275 nm for tadalafil and vardenafil, respectively. Linear relationships were obtained over the concentration range 4-40 and 10-250 ng/mL with a minimum detection of 1 and 3 ng/mL for tadalafil and vardenafil, respectively. Various experimental parameters affecting the fluorescence intensity were carefully studied and optimized. The developed method was applied successfully for the determination of tadalafil and vardenafil in bulk drugs and tablet dosage forms. Moreover, the high sensitivity of the proposed method permitted their determination in spiked human plasma. The developed method was validated in terms of specificity, linearity, lower limit of quantification (LOQ), lower limit of detection (LOD), precision and accuracy. The mean recoveries of the analytes in pharmaceutical preparations were in agreement with those obtained from the comparison methods, as revealed by statistical analysis of the obtained results using Student's t-test and the variance ratio F-test.

Structure based virtual screening to identify selective phosphodiesterase 4B inhibitors.

Phosphodiesterase 4 (PDE4), is a hydrolytic enzyme, is proposed as a promising target in asthma and chronic obstructive pulmonary disease. PDE4B selective inhibitors are desirable to reduce the dose limiting adverse effect associated with non-selective PDE4B inhibitors. To achieve this goal, ligand based pharmacophore modeling and molecular docking approach is employed. Pharmacophore hypotheses for PDE4B and PDE4D are generated using HypoGen algorithm. The best PDE4B pharmacophore hypothesis (Hypo1_PDE4B) consist of one hydrogen-bond acceptor and two ring aromatic features, whereas PDE4D pharmacophore hypothesis (Hypo1_PDE4D) consist of one hydrogen-bond acceptor, one hydrophobic aliphatic, and two ring aromatic features. The validated pharmacophore hypotheses are used in virtual screening to identify selective PDE4B inhibitors. The hits were screened for their estimated activity, FitValue, and quantitative estimation of drug likeness. After molecular docking analysis, ten hits were purchased for in vitro analysis. Out of these, six hits have shown potent and selective inhibitory activity against PDE4B with IC50 values ranging from 2 to 378nM.

Film-coated matrix mini-tablets for the extended release of a water-soluble drug.

Extended release (ER) of water-soluble drugs from hydroxypropylmethylcellulose (HPMC) matrix mini-tablets (mini-matrices) is difficult to achieve due to the large surface area to volume ratio of the mini matrices. Therefore, the aims of this study were to control the release of a water-soluble drug (theophylline) from mini-matrices by applying ER ethylcellulose film coating (Surelease®), and to assess the effects of Surelease®:pore former (Opadry®) ratio and coating load on release rates. Mini-matrices containing 40%w/w HPMC K100M CR were coated with 100:0, 85:15, 80:20, 75:25 or 70:30 Surelease®:Opadry® to different coating weight gains (6-20%). Non-matrix mini-tablets were also produced and coated with 80:20 Surelease®:Opadry® to different coating weight gains. At low coating weight gains, nonmatrix mini-tablets released the entire drug within 0.5 h, while at high coating weight gains only a very small amount (<5%) of drug was released after 12 h. The gel formation of HPMC prevented disintegration of mini-matrices at low coating weight gains but contributed to rupture of the film even at high coating weight gains. As a result, drug release from mini-matrices was slower than that from nonmatrix mini-tablets at low coating weight gains, yet faster at high coating weight gains. An increase in the lag time of drug release from mini-matrices was observed as the concentration of Opadry® reduced or the coating weight gain increased. This study has demonstrated the possibility of extending the release of a water-soluble drug from HPMC mini-matrices by applying ER film coating with appropriate levels of pore former and coating weight gains to tailor the release rate.

Quantitative analyses of CTP-499 and five major metabolites by core-structure analysis.

CTP-499 is a novel oral multi-subtype selective inhibitor of PDEs that is currently in clinical testing, in combination with angiotensin modulators, as a potentially first-in-class treatment for diabetic kidney disease. The compound was discovered and developed by using Concert's proprietary DCE Platform(®) in which deuterium was incorporated at select positions of 1-((S)-5-hydroxyhexyl)-3,7-dimethylxanthine (HDX). CTP-499 metabolizes to five major metabolites: C-21256, D-M2, D-M3, D-M4 and M5, of which all contains deuterium except M5. During in vivo metabolism, however, H/D exchange takes place. As a result, each analyte, except M5, has multiple molecular masses. To accurately quantify the analytes, we developed an LC-MS/MS method focusing on the core structures of the molecules, termed "core-structure analyses". The core-structure analyses method was then validated under GLP guidance in dog, rat and rabbit plasma, with a sample volume of 50 µL. Results demonstrated that this approach accurately quantifies each of the six analytes despite partial exchange of deuterium with hydrogen atoms in the in vivo samples. The validation parameters included accuracy, precision, sensitivity, stability, dilution integrity, hemolysis, matrix effect, selectivity, and recovery. Acceptable intra-run and inter-run assay precision (%CV ≤ 5.5%) and accuracy (90.1-106.7%) were achieved over a linear range of 10-5,000 ng/mL of each analyte. Various stability tests, including bench-top, freeze/thaw, stock solution, and long-term storage, were also performed. All stability results met acceptance criteria. The robustness of the methods was demonstrated by the incurred sample reproducibility (ISR) tests. After validation, the method was successfully used in support of multiple toxicological studies of CTP-499.

Synthesis of core-shell magnetic molecularly imprinted polymers and detection of sildenafil and vardenafil in herbal dietary supplements.

An analytical procedure for selective extraction of sildenafil and vardenafil in herbal dietary supplements (HDSs) has been set up by using the magnetic molecularly imprinted polymers (MMIPs) as the extraction and clean-up materials, followed by high performance liquid chromatography-ultraviolet (HPLC-UV). The MMIPs were prepared by a surface molecular imprinting technique, using Fe(3)O(4) magnetite as a magnetically susceptible component, sildenafil as template molecule, 2-(trifluoromethyl) acrylic acid (TFMAA) as functional monomer, ethylene glycol dimethacrylate (EGDMA) as polymeric matrix components. The MMIPs were characterized by transmission electron microscope (TEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD) and vibrating sample magnetometer (VSM), respectively. The heterogeneity of the MMIPs was modeled with the Freundlich isotherm equation. The resulting MMIPs had high recognition ability and fast binding kinetics for sildenafil. The MMIPs were used as dispersive solid-phase extraction (DSPE) materials to selectively extract sildenafil and vardenafil from HDSs, the contents of sildenafil and vardenafil were found to be 8.05 and 3.86 µg g(-1), respectively, and the average recoveries in spiked HDSs were 70.91-91.75% with a relative standard deviation (R.S.D.) below 7%. The MMIPs were successfully used to selectively enrich and determine sildenafil and vardenafil from HDSs.

Simultaneous determination of yohimbine, sildenafil, vardenafil and tadalafil in dietary supplements using high-performance liquid chromatography-tandem mass spectrometry.

A simple and sensitive method was developed for determination of illegal adulterants (yohimbine, sildenafil, vardenafil and tadalafil) in dietary supplements by HPLC-MS/MS. The separation was achieved on a C(18) column with the mobile phase consisting of acetonitrile and 0.1% acetic acid aqueous solution with a gradient elution at a flow rate of 0.5 mL/min. The analytes were quantified and identified by two characteristic transitions using the multiple-reaction monitoring mode. The recoveries of the analytes ranged from 77.5 to 109.3% with the RSD less than 8.1% (n=6). The method has been successfully applied to screen illegal adulterations of natural dietary supplements.

Comparison and combination of spectroscopic techniques for the detection of counterfeit medicines.

During this study, Fourier transform infrared spectroscopy (FT-IR), near infrared spectroscopy (NIR) and Raman spectroscopy were applied to 55 samples of counterfeit and imitations of Viagra and 39 samples of counterfeit and imitations of Cialis. The aim of the study was to investigate which of these techniques and associations of them were the best for discriminating genuine from counterfeit and imitation samples. Only the regions between 1800-400 cm(-1) and 7000-4000 cm(-1) were used for FT-IR and NIR spectroscopy respectively. Partial least square analysis has been used to allow the detection of counterfeit and imitation tablets. It is shown that for the Viagra samples, the best results were provided by a combination of FT-IR and NIR spectroscopy. On the other hand, the best results for the Cialis samples were provided by the combination of NIR and Raman spectroscopy (1400-1190 cm(-1)). These techniques not only permitted a clear discrimination between genuine and counterfeit or imitation samples but also the distinction of clusters among illegal samples. This might be interesting for forensic investigations by authorities.

New classes of PDE7 inhibitors identified by a fission yeast-based HTS.

Studies of the phosphodiesterase PDE7 family are impeded by there being only one commercially available PDE7 inhibitor, BRL50481. The authors have employed a high-throughput screen of commercial chemical libraries, using a fission yeast-based assay, to identify PDE7 inhibitors that include steroids, podocarpanes, and an unusual heterocyclic compound, BC30. In vitro enzyme assays measuring the potency of BC30 and 2 podocarpanes, in comparison with BRL50481, produce data consistent with those from yeast-based assays. In other enzyme assays, BC30 stimulates the PDE4D catalytic domain but not full-length PDE4D2, suggesting an allosteric site of action. BC30 significantly enhances the anti-inflammatory effect of the PDE4 inhibitor rolipram as measured by release of tumor necrosis factor alpha from activated monocytes. These studies introduce several new PDE7 inhibitors that may be excellent candidates for medicinal chemistry because of the requirements for drug-like characteristics placed on them by the nature of the yeast-based screen.

Colorimetric and electrochemical phosphodiesterase inhibition assays for yessotoxin detection: development and comparison with LC-MS/MS.

This work describes the development and applicability of two functional assays for the detection of yessotoxin (YTX), a polycyclic ether marine toxin produced by dinoflagellates. The assays are based on the interaction between this toxin and the phosphodiesterase (PDE) enzyme and the subsequent measurement of the enzyme activity by colorimetric and electrochemical methods. Firstly, several enzyme substrates were tested in order to select those able to be detected by colorimetry or electrochemistry after enzymatic hydrolysis. The substrates that provided the highest absorbance values and density currents were p-nitrophenyl phenylphosphonate and alpha-naphthyl phosphate, respectively. After optimisation of the experimental parameters, limits of detection of 0.8 and 0.6 microM were attained by colorimetry and electrochemistry, respectively. An inhibitory effect of YTX on the PDE activity was observed. The assays have been applied to the analysis of YTX production by Protoceratium reticulatum cultures, and results were compared with liquid chromatography-tandem mass spectrometry analysis.

Development of an immunoassay for rapid screening of vardenafil and its potential analogues in herbal products based on a group specific monoclonal antibody.

Vardenafil is a phosphodiesterase-5 (PDE-5) inhibitor for the treatment of erectile dysfunction (ED). Undeclared vardenafil and related analogues adulterated in herbal products are a threat to public health. To screen vardenafil and its analogues in herbal matrix rapidly, an immunoassay based on a group specific monoclonal antibody (McAb) was developed. Glutaraldehyde was used to link vardenafil to immunogen and coating-antigen, respectively. Through the assessment of the structural specificity of eight anti-vardenafil McAbs, the McAb of 4B9 was characterized as being specific to the common structure of vardenafil and its analogues. An indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was established based on this McAb, the limit of detection of vardenafil was 5.0 ng mL(-1), the calibration curve was linear from 5.0 to 40 ng mL(-1) (R(2)=0.952) with an IC(50) value of 18.2 ng mL(-1). In the extracts of 20 Chinese traditional drugs, the detection capability (CCbeta) of vardenafil was 0.08 mg g(-1), the recoveries were 76-116% and the coefficients of variation (CV%) were 9.7%-16.2%. The ic-ELISA was in good agreement with LC-UV when detected herbal products containing vardenafil and its analogue. The method is a suitable tool for screening vardenafil and its analogues as illegal additives in herbal products.

[Counterfeit phosphodiesterase type 5 inhibitors--growing safety risks for public health]. / Sfalszowane inhibitory fosfodiesterazy typu 5--narastajace zagrozenie dla zdrowia publicznego.

Counterfeit drugs, medical devises and dietary supplements are inherently dangerous and a growing problem. In Europe the growth of the counterfeit medication market is attributable in part to registration of phosphodiesterase type 5 inhibitors (PDE-5) used for the erectile dysfunction. "Viagra, Levitra and Cialis belong to this group. It has been estimated that up to 2.5 million men in Europe are exposed to an illicit sildenafil, suggesting that there may be as many illegal as legal users of sildenafil. In Europe a strong trend is observed towards increasingly professional counterfeits and imitations of Viagra, Cialis and Levitra, with regard to the appearance of tablets, capsules and packaging. The professional presentation will deceive potential consumers into assuming these products are legal, efficacious and safe. Globally, increased obstacles for counterfeiters are necessary to combat pharmaceutical counterfeiting, including fines and penalties. The worldwide nature of the counterfeit problem requires proper coordination between countries to ensure an adequate enforcement. We described the usefulness of the time-of-flight mass spectrometry with the electrospray ionization (LC-ESI-MS-TOF) and the X-ray powder diffraction method (XRPD) for PDE-5 counterfeit screening from the Polish illegal market.

Screening of Indian aphrodisiac ayurvedic/herbal healthcare products for adulteration with sildenafil, tadalafil and/or vardenafil using LC/PDA and extracted ion LC-MS/TOF.

Ayurvedic/herbal healthcare products are considered safe under the impression that they are derived from natural products. But recently, there have been several reports worldwide on the adulteration of synthetic PDE-5 inhibitors in aphrodisiac herbal formulations. Therefore, the objective of the present study was to explore the presence of synthetic PDE-5 inhibitors (sildenafil, tadalafil and/or vardenafil) in ayurvedic/herbal healthcare products sold in Indian market for aphrodisiac/related uses. In total, 85 herbal formulations (HFs) were included in the study. The formulations were extracted with methanol and subjected to centrifugation. The supernatant was analysed by HPLC and LC-MS/TOF. Early detection of the presence of sildenafil, tadalafil and vardenafil in the herbal samples was done by the study of extracted ion mass chromatograms at the m/z values of respective parent ions, and two prominent fragments of each. In case of sildenafil and tadalafil, adulteration was also detected by comparing the relative retention times (RR(T)) and UV spectra. Further substantiation was done through comparison of accurate mass spectra with those of the two available standards. Of the 85 HFs tested, only one was eventually found to be adulterated with sildenafil. The extent of adulterant in this sample was determined to the therapeutic dose in the formulation. The study thus indicates emergence of the problem of adulteration of Indian herbal products with PDE-5 inhibitors.

Isolation and identification of hydroxythiohomosildenafil in herbal dietary supplements sold as sexual performance enhancement products.

An unknown compound is detected and isolated from two herbal dietary supplements bought on the internet. The structure of the unknown compound is elucidated using ESI-MS/MS, NMR, UV and IR. The compound, named hydroxythiohomosildenafil, is identified as an analogue of sildenafil in which the oxygen atom is substituted with a sulfur atom in the pyrazolopyrimidine moiety, and a hydroxyethyl group instead of a methyl group is attached to the piperazinyl nitrogen. It is the first report of this compound being detected in herbal dietary supplements. The UV, IR and completely assigned NMR data of hydroxythiohomosildenafil is recorded.

High performance liquid chromatography-diode array and electrospray-mass spectrometry analysis of vardenafil, sildenafil, tadalafil, testosterone and local anesthetics in cosmetic creams sold on the Internet web sites.

A simple high-performance liquid chromatography (HPLC) method with ultraviolet diode array (UV-DAD) and electrospray ionisation mass spectrometry (ESI-MS) detection has been developed for the determination of vardenafil, sildenafil, tadalafil, testosterone, procaine, lidocaine, prilocaine, and benzocaine in cosmetic creams sold as promising remedies for male erectile dysfunction and female genitals stimulation. The presence of these substances in commercial cosmetic samples is prohibited. Aliquots (1 g) of the cosmetic creams under investigation were diluted 1:100 in methanol, subjected to ultrasonic treatment, added with benzoic acid as internal standard, and analyzed by HPLC-DAD and HPLC-ESI-MS after a further 1:1000 dilution. The compounds were separated by reversed phase chromatography with water (0.02% trifluoroacetic acid) and acetonitrile gradient elution and detected by UV-DAD at 228, 255 and 290 nm and by ESI-MS positive ionisation mode. Benzoic acid was used as internal standard. Linearity was studied with UV-DAD detection from 2.5-7.8 to 250 microg/g range, depending on the different compounds and with ESI-MS in the 3.3-8.9 to 250 ng/g range. Good determination coefficients (r(2) > or = 0.99) were found in both UV-DAD and ESI-MS. Limits of quantifications ranged between 2.5 and 7.8 microg/g for HPLC-UV-DAD assay and between 3.3 and 8.9 ng/g for HPLC-ESI-MS assay depending on different analyzed substances. At three concentrations spanning the linear dynamic ranges of both UV-DAD and ESI-MS assay, mean recoveries were always higher than 90% for the different analytes and intra-assay and inter-assay precision always better than 15% and 12%. This method was successfully applied to the analysis of substances under investigations present in cosmetic creams, freely sold on the Internet web-sites.

Isolation and structural elucidation of cyclopentynafil and N-octylnortadalafil found in a dietary supplement.

A new sildenafil analogue, cyclopentynafil (1) and a new tadalafil analogue, N-octylnortadalafil (2) were isolated from a dietary supplement illegally marketed for erectile dysfunction. The structures of the sildenafil and tadalafil analogues were elucidated by using HPLC-photodiode array (PDA), LC-MS, high-resolution MS, NMR and circular dichroism (CD). These compounds were determined to be 5-[2-ethoxy-5-(4-cyclopentylpiperazin-1-ylsulfonyl)phenyl]-1-methyl-3-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one and (6R,12aR)-2-octyl-6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydropyrazino[1',2':1,6]pyrido[3,4-b]indole-1,4-dione, respectively. Recently, a large number of phosphodiesterase-5 (PDE-5) inhibitors, including their analogues, have been isolated from dietary supplements, while cyclopentynafil and N-octylnortadalafil are the first compounds reported to be new sildenafil and tadalafil analogues, respectively. Quantitative HPLC analysis showed that the contents of 1 and 2 in the product were about 130 mg/tablet (301 microg/mg) and about 27 mg/tablet (64.1 microg/mg), respectively.

Identification of phosphotyrosine mimetic inhibitors of human tyrosyl-DNA phosphodiesterase I by a novel AlphaScreen high-throughput assay.

Tyrosyl-DNA phosphodiesterase I (Tdp1) resolves topoisomerase I (Top1)-DNA adducts accumulated from natural DNA damage as well as from the action of certain anticancer drugs. Tdp1 catalyzes the hydrolysis of the phosphodiester bond between the catalytic tyrosine residue of topoisomerase I and the DNA 3'-phosphate. Only a limited number of weak inhibitors have been reported for Tdp1, and there is an unmet need to identify novel chemotypes through screening of chemical libraries. Herein, we present an easily configured, highly miniaturized, and robust Tdp1 assay using the AlphaScreen technology. Uninhibited enzyme reaction is associated with low signal, whereas inhibition leads to a gain of signal, making the present assay format especially attractive for automated large-collection high-throughput screening. We report the identification and initial characterization of four previously unreported inhibitors of Tdp1. Among them, suramin, NF449, and methyl-3,4-dephostatin are phosphotyrosine mimetics that may act as Tdp1 substrate decoys. We also report a novel biochemical assay using the SCAN1 Tdp1 mutant to study the mechanism of action of methyl-3,4-dephostatin.

Determination of a new type of phosphodiesterase-5 inhibitor, thioquinapiperifil, in a dietary supplement promoted for sexual enhancement.

A new type of phosphodiesterase-5 (PDE-5) inhibitor, thioquinapiperifil (1), was found in dietary supplements. LC-MS analysis indicated that the supplements contain two major compounds. One was identified as thiodenafil (synonym: thiosildenafil) by direct comparison with the authentic compound. The other showed a molecular weight of 448, and accurate mass measurement showed its elemental composition to be C(24)H(28)N(6)O(1)S(1). Together, the mass and NMR spectrometric data revealed that the compound is an imidazoquinazoline derivative: 3-ethyl-1,3-dihydro-8-[[[2-[4-(hydroxymethyl)-1-piperidinyl]phenyl]methyl]amino]-2H-imidazo[4,5-g]quinazoline-2-thione. This compound had been synthesized as a PDE-5 inhibitor, formerly reported as KF31327 by Kyowa Hakko Kogyo Co., Ltd. Considering this compound's general properties, it has been renamed thioquinapiperifil with the agreement of Kyowa Hakko Kogyo Co., Ltd. The detection of imidazoquinazoline-type compounds in dietary supplements has not been reported. Quantitative analysis showed that the contents of 1 and thiodenafil in the products were about 13-15 mg/tablet (43-48 microg/mg) and about 0.4 mg/tablet (1 microg/mg), respectively.