Predictors of Systemic Exposure to Topical Crisaborole: A Nonlinear Regression Analysis.

Crisaborole ointment, 2%, is a nonsteroidal phosphodiesterase 4 inhibitor for the treatment of mild to moderate atopic dermatitis. Results from 2 randomized, double-blind, vehicle-controlled phase 3 studies showed that twice-daily crisaborole in children and adults with mild to moderate atopic dermatitis was efficacious and well tolerated. Initial pharmacokinetics (PK) studies of crisaborole indicated absorption with measurable systemic levels of crisaborole. The current analysis was conducted to correlate steady-state systemic exposure parameters with ointment dose and identify covariates impacting PK parameters in healthy participants and patients with atopic dermatitis or psoriasis. A nonlinear regression analysis was conducted using ointment dose and noncompartmental PK parameters at steady state (area under the curve [AUCss ] and maximum concentration [Cmax,ss ]). PK data were available from 244 participants across 6 clinical studies (AUCss , N = 239; Cmax,ss , N = 241). Disease condition had the greatest impact on slope in both models, corresponding to 2.5-fold higher AUCss and Cmax,ss values at a given ointment dose in patients with atopic dermatitis or psoriasis relative to healthy participants. Disease severity, race/ethnicity, and sex had marginal effects on AUCss and Cmax,ss . Systemic exposures were similar across age groups ≥2 years of age when the same percentage of body surface area (%BSA) was treated. Predictive performance plots for AUCss and Cmax,ss for different age groups demonstrated that the models adequately describe the observed data. Model predictions indicated that systemic exposure to crisaborole in pediatric patients (2-17 years) is unlikely to exceed systemic exposure in adults (≥18 years), even at the highest possible ointment dose corresponding to a %BSA of 90.

Roflumilast analogs with improved metabolic stability, plasma protein binding, and pharmacokinetic profile.

With the aim of studying their in vitro and in vivo pharmacokinetics, new chromatographic methods were developed for the determination of three new roflumilast synthetic analogs (I-III) as PDE-4B inhibitors in rat liver S9 fraction, phosphate buffered saline, pH 7.4, and human and rat plasma. The developed high performance liquid chromatography-ultra violet (HPLC-UV) methods were performed on a Zorbax Eclipse C8 column and UV detection was carried out at 215 nm. The three compounds were tested for their metabolic stability and were found to be metabolically more stable than roflumilast especially the 2-mercaptobenzothiazol-6-yl analog (III) which displayed an in vitro half-life time (247.55 minutes) higher than that of roflumilast (12.29 minutes) and a low in vitro clearance of 5.67 mL/min./kg. Possible phase I metabolites were investigated using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) showing hydroxylation of the unsubstituted benzothiazol-2-yl (I) and benzothiazole-6-yl (II) analogs and a cleaved benzothiazole metabolite of the 2-mercaptobenzothiazol-6-yl analog (III). Plasma protein binding affinity was tested using equilibrium membrane dialysis method showing a very high percentage (more than 95%) of plasma protein binding of compounds I and II where compound III exhibited lower percentage (53.71%) demonstrating its accessibility for tissue distribution. Also, a UPLC-MS/MS method was developed using an Acquity UPLC BEH shield RP C18 column to be applied to an in vivo pharmacokinetic study in rats following a subcutaneous dose (1 mg/kg). Compounds I-III showed improved in vivo pharmacokinetic parameters especially compound III which displayed a half-life 3-fold greater than roflumilast (21 hours) and a Cmax value of 113.958 ng/mL. Accordingly, this new chemical entity should be subjected to further investigation as it can be a good drug candidate for treating chronic obstructive pulmonary disease.

Phosphodiesterase 4 inhibition reduces lung fibrosis following targeted type II alveolar epithelial cell injury.

Fibrosis of the lung constitutes a major clinical challenge and novel therapies are required to alleviate the associated morbidity and mortality. Investigating the antifibrotic efficacy of drugs that are already in clinical practice offers an efficient strategy to identify new therapies. The phosphodiesterase 4 (PDE4) inhibitors, approved for the treatment of chronic obstructive pulmonary disease, harbor therapeutic potential for pulmonary fibrosis by augmenting the activity of endogenous antifibrotic mediators that signal through cyclic AMP. In this study, we tested the efficacy of several PDE4 inhibitors including a novel compound (Compound 1) in a murine model of lung fibrosis that results from a targeted type II alveolar epithelial cell injury. We also compared the antifibrotic activity of PDE4 inhibition to the two therapies that are FDA-approved for idiopathic pulmonary fibrosis (pirfenidone and nintedanib). We found that both preventative (day 0-21) and therapeutic (day 11-21) dosing regimens of the PDE4 inhibitors significantly ameliorated the weight loss and lung collagen accumulation that are the sequelae of targeted epithelial cell damage. In a therapeutic protocol, the reduction in lung fibrosis with PDE4 inhibitor administration was equivalent to pirfenidone and nintedanib. Treatment with this class of drugs also resulted in a decrease in plasma surfactant protein D concentration, a reduction in the plasma levels of several chemokines implicated in lung fibrosis, and an in vitro inhibition of fibroblast profibrotic gene expression. These results motivate further investigation of PDE4 inhibition as a treatment for patients with fibrotic lung disease.

Determination of a PDE4 inhibitor Hemay005 in human plasma and urine by UPLC-MS/MS and its application to a PK study.

AIM: Hemay005 is a novel small-molecule inhibitor of phosphodiesterase-4 developed for the treatment of psoriasis. Measurement of Hemay005 in biological samples is critical for evaluation of its pharmacokinetics in clinical studies. Methodology & results: Plasma and urine samples were extracted and then chromatographed on an Acquity UPLC HSS T3 column with a gradient elution. Detection was performed on a Xevo TQ-S tandem mass spectrometer using negative ESI. CONCLUSION: For the first time, a sensitive and robust ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was established and validated for the quantitative determination of Hemay005 in human plasma and urine, and it was successfully applied to evaluate the pharmacokinetics of Hemay005 in healthy subjects in a first-in-human study.

A dried blood spot assay with UPLC-MS/MS for the simultaneous determination of E6005, a phosphodiesterase 4 inhibitor, and its metabolite in human blood.

E6005, a novel phosphodiesterase 4 inhibitor, is currently under clinical development for the treatment of atopic dermatitis. To support pediatric clinical trials, the dried blood spot assay for simultaneous determination of E6005 and its main metabolite, ER-392710 (M11), has been developed using ultra-performance liquid chromatography with tandem mass spectrometry. E6005 and M11, in 25 µL blood spotted onto FTA™ DMPK-C cards, were extracted by water/acetonitrile (1:1, v/v), and then chromatographed on a reversed phase column under gradient elution. The mass transitions, m/z 473.1 → 163.0 for E6005 and m/z 459.1 → 149.0 for M11, with corresponding stable isotope internal standard, m/z 477.2 → 167.0, and m/z 463.2 → 153.0, were monitored. E6005 and M11 were quantifiable from 1 to 200 ng/mL as free base. Accuracy and precision of the two analytes in the intra- and inter-batch reproducibility were within ±8.0% and 15.7%, respectively. Extraction recoveries of the analytes were 73% or more and hematocrit ranging from 26.9% to 51.8% did not impact the analytes' accuracy. Various stability assessments, including possible conversion of E6005 to M11, were thoroughly performed, and bench-top stability was ensured up to 160 days. The DBS method was applied to determine E6005 and M11 concentrations in blood samples supporting a pediatric clinical trial.

A simple and sensitive HPLC-MS/MS method for quantification of eggmanone in rat plasma and its application to pharmacokinetics.

Allosteric phosphodiesterase 4 (PDE4) inhibitors are highly sought after due to their important anti-inflammatory and anti-cancer therapeutic effects. We recently identified Eggmanone, an extraordinarily selective allosteric PDE4 inhibitor displaying favorable drug properties. However, a specific analytic method of Eggmanone in serum and its pharmacokinetics have not been reported yet. In this study, we developed a rapid and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS/MS) method to determine Eggmanone concentrations in rat plasma. This assay method was validated in terms of specificity, linearity, sensitivity, accuracy, precision, matrix effect, recovery and stability, and was applied to a pharmacokinetic study in rats following intravenous injection of Eggmanone at doses of 1 and 3 mg/kg. The lower limit of quantification (LLOQ) of this assay was 5 ng/mL and the linear calibration curve was acquired with R2 > 0.99 between 5 and 1000 ng/m. The intra-day and inter-day precision was evaluated with the coefficient of variations less than 11.09%, whereas the mean accuracy ranged from 98.38% to 105.13%. The assay method exhibited good recovery and negligible matrix effect. The samples were stable under all the experimental conditions. The plasma concentrations of Eggmanone were detected and quantified over 24 h with the terminal elimination half-live of 3.57 ±â€¯1.80 h and 5.92 ±â€¯3.34 h for the low dose (1 mg/kg) and high dose (3 mg/kg) respectively. In summary, the present method provides a robust, fast and sensitive analytical approach for quantification of Eggmanone in plasma and was successfully applied to a pharmacokinetic study in rats.

Simultaneous determination of roflumilast and its metabolite in human plasma by LC-MS/MS: Application for a pharmacokinetic study.

Roflumilast had shown good efficacy and safety in Caucasian COPD patients after oral administration of 0.5mg. The main active metabolite of it is roflumilast N-oxide. A reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantitation method was developed for the simultaneous determination of them in human plasma with rather low limits of quantitation for roflumilast (0.02ng/mL) and roflumilast N-oxide (0.04ng/mL). Human plasma samples were prepared by solid phase extraction (SPE), which ensured high recovery and slight matrix effect for the both analytes. This method showed good linearity, accuracy, precision and stability in the range of 0.02-10ng/mL and 0.04-50ng/mL for roflumilast and roflumilast N-oxide respectively. The developed method was successfully applied for the pharmacokinetic research in Chinese healthy volunteers after oral administration of 0.25mg, 0.375mg and 0.5mg of roflumilast tablet.

OPA-15406, a novel, topical, nonsteroidal, selective phosphodiesterase-4 (PDE4) inhibitor, in the treatment of adult and adolescent patients with mild to moderate atopic dermatitis (AD): A phase-II randomized, double-blind, placebo-controlled study.

BACKGROUND: Peripheral leukocytes in patients with atopic dermatitis (AD) have elevated phosphodiesterase-4 activity, which is associated with production of proinflammatory mediators. OPA-15406 is a phosphodiesterase-4 inhibitor with high selectivity for phosphodiesterase-4-B. OBJECTIVES: We sought to assess effectiveness and tolerability of topical OPA-15406 in patients with AD. METHODS: This was a randomized, double-blind, vehicle-controlled, phase-II study. Patients 10 to 70 years of age with mild or moderate AD received topical OPA-15406 0.3% (n = 41), OPA-15406 1% (n = 43), or vehicle (n = 37) twice daily for 8 weeks. RESULTS: The primary end point, Investigator Global Assessment of Disease Severity score of 0 or 1 with greater than or equal to 2-grade reduction, was met at week 4 in the OPA-15406 1% group (P = .0165 vs vehicle). Mean percentage improvement from baseline Eczema Area and Severity Index score for OPA-15406 1% was notable in week 1 (31.4% vs 6.0% for vehicle; P = .0005), even larger in week 2 (39.0% vs 3.0%; P = .0001), and persisted for 8 weeks. Visual analog scale pruritus scores improved from moderate to mild within the first week in the OPA-15406 1% group (36.4% mean change; P = .0011). OPA-15406 levels in blood were negligible. Incidence of adverse events was low, with most events mild in intensity. LIMITATIONS: Further confirmatory phase-III studies are required. CONCLUSION: OPA-15406 ointment may provide an effective therapeutic modality for patients with mild to moderate AD.

Effect of topical phosphodiesterase 4 inhibitor E6005 on Japanese children with atopic dermatitis: Results from a randomized, vehicle-controlled exploratory trial.

This exploratory study was designed to evaluate the safety and efficacy profile of the topical phosphodiesterase 4 inhibitor E6005 in Japanese children with mild-to-moderate atopic dermatitis. The present randomized, multicenter study included 62 patients who were treated with 0.05% E6005, 0.2% E6005 or vehicle ointment twice daily for 2 weeks. Safety and pharmacokinetics were assessed with a focus on the occurrence of adverse events and the whole blood concentrations of E6005 and its metabolite. Exploratory efficacy evaluations included assessments of lesion severity and pruritus score. The 2-week application of topical E6005 was safe and well tolerated with no cutaneous adverse events. The whole blood concentration of E6005 was quantified in only one subject receiving 0.2% E6005 treatment, while its major metabolite was undetectable. The 0.2% E6005 group showed a greater decrease in the severity score than the vehicle group (-45.94% vs -32.26%), although this difference was not statistically significant. Similarly, the treatment success rate according to the investigator's global assessment of the total application sites was higher in the 0.2% E6005 group than in the vehicle group (34.4% vs 20.0%). Moreover, the 0.2% E6005 group showed a greater decrease in the pruritus score than the vehicle group (-37.5% vs -6.7%) in a predefined subpopulation. The efficacy of 0.05% E6005 treatment was comparable to that of vehicle treatment. These results suggest that topical 0.2% E6005 treatment is safe and effective in children with atopic dermatitis, although further large confirmatory clinical trials are warranted.

Determination of a novel phosphodiesterase-4 inhibitor chlorbipram in mouse plasma and brain by UFLC-MS/MS: Application in pharmacokinetic studies after intravenous administration.

In this study, we evaluated a simple and sensitive method for determination of a novel phosphodiesterase-4 (PDE4) inhibitor, chlorbipram, in mouse plasma and brain using ultra-fast liquid chromatography-tandem mass spectrometry (UFLC-MS/MS). Separation was achieved using an Acquity UPLC BEH C18 column (50mm×2.1mm, particle size 1.7µm) with a gradient mobile phase consisting of water and methanol at a flow rate of 0.25ml/min. Detection was performed in the multiple reaction monitoring (MRM) mode using electrospray ionization (ESI) in the positive ion mode. The liquid-liquid extraction method with ethyl acetate was used for both pretreatment of plasma and brain homogenates. The calibration curves of chlorbipram showed good linearity over the concentration range of 0.5-200ng/ml (R(2)>0.994) for mouse plasma and over the range of 0.25-100ng/ml (R(2)>0.994) for mouse brain homogenate. The extraction recovery was in the range of 78.3-84.8% for chlorbipram and the internal standard (IS) ZXI14 in two different biological matrices. The intra- and inter-day precision values were less than 13.0% and the accuracy ranged from 97.8% to 106.0% for quality control samples. No noteworthy matrix effects and instability were observed for chlorbipram. This validated method was successfully applied to a pharmacokinetic study of chlorbipram in mice after intravenous administration. The results show that this novel drug crosses the blood-brain barrier and provides the basis for further studies on chlorbipram.

In vitro and in vivo metabolism of CHF 6001, a selective phosphodiesterase (PDE4) inhibitor.

1. The metabolism of CHF 6001, a novel PDE4 inhibitor, was determined in vitro in mouse, rat, dog, monkey and human microsomes and hepatocytes and in vivo in plasma, urine, feces and bile of rats after intravenous and intratracheal administration. 2. The behavior of CHF 6001 in microsomes and hepatocytes changed across species. CYP3A4/5 isoenzymes were identified to be the primary enzymes responsible for the metabolism of CHF 6001 in human liver microsomes. 3. In the rat, CHF 6001 was found extensively metabolized in urine, feces and bile, but not in plasma, where CHF 6001 was the main compound present. The metabolite profiles were different in the four biological matrices from both qualitative and quantitative point of view. 4. CHF 6001 was metabolized through hydrolysis with the formation of the alcohol CHF 5956, loss of a chlorine atom, loss of the N-oxide, hydroxylation, loss of the cyclopropylmethyl group in the alcohol moiety, conjugation with glucuronic acid, glutathione and cysteine-glycine. 5. The major metabolite present in the bile was isolated and characterized by nuclear magnetic resonance analysis. It derived from CHF 6001 through contraction of the pyridine-N-oxide ring to N-hydroxy pyrrole and conjugation with glucuronic acid.

Determination of a novel phosphodiesterase4 inhibitor, 3-[1-(3cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid (PDE-423) in rat plasma using liquid chromatography-tandem mass spectrometry.

A method for determining a novel phosphodiesterase-4 inhibitor, 3-[1-(3cyclopropylmethoxy-4-difluoromethoxybenzyl)-1H-pyrazol-3-yl]-benzoic acid (PDE-423), in rat plasma was developed and validated using liquid chromatography-tandem mass spectrometry for further pharmacokinetic study for development as a novel anti-asthmatic drug. PDE-423 in the concentration range of 0.02-10 µg/mL was linear with a correlation coefficient of >0.99, and the mean intra- and inter-assay precisions of the assay were 7.50 and 3.86%, respectively. The validated method was used successfully for a pharmacokinetic study of PDE-423 in rats.

Determination of ASP3258, a novel phosphodiesterase type 4 inhibitor, in rat plasma by high-performance liquid chromatography with fluorescence detection and its application to pharmacokinetic study.

The potent phosphodiesterase 4 inhibitor ASP3258 contains a carboxylic acid moiety and a naphthyridine ring and is a novel therapeutic agent for asthma and chronic obstructive pulmonary disease. To support the drug development of ASP3258, we developed and validated a simple method for its determination in rat plasma. Following the addition of the analog AS1406604-00 as an internal standard, plasma samples were processed using C18 -bonded solid-phase extraction cartridges under acidic conditions and injected into a high-performance liquid chromatography system with fluorescence detection. Chromatographic separation was achieved on a Shiseido Capcell Pak C18 UG120 column (3.0 × 150 mm, 5 µm) with a mobile phase consisting of acetonitrile-0.5% acetic acid (50:50, v/v). HPLC eluent was monitored with a fluorescence detector set at a wavelength of 315 nm for excitation and 365 nm for emission. The calibration curve was linear over a range of 2.5-250 ng/mL. Validation data demonstrated that the method is selective, sensitive and accurate. In addition, the present method was successfully applied to rat plasma samples from a pharmacokinetic study.

Effects of rolipram and roflumilast, phosphodiesterase-4 inhibitors, on hypertension-induced defects in memory function in rats.

Hypertension (HT) is a prevailing risk factor for cognitive impairment, the most common cause of vascular dementia; yet, no possible mechanism underlying the cognitive impairment induced by hypertension has been identified so far. Inhibition of PDE-4 has been shown to increase phosphorylation of cAMP-response element binding protein in the hippocampus and enhance the memory performance. Here, we examined the effects of PDE-4 inhibitors, rolipram and roflumilast, on the impairment of learning and memory observed in hypertensive rats. We used 2k-1c hypertensive model to induce learning and memory defects. In addition, mRNA expression of PDE-4 sub-types A-D was also assessed in the hippocampus tissue. Systolic blood pressure (SBP) was measured by tail-cuff method was significantly increased in 2k-1c rats when compared to sham operated rats; this effect was reversed by clonidine, whereas, PDE-4 inhibitors did not. PDE-4 inhibitors significantly reversed time induced memory deficit in novel object recognition task (NORT). Further, the retention latency on the second day in the elevated plus maze model was significantly shortened after repeated administration of rolipram and roflumilast. Plasma and brain concentrations of rolipram, roflumilast and roflumilast N-oxide were also measured after the NORT and showed linear increase in plasma and brain concentrations. The PDE4B and PDE4D gene expression was significantly enhanced in hypertensive rats compared with sham operated however PDE4A and PDE4C remained unaltered. Repeated treatment with PDE-4 inhibitors caused down regulation of PDE4B and PDE4D in hypertensive rats. These results suggest that inhibition of PDE-4 ameliorates HT-induced impairment of learning and memory functions.

Novel Roflumilast analogs as soft PDE4 inhibitors.

PDE4 inhibitors are of high interest for treatment of a wide range of inflammatory or autoimmune diseases. Their potential however has not yet been realized due to target-associated side effects, resulting in a low therapeutic window. We herein report the design, synthesis and evaluation of novel PDE4 inhibitors containing a γ-lactone structure. Such molecules are designed to undergo metabolic inactivation when entering circulation, thereby limiting systemic exposure and reducing the risk for side effects. The resulting inhibitors were highly active on both PDE4B1 and PDE4D2 and underwent rapid degradation in human plasma by paraoxonase 1. In contrast, their metabolites displayed markedly reduced permeability and/or on-target activity.

Nanostructured lipid carriers containing a high percentage of a Pluronic copolymer increase the biodistribution of novel PDE4 inhibitors for the treatment of traumatic hemorrhage.

Although the application of nanotechnology to drug therapy has been widely investigated, very few nanomedicine-based treatments for traumatic hemorrhage have been reported so far. The aim of this work was to develop nanostructured lipid carriers (NLCs) loaded with phosphodiesterase 4 (PDE4) inhibitors to treat acute inflammation in peripheral organs. The pharmacokinetics and biodistribution of DSM-RX78 and EFB-1, two novel PDE4 inhibitors, were examined using rats as an animal model. Entrapment by NLCs resulted in sustained drug release. The plasma concentrations of DSM-RX78 and EFB-1 in NLCs were lower, and their half-lives were much shorter in the NLC condition than in the control condition. PDE4 inhibitors delivered in NLCs accumulated with high abundance in many organs, especially the brain and lungs. Polyethylene glycol (PEG) coating on the particulate surface (P-NLCs) significantly reduced brain delivery of the drugs. P-NLCs enhanced drug distribution to the lungs by 5-fold compared to free control. In vivo real-time imaging confirmed rapid escape of nanoparticles from the blood circulation. Histological examination and aminotransferase measurement revealed that P-NLCs containing EFB-1 improved hemorrhagic shock-induced injuries in the lungs, intestines, and liver. P-NLCs even reversed acute lung inflammation to the level observed in an uninjured condition. Our results indicate that NLC-based delivery of PDE4 inhibitors is a candidate treatment for traumatic hemorrhage.

The impact of co-administration of ketoconazole and rifampicin on the pharmacokinetics of apremilast in healthy volunteers.

AIMS: Two clinical studies were conducted to determine possible drug-drug interactions between apremilast and a strong CYP3A4 inhibitor, ketoconazole, or a potent CYP3A4 inducer, rifampicin. The main objectives of these two studies were to evaluate the impact of multiple doses of ketoconazole on the pharmacokinetics of apremilast and its metabolites, and the effect of multiple oral doses of rifampicin on the pharmacokinetics of apremilast. METHODS: These single centre, open label, sequential treatment studies in healthy subjects included two treatment periods for ketoconazole and three treatment periods for rifampicin. Apremilast was administered as a 20 mg (ketoconazole study) or 30 mg (rifampicin study) single oral dose. RESULTS: Ketoconazole increases overall exposure (AUC(0,∞)) of apremilast by ≈36% (2827 vs. 2072 ng ml(-1) h, 90% CI = 126.2, 147.5) and peak exposure (Cmax ) by 5% (247 vs. 236 ng ml(-1) ). Multiple doses of rifampicin increase apremilast clearance ≈3.6-fold and decrease apremilast mean AUC(0,∞) by ≈72% (3120 vs. 869 ng ml(-1) h, 90% CI = 25.7, 30.4) and Cmax (from 290 vs. 166 ng ml(-1) ) relative to that of apremilast given alone. A 30 min intravenous infusion of rifampicin 600 mg had negligible effects on the overall exposure (AUC(0,∞)) of apremilast (2980 vs. 3120 ng ml(-1) h, 90% CI = 88.0, 104.1). CONCLUSION: Ketoconazole slightly decreased apremilast clearance, resulting in a small increase in AUC which is probably not meaningful clinically. However, the effect of CYP3A4 induction by rifampicin on apremilast clearance is much more pronounced than that of CYP3A4 inhibition by ketoconazole. Strong CYP3A4 inducers may result in a loss of efficacy of apremilast because of decreased drug exposure.

Determination of 2-aryl-7(3',4'-dialkoxyphenyl)-pyrazolo [1,5-alpha] pyrimidine, a novel phosphodiesterase-4 inhibitor, in rat plasma by liquid chromatography-tandem mass spectrometry.

A method for assaying a novel phosphodiesterase-4 inhibitor, 2-aryl-7(3',4'-dialkoxyphenyl)-pyrazolo [1,5-alpha] pyrimidine (PDE-310), was developed and validated in rat plasma using liquid chromatography-tandem mass spectrometry (LC-MS-MS). Rat plasma samples were processed by liquid-liquid extraction with ethyl acetate and injected onto the LC-MS-MS system for quantification. PDE-310 and imipramine (i.e., internal standard) were separated using a Gemini C18 column with mixture of acetonitrile and 0.1% formic acid (70:30, v/v) as the mobile phase. The ion transitions monitored were m/z 425.0 → 331.0 for PDE-310 and m/z 281.3 → 86.1 for imipramine in the multiple-reaction monitoring mode. The detector response was specific and linear for PDE-310 concentrations in the range of 0.1-50 µg/mL. The intra-day and inter-day precision and accuracy of the method were determined to be within the acceptance criteria for assay validation guidelines. The recoveries were approximately 85.7 and 88.2% from rat plasma for PDE-310 and imipramine, respectively. PDE-310 was stable under various processing and handling conditions. PDE-310 concentrations were readily measured in rat plasma samples up to 8 h after an intravenous administration of PDE-310, suggesting that the assay is practically useful.

Early rapid identification of in vivo rat metabolites of AN6414, a novel boron-containing PDE4 inhibitor by QTRAP LC/MS/MS to support drug discovery.

There is an increasing interest in in vivo metabolite identification in early drug discovery in order to (i) give a more complete picture of metabolic profile in investigational animal models, (ii) propose phase I and phase II metabolites using the same pharmacokinetic/toxicokinetic study samples, (iii) expose metabolically labile groups where chemical modifications could improve stability, and (iv) enable early safety assessment of metabolites. In the early discovery stage of our anti-inflammatory program, one novel benzoxaborole, AN6414, exhibiting both PDE4 enzyme and TNFα inhibition activities, became our primary candidate for further investigation. The traditional metabolite identifications usually require high dosed samples with long data scans and analysis. In this study, we conducted quick and more selective core-structure related precursor scans followed by daughter ion scans and identified a total of 10 major phase I and phase II metabolites using rat plasma samples from a toxicokinetic study at an oral dosing of 30 mg/kg. Plasma samples were treated with solid phase extraction (SPE) prior to LC/MS/MS. An AB SCIEX API 4000 QTRAP mass spectrometer coupled with a Shimadzu LC system was used for LC/MS/MS analysis. We found the major metabolites of AN6414 to be oxidative deboronation, protodeboronation, oxidation products and their sulfate-conjugated species. This analysis drove analoging efforts which improved the pharmacokinetic profile, namely, lowering clearance and increasing exposure relative to AN6414. Toxicity predictions by the software program DEREK suggest the identified potential metabolites to be safe.

Downregulation of brain phosphodiesterase type IV measured with 11C-(R)-rolipram positron emission tomography in major depressive disorder.

BACKGROUND: Phosphodiesterase type IV (PDE4), an important component of the cyclic adenosine monophosphate (cAMP) cascade, selectively metabolizes cAMP in the brain to the inactive monophosphate. Basic studies suggest that PDE4 mediates the effects of several antidepressants. This study sought to quantify the binding of 11C-(R)-rolipram, a PDE4 inhibitor, as an indirect measure of this enzyme's activity in the brain of individuals with major depressive disorder (MDD) compared with healthy control subjects. METHODS: 11C-(R)-Rolipram brain positron emission tomography scans were performed in 28 unmedicated MDD subjects and 25 age- and gender-matched healthy control subjects. Patients were moderately depressed and about one half were treatment-naive. 11C-(R)-Rolipram binding in the brain was measured using arterial 11C-(R)-rolipram levels to correct for the influence of cerebral blood flow. RESULTS: Major depressive disorder subjects showed a widespread, approximately 20% reduction in 11C-(R)-rolipram binding (p = .002), which was not caused by different volumes of gray matter. Decreased rolipram binding of similar magnitudes was observed in most brain areas. Rolipram binding did not correlate with the severity of depressive or anxiety symptoms. CONCLUSIONS: This study is the first to demonstrate that brain levels of PDE4, a critical enzyme that regulates cAMP, are decreased in unmedicated individuals with MDD in vivo. These results are in line with human postmortem and rodent studies demonstrating downregulation of the cAMP cascade in MDD and support the hypothesis that agents such as PDE4 inhibitors, which increase activity within the cAMP cascade, may have antidepressant effects.