Suspected-target and non-targeted screenings of phosphodiesterase 5 inhibitors in herbal remedies using liquid chromatography–quadrupole time-of-flight–mass spectrometry

The lucrative market of herbal remedies spurs rampant adulteration, particularly with pharmaceutical drugs and their unapproved analogues. A comprehensive screening strategy is, therefore, warranted to detect these adulterants and, accordingly, to safeguard public health. This study uses the data-dependent acquisition of liquid chromatography-quadrupole time-of-flight–mass spectrometry (LC–QTOF–MS) to screen phosphodiesterase 5 (PDE5) inhibitors in herbal remedies using suspected-target and non-targeted strategies. For the suspected-target screening, we used a library comprising 95 PDE5 inhibitors. For the non-targeted screening, we adopted top-down and bottom-up approaches to flag novel PDE5 inhibitor analogues based on common fragmentation patterns. LC–QTOF–MS was optimised and validated for capsule and tablet dosage forms using 23 target analytes, selected to represent different groups of PDE5 inhibitors. The method exhibited excellent specificity and linearity with limit of detection and limit of quantification of <40 and 80 ng/mL, respectively. The accuracy ranged from 79.0% to 124.7% with a precision of <14.9% relative standard deviation. The modified, quick, easy, cheap, effective, rugged, and safe extraction provided insignificant matrix effect within −9.1%–8.0% and satisfactory extraction recovery of 71.5%–105.8%. These strategies were used to screen 52 herbal remedy samples that claimed to enhance male sexual performance. The suspected-target screening resulted in 33 positive samples, revealing 10 target analytes and 2 suspected analytes. Systematic MS and tandem MS interrogations using the non-targeted screening returned insignificant signals, indicating the absence of potentially novel analogues. The target analytes were quantified from 0.03 to 121.31 mg per dose of each sample. The proposed strategies ensure that all PDE5 inhibitors are comprehensively screened, providing a useful tool to curb the widespread adulteration of herbal remedies.     

Recent applications of phosphodiesterase (PDE5) inhibition assays for detecting adulterated sexual enhancement products

Consumer products marketed for sexual enhancement are frequently adulterated with erectile dysfunction (ED) drugs and analogs; consuming these undisclosed adulterants can pose significant health hazards. Although ED drugs/analogs have unpredictable and diverse structures that pose challenges for detecting them, they all share the ability to inhibit phosphodiesterase-5 (PDE5) activity, a pharmacological mechanism responsible for their effects. Consequently, several PDE5 inhibition assays have been recently applied as screening methods to detect ED drug/analogs in products. Here, the successes and challenges are highlighted for screening sexual enhancement products by PDE5 inhibition assays.     

Development of a detector tube for screening tadalafil and its analogues in adulterated sexual enhancement products

Sexual enhancement products adulterated with phosphodiesterase 5 inhibitors (PDE-5i) pose a serious public health concern. Tadalafil and its analogues (Tds) are PDE-5i frequently detected as adulterants. In this study, a Td detector tube for the rapid detection of Tds was developed based on the color change reaction between sulfuric acid and Tds. The specificity of this test method was evaluated using 13 Tds, all of which elicited positive results. Additionally, 30 commonly found adulterants in dietary supplements, 11 active pharmaceutical ingredients of psychotropic drugs and 18 food ingredients were tested and obtained no false-positive results, except levomepromazine. The test tube accurately detected the presence or absence of Tds in 54 commercially available products. The visual detection limit was 2–50 and 5–20 μg/ml for Tds and tadalafil-spiked samples with matrix, respectively. The applicability of the developed detector tube to a semiquantitative test using digital image analyses were investigated using red, green, and blue color values. The results of the recovery test suggested that the tube test was affected by the dark-colored matrix. The results of semiquantitative analyses of tadalafil for five marketed products were consistent with the liquid chromatographic quantification results, except for the blue value. The detector tube developed in this study can facilitate with the rapid screening of Tds in adulterated sexual enhancement products.     

Suitability evaluation of new endogenous biomarkers for the identification of nitrite‐based urine adulteration in mass spectrometry methods

Urine adulteration to circumvent positive drug testing is a fundamental challenge for toxicological laboratories all over the world. Untargeted mass spectrometry (MS) methods used in metabolomics had previously revealed uric acid (UA), histidine, methylhistidine, and their oxidation products, for example 5‐hydroxyisourate (HIU) as potential biomarkers for urine adulteration using potassium nitrite (KNO₂). These markers should be further evaluated for their reliability, stability, and routine applicability. Influence of KNO₂ concentration, urinary pH, reaction time, and stability at room temperature, 4°C, and ? 20°C was determined in urine under varying conditions. Analysis was performed after protein precipitation with acetonitrile by liquid chromatography–high resolution mass spectrometry (LC–HRMS). Receiver operating characteristics (ROC) analysis was applied for cut‐off evaluation after biomarker quantification (n = 100 per group). Blinded measurements (n = 50) were performed to check the general applicability to identify adulterated samples under routine conditions. The higher the adulterant concentration, the lower the concentrations of histidine, methylhistidine, and UA. In return, amounts of their oxidation products increased. Highest changes were observed under weak acid conditions (pH 4–5). Storage at ?20°C ensured sufficient stability for all oxidative markers over one month. ROC evaluated biomarker performance and application to unknown samples revealed satisfying results, with HIU as the most suitable biomarker (positive predictive value (PPV) 100%), followed by UA (PPV 93%). HIU and UA proved suitable markers to identify urine adulteration using KNO₂ and are ready for implementation into routine MS procedures.     

The rapid and sensitive detection of edible bird's nest (Aerodramus fuciphagus) in processed food by a loop-mediated isothermal amplification (LAMP) assay

Edible bird's nest (EBN) is a well-known and precious traditional Chinese herbal material (CHM). Because of this, preventing the adulteration of EBN efficiently and precisely is crucial to protect consumers' interests and health. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed for the detection of EBN using specifically designed LAMP primers. The results demonstrated that the identification of EBN by LAMP assay was specific and rapid (within 1 h). It had no cross-reaction with EBN adulterants, including white fungus, egg white and pig skin, in different ratios. The relative detection limit was 0.01% EBN in the adulterants. Moreover, the sensitivity of LAMP in authenticating EBN was 10^?8 μg, it showed higher sensitivity than that of conventional PCR with 10⁵ fold. When genomic DNAs extracted from boiled or steamed EBN samples were used as templates, LAMP for EBN detection was not affected and was reproducible after heat processing. In conclusion, the LAMP assay established herein could be applicable for authenticating EBN and for identifying commercial EBN products in herbal markets.     

Evaluation of endogenous urinary biomarkers for indirect detection of urine adulteration attempts by five different chemical adulterants in mass spectrometry methods

Reliable detection of urine adulteration attempts to circumvent positive drug testing represents a critical step for laboratories in abstinence control settings. An ideal workflow for high‐throughput testing would involve simultaneous detection of adulteration attempts in the same run with drug detection. Monitoring of degraded or oxidized endogenous urinary compounds as indirect markers has been previously evaluated for that purpose exemplified for the adulterant potassium nitrite (KNO₂). Fifteen, previously identified endogenous markers should now be evaluated for their general applicability to detect adulteration attempts for the adulterants hypochlorite‐based bleach (NaOCl), peroxidase and peroxide (H₂O₂), pyridinium chlorochromate (PCC), and iodine (I₂). Initial experiments revealed similar results for the tested adulterants regarding degradation of indolylacryloylglycine (IAG), uric acid (UA), or UA derivatives. 5‐Hydroxyisourate (HIU), the oxidation product of UA, was however only formed by KNO₂, PCC, and H₂O₂. Amino acids showed larger adulterant‐dependent differences. All reactions were shown to be influenced by the adulterant concentration and the urinary pH with large variances depending on compound and adulterant. Except for HIU/PCC, all markers were stable within +/? 30% variation for all adulterants at ?20°C. Receiver operating characteristics indicated best sensitivity and specificity over all adulterants for IAG (specificity 0.9, sensitivity 1.0) and UA (specificity 1.0, sensitivity 0.9). HIU gave best results for KNO₂, PCC, and H₂O₂ and N‐acetylneuraminic acid for PCC and H₂O₂, respectively. When integrating a limited number of targets into existing screening methods, monitoring of UA, IAG, N‐acetylneuraminic acid, and HIU is recommended.     

Rapid screening of sulfonamides in dietary supplements based on extracted common ion chromatogram and neutral loss scan by LC-Q/TOF-mass spectrometry

There is an increasing amount of dietary supplements that are adulterated with diuretics and anti-diabetic drugs; this has become a global problem due to the wide distribution of dietary supplements and the serious negative health effects of the adulterants. In this study, a rapid screening method was developed for detection and confirmation of 35 sulfonamides in supplements by ultra-high performance liquid chromatography quadrupole/time of flight mass spectrometry. For effective extraction of sulfonamides from dietary supplements, four extraction protocols including HLB and WAX solid-phase extraction, Quick Easy Cheap Effective Rugged and Safe method, and pH-controlled liquid–liquid extraction were evaluated, and pH-controlled liquid–liquid extraction method was shown to be the most effective with high recovery efficiency and low matrix effect. Rapid separation of 35 sulfonamides was achieved with the UHPLC C18 column (150 × 2.1 mm, 1.7 um) within 7 min using ammonium acetate aqueous solution (pH 8) and acetonitrile as the mobile phase. From the MS/MS spectra of sulfonamides, common ions (m/z 77.9650 [SO₂N]^- and m/z 79.9812 [SO₂NH₂]^-) and neutral molecule loss fragments (HCl and SO₂) were observed according to their structural characteristics. Extracted common ion chromatograms and neutral loss scan of these characteristic fragments could effectively apply for rapid screening of sulfonamides in various types of supplements. A reduced mass tolerance window of ±5 ppm was useful for detecting targeted and untargeted sulfonamides and could avoid false positive and false negative results. Overall calibration curves within dynamic range for all targets were shown to be linear with a correlation coefficient R² > 0.995 and limits of detection ranged from 0.04 to 11.18 ng/g for all sulfonamides. The established method was successfully applied for screening and confirmation of sulfonamides in various supplements. The developed method will be helpful for the identification of sulfonamide diuretics and anti-diabetics in dietary supplements, promoting public health and consumer safety.     

Collision-induced dissociation pathways of H<Subscript>1</Subscript>-antihistamines by electrospray ionization quadrupole time-of-flight mass spectrometry

Over the past decades, mass spectrometry technologies have been developed to obtain mass accuracies of one ppm or less. Of the newly developed technologies, quadrupole time-of–flight mass spectrometry (Q-TOF–MS) has emerged as being well suited to routine and high-throughput analyses of pharmaceuticals. Dietary supplements and functional foods have frequently been found to be contaminated with pharmaceuticals. In our continuous efforts to develop methodologies to protect public health against adulterated dietary supplements, we have constructed a mass spectral database for 21 H₁-antihistamines encountered as adulterants by using liquid chromatography-electrospray ionization (LC-ESI)/Q-TOF–MS, and have proposed their possible collision-induced dissociation pathways. This database will be very useful for the rapid and accurate detection of H₁-antihistamines (known) and their analogues (unknown) illegally added to dietary supplements as well as in other sample matrices.     

Repurposing Human PDE4 Inhibitors for Neglected Tropical Diseases. Evaluation of Analogs of the Human PDE4 Inhibitor GSK‐256066 as Inhibitors of PDEB1 of Trypanosoma brucei

Cyclic nucleotide phosphodiesterases (PDEs) have been identified as important enzyme targets for drug development in both humans and Trypanosoma brucei, the causative agent of human African trypanosomiasis. With this in mind, we recently reported the profiling of a range of human phosphodiesterase inhibitors, showing that human PDE4 inhibitors tend to display the best potency against the trypanosomal phosphodiesterase TbrPDEB1. Among these was GSK‐256066, a potent inhibitor of human PDE4 and a weak inhibitor of TbrPDEB1. In this report, we describe the results of a structure–activity relationship study of this chemotype, leading to the discovery of analogs with improved potency against TbrPDEB1 and micromolar inhibition of T. brucei cellular growth. We rationalize the potency trends via molecular docking of the new inhibitors into a recently reported apo structure of TbrPDEB1. The studies in this article will inform future efforts in repurposing human PDE inhibitors as antitrypanosomal agents.     

Discovery of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl)acetonitriles as phosphodiesterase 4 inhibitors with anti‐neuroinflammation potential based on three‐dimensional quantitative structure–activity relationship study

Phosphodiesterase 4 (PDE4) inhibitors with potential activities for CNS disorders provide a new therapeutic strategy for depression. To discover PDE4 inhibitors with anti‐neuroinflammation activities, reliable three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) models on our previous reported catecholic PDE4 inhibitors was built with a statistically significant cross‐validated coefficient (q²), conventional coefficient (r²), and good predictive capabilities based on the molecular docking results, using comparative molecular field analysis (CoMFA) and comparative molecular similarity index analysis (CoMSIA) methods. Based on the analysis of CoMFA and CoMSIA contour maps, a series of 2‐(3,4‐dialkoxyphenyl)‐2‐(substituted pyridazin‐3‐yl) acetonitriles 16a–i was designed and synthesized. Among these compounds, compound 16a exhibited good inhibitory activities toward PDE4B1 and PDE4D7 with mid‐nanomolar IC₅₀ values and potential anti‐neuroinflammation activity in BV‐2 cells. Docking simulation of compound 16a in the PDE4 catalytic domain activity pocket revealed that compound 16a maybe assumed a “V‐shaped” conformation, extending the side chain to S‐pocket.     

Inhibition of cGMP phosphodiesterase 5 suppresses matrix metalloproteinase‐2 production in pulmonary artery smooth muscles cells

1. It has been shown that the beneficial effects of phosphodiesterase (PDE) 5 inhibition on pulmonary hypertension (PH) are associated with the induction of vascular relaxation and suppression of the proliferation of pulmonary artery smooth muscle cells (PASMC). In the present study, we investigated whether PDE5 inhibition affects the production and/or secretion of matrix metalloproteinases (MMPs) in PASMC, resulting in extracellular matrix remodelling in the pulmonary vasculature and, thus, the development of PH. 2. Primary cultured PASMC were stimulated with endothelin (ET)‐1 and MMP‐2 production and RhoA activation were then determinded using gelatin zymography and a GTP‐bound RhoA assay, respectively. The effects of the selective PDE5 inhibitor sildenafil and subsequent protein kinase G‐specific inhibitor Rp‐8Br‐cGMPs on MMP‐2 production and RhoA activation were further exmamined. 3. Endothelin‐1 (1–1000 nmol/L) concentration‐dependently stimulated MMP‐2 production and/or secretion in primary cultured PASMC, with 100 nmol/L ET‐1 causing a 2.41‐fold increase in MMP‐2 production compared with control (P < 0.01). This increase in MMP‐2 production was accompanied by RhoA activation, which was abolished by preincubation of cells with 10 μmol/L Y27632, an inhibitor of Rho‐associated kinase (ROCK). Furthermore, 10 μmol/L Y27632 abolished the ET‐1‐induced production of MMP‐2. 4. The selective PDE5 inhibitor sildenafil (0.1–1 μmol/L) concentration‐dependently reduced the increased MMP‐2 production induced by 100 nmol/L ET‐1. Specifically, in the presence of 1 μmol/L sildenafil, the 100 nmol/L ET‐1‐induced increase in MMP‐2 production was only increased 1.3‐fold over that of the control (P < 0.01 vs 100 nmol/L ET‐1‐stimulated cells). 5. Suppression of RhoA activation was found to mediate the inhibitory effect of sildenafil on ET‐1‐induced increases in MMP‐2 production. Furthermore, the protein kinase G‐specific inhibitor Rp‐8Br‐cGMPs reversed the inhibitory effects of sildenafil on RhoA activation and MMP‐2 production. 6. The results of the present study indicate that PDE5 inhibition suppresses RhoA/ROCK‐mediated MMP‐2 production by PASMC, which may contribute to the regulation of pulmonary vascular remodelling. Thus, PDE5 inhibition may benefit patients with PH through multiple mechanisms of action.     

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.     

Discovery of a highly specific 18F-labeled PET ligand for phosphodiesterase 10A enabled by novel spirocyclic iodonium ylide radiofluorination

As a member of cyclic nucleotide phosphodiesterase (PDE) enzyme family, PDE10A is in charge of the degradation of cyclic adenosine (cAMP) and guanosine monophosphates (cGMP). While PDE10A is primarily expressed in the medium spiny neurons of the striatum, it has been implicated in a variety of neurological disorders. Indeed, inhibition of PDE10A has proven to be of potential use for the treatment of central nervous system (CNS) pathologies caused by dysfunction of the basal ganglia–of which the striatum constitutes the largest component. A PDE10A-targeted positron emission tomography (PET) radioligand would enable a better assessment of the pathophysiologic role of PDE10A, as well as confirm the relationship between target occupancy and administrated dose of a given drug candidate, thus accelerating the development of effective PDE10A inhibitors. In this study, we designed and synthesized a novel ¹⁸F-aryl PDE10A PET radioligand, codenamed [¹⁸F]P10A-1910 ([¹⁸F]9), in high radiochemical yield and molar activity via spirocyclic iodonium ylide-mediated radiofluorination. [¹⁸F]9 possessed good in vitro binding affinity (IC₅₀ = 2.1 nmol/L) and selectivity towards PDE10A. Further, [¹⁸F]9 exhibited reasonable lipophilicity (logD = 3.50) and brain permeability (P_app > 10 × 10^?6 cm/s in MDCK-MDR1 cells). PET imaging studies of [¹⁸F]9 revealed high striatal uptake and excellent in vivo specificity with reversible tracer kinetics. Preclinical studies in rodents revealed an improved plasma and brain stability of [¹⁸F]9 when compared to the current reference standard for PDE10A-targeted PET, [¹⁸F]MNI659. Further, dose–response experiments with a series of escalating doses of PDE10A inhibitor 1 in rhesus monkey brains confirmed the utility of [¹⁸F]9 for evaluating target occupancy in vivo in higher species. In conclusion, our results indicated that [¹⁸F]9 is a promising PDE10A PET radioligand for clinical translation.     

Identification and in vivo evaluation of a fluorine‐18 rolipram analogue, [18F]MNI‐617, as a radioligand for PDE4 imaging in mammalian brain

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non‐invasive way, as has been shown using the carbon‐11 labeled PDE4 inhibitor R‐(?)‐rolipram. Herein we describe a small series of rolipram analogs that contain fluoro‐ or iodo‐substituents that could be used as fluorine‐18 PET or iodine‐123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4‐(fluoromethyl) derivative of rolipram, MNI‐617, was identified, with a five‐fold increase in affinity for PDE4 (K_d = 0.26 nM) over R‐(?)‐rolipram (K_d = 1.6 nM). A deutero‐analogue d₂‐[¹⁸F]MNI‐617 was radiolabeled and produced in 23% yield with high (>5 Ci/µmol) specific activity and evaluated in non‐human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.     

Structure‐based discovery of new selective small‐molecule sirtuin 5 inhibitors

Human sirtuin 5 (SIRT5) is a protein deacylase regulating metabolic pathways and stress responses and is implicated in metabolism‐related diseases. Small‐molecule inhibitors for SIRT5 are sought as chemical tools and potential therapeutics. Herein, we proposed a customized virtual screening approach targeting catalytically important and unique residues Tyr102 and Arg105 of SIRT5. Of the 20 tested virtual screening hits, six compounds displayed marked inhibitory activities against SIRT5. For the hit compound 19 , a series of newly synthesized (E)‐2‐cyano‐N‐phenyl‐3‐(5‐phenylfuran‐2‐yl)acrylamide derivatives/analogues were carried out structure–activity relationship analyses, resulting in new more potent inhibitors, among which 37 displayed the most potent inhibition to SIRT5 with an IC₅₀ value of 5.59 ± 0.75 μM. The biochemical studies revealed that 37 likely acts via competitive inhibition with the succinyl‐lysine substrate, rather than the NAD⁺ cofactor, and it manifested substantial selectivity for SIRT5 over SIRT2 and SIRT6. This study will aid further efforts to develop new selective SIRT5 inhibitors as tools and therapeutics.     

The Texture of Psychoactive Illicit Drugs in Iran: Adulteration with Lead and other Active Pharmaceutical Ingredients

ABSTRACT

Psychoactive illicit drugs are widely used all over the world. Due to the high demand for illicit drugs, adulteration of substances with poisonous and active pharmaceutical ingredients is a common phenomenon in some countries. Lead and other active pharmaceutical ingredients are among adulterants added to illicit drugs intentionally. In the present study, we analyzed four major abused street drugs in Iran’s drug black market (opium, Iranian crack, ecstasy tablets, and crystal methamphetamine) to assess active pharmaceutical ingredients and determine a quantitative assay of lead. A total of 40 psychoactive drugs were analyzed using high-performance liquid chromatography, gas chromatography/mass spectrometry, and flame atomic absorption spectroscopy. The results demonstrated that psychoactive drugs were adulterated with different drug categories, such as tramadol, ketamine, methadone, acetaminophen, and caffeine. Lead was found in all analyzed samples, ranging from 9–90 ppm. The smallest lead level was detected in methamphetamine samples. Iranian crack samples contained the highest amount of lead. Psychoactive illicit drugs were adulterated with different drug classes and also lead. Lead-adulterated psychoactive drugs are among the new sources of exposure to lead, while illicit drugs’ contamination with different drugs may present a health hazard for drug-abusing patients.     

The mechanism of attenuation of epithelial‐mesenchymal transition by a phosphodiesterase 5 inhibitor via renal klotho expression

Phosphodiesterase‐5 (PDE‐5) inhibitors induces vasodilation in several organs by blocking cyclic GMP (guanosine monophosphate) degradation. However, the existence of alternative mechanism of action in case of an impaired nitric oxide (NO) system remains controversial. Previous studies suggested that decreased NO bioavailability may result in the downregulation of klotho expression, but the relationship between klotho and NO remains obscure. Therefore, we investigated whether a PDE‐5 inhibitor could preserve epithelial–mesenchymal transition (EMT) and relationship exists between the NO and renal klotho expression. Ten‐week‐old SD rats (N = 24, 200 g, male) were divided (N = 6) into four groups, which received: A LSD, L‐NAME 1 mg/mL in drinking water, Udenafil 5 mg/kg subcutaneously and both for 4 weeks. Urine nitrate/nitrite, NGAL (Neutrophil gelatinase‐associated lipocalin), and cGMP were measured using ELISA. Kidney was subjected to evaluate PCNA (proliferative cell nuclear antigen), α‐SMA (smooth muscle cell antigen), E‐cadherin, and klotho expression. Urine cGMP decreased after treatment of PDE‐5 inhibitor compared with control due to blocking degradation of cGMP (P < .05, control vs Udenafil and L‐NAME with Udenafil groups). Urine NGAL increased after treating of L‐NAME and attenuated after using PDE‐5 inhibitor (P < .05, control vs L‐NAME and L‐NAME with Udenafil). PCNA, α‐SMA, and E‐cadherin (EMT markers) increased after L‐NAME treatment and normalized after using PDE‐5 inhibitor. Klotho expression showed trend to increase in the L‐NAME with PDE‐5 inhibitor group compared with the L‐NAME group, however, eNOS expression did not change after treatment of L‐NAME or PDE‐5 inhibitor compared with control. PDE‐5 inhibitor alleviates EMT in the kidney via klotho modulation independent of the NO system.     

Detection and characterization of synthetic steroidal and non‐steroidal anti‐inflammatory drugs in Indian ayurvedic/herbal products using LC‐MS/TOF

It is claimed that ayurvedic/herbal healthcare products (AHPs) are safe because of their natural origin. However, several reports exist of adulteration of AHPs with synthetic drugs. In this study, a generalized strategy was developed using LC‐MS/TOF for the detection and verification of steroidal and anti‐inflammatory drugs in 58 AHPs collected from various parts of India. The strategy involved recording of mass spectral information for standard drugs—including ionization mode (ESI/APCI ? ve or + ve), mass spectrum, accurate mass, identification of qualifier fragments (two), extracted ion chromatograms (EICs), isotopic pattern and determination of UV max (nm)—through UV‐PDA studies. Adulteration was then detected in AHPs primarily through comparison of EICs at accurate m/z for molecular ion peaks and R_T matching with the standard. It was confirmed by spiking with the standards, and matching mass spectrum, accurate mass, R_T of qualifier fragments, isotopic pattern and UV spectrum of the standards with the adulterant peaks in AHPs. Dexamethasone and diclofenac were detected as adulterants in ten AHPs whereas one AHP tested positive for piroxicam and another for dexamethasone. All the adulterated products were sold by the healthcare practitioners, while no product marketed by manufacturers or chemist shops had this problem. The study showed that LC‐MS/TOF‐based screening could be used as a rapid approach to monitor adulteration of steroids and anti‐inflammatory drugs in AHPs. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis and evaluation of [123I]‐indomethacin derivatives as COX‐2 targeted imaging agents

A novel series of iodinated indomethacin derivatives was synthesized, and evaluated as selective inhibitors of COX‐2. Two candidate compounds N‐(p‐iodobenzyl)‐2‐(1‐(p‐chlorobenzoyl)‐5‐methoxy‐2‐methyl‐1H‐indol‐3‐yl)acetamide (3) and 1‐(p‐iodobenzyl)‐5‐methoxy‐2‐methyl‐3‐indoleacetic acid (9) possessed optimum properties suitable for potential in vivo imaging. Arylstannane precursors for radioiododestannylation were synthesized in 70–85% yield from the iodo compounds by reaction with hexabutylditin and tetrakis(triphenylphosphine)palladium(0) in refluxing dioxane. Radioiododestannylation was conducted by reaction with carrier‐added Na[¹²³I] in the presence of Chloramine‐T in an EtOAc/H₂O binary system under acidic conditions (pH 3.5), allowing direct isolation of the labeled products by separation of the organic phase. Radioiodinated products [¹²³I]3 and [¹²³I]9 were recovered in a decay‐corrected radiochemical yield of 86–87% and radiochemical purity of 98–99%. Copyright © 2009 John Wiley & Sons, Ltd.