Head-to-head comparison of (R)-[11C]verapamil and [18F]MC225 in non-human primates, tracers for measuring P-glycoprotein function.

PURPOSE: P-glycoprotein (P-gp) function is altered in several brain disorders; thus, it is of interest to monitor the P-gp function in vivo using PET. (R)-[11C]verapamil is considered the gold standard tracer to measure the P-gp function; however, it presents some drawbacks that limit its use. New P-gp tracers have been developed with improved properties, such as [18F]MC225. This study compares the characteristics of (R)-[11C]verapamil and [18F]MC225 in the same subjects. METHODS: Three non-human primates underwent 4 PET scans: 2 with (R)-[11C]verapamil and 2 with [18F]MC225, at baseline and after P-gp inhibition. The 30-min PET data were analyzed using 1-Tissue Compartment Model (1-TCM) and metabolite-corrected plasma as input function. Tracer kinetic parameters at baseline and after inhibition were compared. Regional differences and simplified methods to quantify the P-gp function were also assessed. RESULTS: At baseline, [18F]MC225 VT values were higher, and k2 values were lower than those of (R)-[11C]verapamil, whereas K1 values were not significantly different. After inhibition, VT values of the 2 tracers were similar; however, (R)-[11C]verapamil K1 and k2 values were higher than those of [18F]MC225. Significant regional differences between tracers were found at baseline, which disappeared after inhibition. The positive slope of the SUV-TAC was positively correlated to the K1 and VT of both tracers. CONCLUSION: [18F]MC225 and (R)-[11C]verapamil show comparable sensitivity to measure the P-gp function in non-human primates. Moreover, this study highlights the 30-min VT as the best parameter to measure decreases in the P-gp function with both tracers. [18F]MC225 may become the first radiofluorinated tracer able to measure decreases and increases in the P-gp function due to its higher baseline VT.

In Vivo Induction of P-Glycoprotein Function can be Measured with [18F]MC225 and PET.

P-Glycoprotein (P-gp) is an efflux pump located at the blood-brain barrier (BBB) that contributes to the protection of the central nervous system by transporting neurotoxic compounds out of the brain. A decline in P-gp function has been related to the pathogenesis of neurodegenerative diseases. P-gp inducers can increase the P-gp function and are considered as potential candidates for the treatment of such disorders. The P-gp inducer MC111 increased P-gp expression and function in SW480 human colon adenocarcinoma and colo-320 cells, respectively. Our study aims to evaluate the P-gp inducing effect of MC111 in the whole brain in vivo, using the P-gp tracer [18F]MC225 and positron emission tomography (PET). Eighteen Wistar rats were treated with either vehicle solution, 4.5 mg/kg of MC111 (low-dose group), or 6 mg/kg of MC111 (high-dose group). Animals underwent a 60 min dynamic PET scan with arterial-blood sampling, 24 h after treatment with the inducer. Data were analyzed using the 1-tissue-compartment model and metabolite-corrected plasma as the input function. Model parameters such as the influx constant (K1) and volume of distribution (VT) were calculated, which reflect the in vivo P-gp function. P-gp and pregnane xenobiotic receptor (PXR) expression levels of the whole brain were assessed using western blot. The administration of MC111 decreased K1 and VT of [18F]MC225 in the whole brain and all of the selected brain regions. In the high-dose group, whole-brain K1 was decreased by 34% (K1-high-dose = 0.20 ± 0.02 vs K1-control = 0.30 ± 0.02; p < 0.001) and in the low-dose group by 7% (K1-low-dose = 0.28 ± 0.02 vs K1-control = 0.30 ± 0.02; p = 0.42) compared to controls. Whole-brain VT was decreased by 25% in the high-dose group (VT-high-dose = 5.92 ± 0.41 vs VT-control = 7.82 ± 0.38; p < 0.001) and by 6% in the low-dose group (VT-low-dose = 7.35 ± 0.38 vs VT-control = 7.82 ± 0.37; p = 0.38) compared to controls. k2 values did not vary after treatment. The treatment did not affect the metabolism of [18F]MC225. Western blot studies using the whole-brain tissue did not detect changes in the P-gp expression, however, preliminary results using isolated brain capillaries found an increasing trend up to 37% in treated rats. The decrease in K1 and VT values after treatment with the inducer indicates an increase in the P-gp functionality at the BBB of treated rats. Moreover, preliminary results using brain endothelial cells also sustained the increase in the P-gp expression. In conclusion, the results verify that MC111 induces P-gp expression and function at the BBB in rats. An increasing trend regarding the P-gp expression levels is found using western blot and an increased P-gp function is confirmed with [18F]MC225 and PET.

Pharmacokinetic Modeling of (R)-[11C]verapamil to Measure the P-Glycoprotein Function in Nonhuman Primates.

(R)-[11C]verapamil is a radiotracer widely used for the evaluation of the P-glycoprotein (P-gp) function at the blood-brain barrier (BBB). Several studies have evaluated the pharmacokinetics of (R)-[11C]verapamil in rats and humans under different conditions. However, to the best of our knowledge, the pharmacokinetics of (R)-[11C]verapamil have not yet been evaluated in nonhuman primates. Our study aims to establish (R)-[11C]verapamil as a reference P-gp tracer for comparison of a newly developed P-gp positron emission tomography (PET) tracer in a species close to humans. Therefore, the study assesses the kinetics of (R)-[11C]verapamil and evaluates the effect of scan duration and P-gp inhibition on estimated pharmacokinetic parameters. Three nonhuman primates underwent two dynamic 91 min PET scans with arterial blood sampling, one at baseline and another after inhibition of the P-gp function. The (R)-[11C]verapamil data were analyzed using 1-tissue compartment model (1-TCM) and 2-tissue compartment model fits using plasma-corrected for polar radio-metabolites or non-corrected for radio-metabolites as an input function and with various scan durations (10, 20, 30, 60, and 91 min). The preferred model was chosen according to the Akaike information criterion and the standard errors (SE %) of the estimated parameters. 1-TCM was selected as the model of choice to analyze the (R)-[11C]verapamil data at baseline and after inhibition and for all scan durations tested. The volume of distribution (VT) and the efflux constant k2 estimations were affected by the evaluated scan durations, whereas the influx constant K1 estimations remained relatively constant. After P-gp inhibition (tariquidar, 8 mg/kg), in a 91 min scan duration, the whole-brain VT increased significantly up to 208% (p < 0.001) and K1 up to 159% (p < 0.001) compared with baseline scans. The k2 values decreased significantly after P-gp inhibition in all the scan durations except for the 91 min scans. This study suggests the use of K1, calculated with 1-TCM and using short PET scans (10 to 30 min), as a suitable parameter to measure the P-gp function at the BBB of nonhuman primates.

Structure-activity relationship study towards non-peptidic positron emission tomography (PET) radiotracer for gastrin releasing peptide receptors: Development of [18F] (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide.

Gastrin-releasing peptide receptors (GRP-Rs, also known as bombesin 2 receptors) are overexpressed in a variety of human cancers, including prostate cancer, and therefore they represent a promising target for in vivo imaging of tumors using positron emission tomography (PET). Structural modifications of the non-peptidic GRP-R antagonist PD-176252 ((S)-1a) led to the identification of the fluorinated analog (S)-3-(1H-indol-3-yl)-N-[1-[5-(2-fluoroethoxy)pyridin-2-yl]cyclohexylmethyl]-2-methyl-2-[3-(4-nitrophenyl)ureido]propionamide ((S)-1m) that showed high affinity and antagonistic properties for GRP-R. This antagonist was stable in rat plasma and towards microsomal oxidative metabolism in vitro. (S)-1m was successfully radiolabeled with fluorine-18 through a conventional radiochemistry procedure. [18F](S)-1m showed high affinity and displaceable interaction for GRP-Rs in PC3 cells in vitro.

Measurements of serum non-ceruloplasmin copper by a direct fluorescent method specific to Cu(II).

BACKGROUND: Meta-analyses indicated the breakdown of copper homeostasis in the sporadic form of Alzheimer's disease (AD), comprising copper decreases within the brain and copper increases in the blood and the pool not bound to ceruloplasmin (non-Cp Cu, also known in the literature as "free" copper). The calculated non-Cp Cu (Walshe's) index has many limitations. METHODS: A direct fluorescent method for non-Cp Cu detection has been developed and data are presented herein. The study included samples from 147 healthy subjects, 36 stable mild cognitive impairment (MCI) and 89 AD patients, who were tested for non-Cp Cu through the direct method, total serum copper, ceruloplasmin concentration and o-dianisidine ceruloplasmin activity. The indirect non-Cp Cu Walshe's index was also calculated. RESULTS: The direct method was linear (0.9-5.9 µM), precise (within-laboratory coefficient variation of 9.7% for low and 7.1% for high measurements), and had a good recovery. A reference interval (0-1.9 µM) was determined parametrically in 147 healthy controls (27-84 years old). The variation of non-Cp Cu was evaluated according to age and sex. Non-Cp Cu was 1.5 times higher in AD patients (regarding the upper value of the reference interval) than in healthy controls. Healthy, MCI and AD subjects were differentiated through the direct non-Cp Cu method [areas under the curve (AUC)=0.755]. Considering a 95% specificity and a 1.91 µmol/L cut-off, the sensitivity was 48.3% (confidence interval 95%: 38%-58%). The likelihood ratio (LR) was 9.94 for positive test results (LR+) and 0.54 for negative test result (LR-). CONCLUSIONS: The direct fluorescent test reliably and accurately measures non-Cp Cu, thereby determining the probability of having AD.

Radiosynthesis and in vivo Evaluation of Carbon-11 (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide: An Attempt to Visualize Brain Formyl Peptide Receptors in Mouse Models of Neuroinflammation.

Here, we describe the very first attempt to visualize in vivo formyl peptide receptors (FPRs) in mouse brain by positron emission tomography (PET). FPRs are expressed in microglial cells where they mediate chemotactic activity of ß-amyloid peptide in Alzheimer disease and, thus, are involved in neuroinflammatory processes. To this purpose, we have selected (2S)-3-(1H-Indol-3-yl)-2-{[(4-methoxyphenyl)carbamoyl]amino}-N-{[1-(5-methoxypyridin-2-yl)cyclohexyl]methyl}propanamide ((S)-1), that we have previously identified as a potent non-peptidic FPR agonist. (S)-[(11) C]-1 has been prepared in high radiochemical yield. (S)-[(11) C]-1 showed very low penetration of blood-brain barrier and, thus, was unable to accumulate into the brain. In addition, (S)-[(11) C]-1 was not able to label FPRs receptors in brain slices of PS19 and APP23 mice, two animal models of Alzheimer disease. Although (S)-[(11) C]-1 was not suitable to visualize FPRs in the brain, this study provides useful information for the design and characterization of future potential PET radioligands for visualization of brain FPRs by PET.

Functionalized Coumarine Fragment to Obtain Fluorescent and Selective P-Glycoprotein Ligands.

Starting from our lead compound MC70 displaying high P-glycoprotein (P-gp) inhibition activity but low selectivity, a new class of coumarine derivatives was studied to develop selective and fluorescent P-gp ligands. In this series, the biphenyl moiety of MC70 was replaced with the coumarine fluorophore as a bioisostere of the biphenyl nucleus in order to improve the selectivity toward P-gp and the fluorescent properties for in vitro studies. Moreover, the presence and position of substituents on the coumarine nucleus were probed to develop suitable fluorescent probes to study the expression and activity of P-gp in living cells. The best result was found for compound 4c, which exerts a good P-gp activity profile (EC50 = 13 µM) as substrate and a high selectivity toward the pump since it is inactive toward MRP1.

Synthesis and Preclinical Evaluation of Three Novel Fluorine-18 Labeled Radiopharmaceuticals for P-Glycoprotein PET Imaging at the Blood-Brain Barrier.

P-Glycoprotein (P-gp), along with other transporter proteins at the blood-brain barrier (BBB), limits the entry of many pharmaceuticals into the brain. Altered P-gp function has been found in several neurological diseases. To study the P-gp function, many positron emission tomography (PET) radiopharmaceuticals have been developed. Most P-gp radiopharmaceuticals are labeled with carbon-11, while labeling with fluorine-18 would increase their applicability due to longer half-life. Here we present the synthesis and in vivo evaluation of three novel fluorine-18 labeled radiopharmaceuticals: 4-((6,7-dimethoxy-3,4-dihydroisoquinolin-2(1H)-yl)methyl)-2-(4-fluorophenyl)oxazole (1a), 2-biphenyl-4-yl-2-fluoroethoxy-6,7-dimethoxy-1,2,3,4-tetrahydro-isoquinoline (2), and 5-(1-(2-fluoroethoxy))-[3-(6,7-dimethoxy-3,4-dihydro-1H-isoquinolin-2-yl)-propyl]-5,6,7,8-tetrahydronaphthalen (3). Compounds were characterized as P-gp substrates in vitro, and Mdr1a/b((-/-))Bcrp1((-/-)) and wild-type mice were used to assess the substrate potential in vivo. Comparison was made to (R)-[(11)C]verapamil, which is currently the most frequently used P-gp substrate. Compound [(18)F]3 was performing the best out of the new radiopharmaceuticals; it had 2-fold higher brain uptake in the Mdr1a/b((-/-))Bcrp1((-/-)) mice compared to wild-type and was metabolically quite stable. In the plasma, 69% of the parent compound was intact after 45 min and 96% in the brain. Selectivity of [(18)F]3 to P-gp was tested by comparing the uptake in Mdr1a/b((-/-)) mice to uptake in Mdr1a/b((-/-))Bcrp1((-/-)) mice, which was statistically not significantly different. Hence, [(18)F]3 was found to be selective for P-gp and is a promising new radiopharmaceutical for P-gp PET imaging at the BBB.

Arylpiperazine agonists of the serotonin 5-HT1A receptor preferentially activate cAMP signaling versus recruitment of ß-arrestin-2.

G protein-coupled receptors (GPCRs) mediate biological signal transduction through complex molecular pathways. Therapeutic effects of GPCR-directed drugs are typically accompanied by unwanted side effects, owing in part to the parallel engagement of multiple signaling mechanisms. The discovery of drugs that are 'functionally selective' towards therapeutic effects, based on their selective control of cellular responses through a given GPCR, is thus a major goal in pharmacology today. In the present study, we show that several arylpiperazine ligands of the serotonin 5-HT1A receptor (5-HT1AR) preferentially activate 3',5'-cyclic adenosine monophosphate (cAMP) signaling versus ß-arrestin-2 recruitment. The pharmacology of these compounds is thus qualitatively different from the endogenous agonist serotonin, indicating functional selectivity of 5-HT1AR-mediated response pathways. Preliminary evidence suggests that phosphorylation of extracellular signal-regulated kinases 1/2 (ERK1/2) downstream of 5-HT1AR is a substrate of functionally selective signaling by partial agonists. We propose that the compounds described in the present study are useful starting points for the development of signaling pathway-selective drugs targeting 5-HT1AR.

Novel 3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]propanamides as selective agonists of human formyl-peptide receptor 2.

N-Formyl peptide receptors (FPRs) are G protein-coupled receptors (GPCRs) that play critical roles in inflammatory reactions, and FPR-specific interactions can possibly be used to facilitate the resolution of pathological inflammatory reactions. We here report the synthesis and biological evaluation of six pairs of chiral ureidopropanamido derivatives as potent and selective formyl peptide receptor-2 (FPR2) agonists that were designed starting from our lead agonist (S)-3-(1H-indol-3-yl)-2-[3-(4-methoxyphenyl)ureido]-N-[[1-(5-methoxy-2-pyridinyl)cyclohexyl]methyl]propanamide ((S)-9a). The new compounds were obtained in overall yields considerably higher than (S)-9a. Several of the new compounds showed agonist properties comparable to that of (S)-9a along with higher selectivity over FPR1. Molecular modeling was used to define chiral recognition by FPR2. In vitro metabolic stability of selected compounds was also assessed to obtain preliminary insight on drug-like properties of this class of compounds.

Selective agonists for serotonin 7 (5-HT7) receptor and their applications in preclinical models: an overview.

The serotonin 7 (5-HT7) receptor was the last serotonin receptor subtype to be discovered in 1993. This receptor system has been implicated in several central nervous system (CNS) functions, including circadian rhythm, rapid eye movement sleep, thermoregulation, nociception, memory and neuropsychiatric symptoms and pathologies, such as anxiety, depression and schizophrenia. In 1999, medicinal chemistry efforts led to the identification of SB-269970, which became the gold standard selective 5-HT7 receptor antagonist, and later of various selective agonists such as AS-19, LP-44, LP-12, LP-211 and E-55888. In this review, we summarize the preclinical pharmacological studies performed using these agonists, highlighting their strengths and weaknesses. The data indicate that 5-HT7 receptor agonists can have neuroprotective effects against N-methyl-d-aspartate-induced toxicity, modulate neuronal plasticity in rats, enhance morphine-induced antinociception and alleviate hyperalgesia consecutive to nerve lesion in neuropathic animals.

Design, synthesis, radiolabeling and in vivo evaluation of potential positron emission tomography (PET) radioligands for brain imaging of the 5-HT7 receptor.

Here we describe the design, synthesis, and pharmacological evaluation of a set of compounds structurally related to the high affinity serotonin 5-HT7 receptor agonist N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (6, LP-211). Specific structural modifications were performed in order to maintain affinity for the target receptor and to improve the selectivity over 5-HT1A and adrenergic α1 receptors. The synthesized compounds have chemical features that could enable labeling with a positron emitter radioisotope (carbon-11 or fluorine-18) and lipophilicity within the range considered optimal for brain penetration and low non-specific binding. 4-[2-(4-Methoxyphenyl)phenyl]-N-(pyridin-4-ylmethyl)piperazinehexanamide (23a) and N-pyridin-4-ylmethyl-3-[4-[2-(4-methoxyphenyl)phenyl]piperazin-1-yl]ethoxy]propanamide (26a) were radiolabeled on the methoxy group with carbon-11. Positron emission tomography (PET) analysis revealed that [(11)C]-23a and [(11)C]-26a were P-glycoprotein (P-gp) substrates and rapidly metabolized, resulting in poor brain uptake. These features were not predicted by in vitro tests.

Design, synthesis, lipophilic properties, and binding affinities of potential ligands in positron emission tomography (PET) for visualization of brain dopamine D4 receptors.

We report the synthesis of compounds structurally related to the high-affinity dopamine D4 receptor ligand N-{2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-methoxybenzamide (1e). All compounds were specifically designed as potential PET radioligands for brain D4 receptor visualization, having lipophilicity within a range for brain uptake and weak non-specific binding (0.75100-fold), but its D4 receptor affinity was suboptimal for imaging of brain D4 receptors (Ki =30 nM).

Crystallographic study of PET radio-tracers in clinical evaluation for early diagnosis of Alzheimers.

The title compound, C24H25NO3·2CH3OH, which crystallized as a methanol disolvate, has applications as a PET radiotracer in the early diagnosis of Alzheimer's disease. The dihedral angle between the biphenyl rings is 8.2 (2)° and the heterocyclic ring adopts a half-chair conformation with the N atom adopting a pyramidal geometry (bond-angle sum = 327.6°). The C atoms of both meth-oxy groups lie close to the plane of their attached ring [deviations = 0.107 (6) and 0.031 (6) Å]. In the crystal, the components are linked by O-H⋯O and O-H⋯N hydrogen bonds, generating [010] chains. C-H⋯O inter-actions are also observed.

Nature-Inspired 1-Phenylpyrrolo[2,1-a]isoquinoline Scaffold for Novel Antiproliferative Agents Circumventing P-Glycoprotein-Dependent Multidrug Resistance.

Previous studies have shown that some lamellarin-resembling annelated azaheterocyclic carbaldehydes and related imino adducts, sharing the 1-phenyl-5,6-dihydropyrrolo[2,1-a]isoquinoline (1-Ph-DHPIQ) scaffold, are cytotoxic in some tumor cells and may reverse multidrug resistance (MDR) mediated by P-glycoprotein (P-gp). Herein, several novel substituted 1-Ph-DHPIQ derivatives were synthesized which carry carboxylate groups (COOH, COOEt), nitrile (CN) and Mannich bases (namely, morpholinomethyl derivatives) in the C2 position, as replacements of the already reported aldehyde group. They were evaluated for antiproliferative activity in four tumor cell lines (RD, HCT116, HeLa, A549) and for the ability of selectively inhibiting P-gp-mediated MDR. Lipophilicity descriptors and molecular docking calculations helped us in rationalizing the structure-activity relationships in the P-gp inhibition potency of the investigated 1-Ph-DHPIQs. As a main outcome, a morpholinomethyl Mannich base (8c) was disclosed which proved to be cytotoxic to all the tested tumor cell lines in the low micromolar range (IC50 < 20 µM) and to inhibit in vitro the efflux pumps P-gp and MRP1 responsible for MDR, with IC50s of 0.45 and 12.1 µM, respectively.

Towards metabolically stable 5-HT7 receptor ligands: a study on 1-arylpiperazine derivatives and related isosters.

Serotonin 7 (5-hydroxytryptamine7 or 5-HT7) is the most recently identified serotonin receptor. It is involved in mood disorders and is studied as a target for antidepressants. Here, we report on the structural manipulation of the 5-HT7 receptor ligand 4-[2-(3-methoxyphenyl)ethyl]-1-(2-methoxyphenyl)piperazine (1a) aimed at obtaining 5-HT7 receptor ligands endowed with good in vitro metabolic stability. A set of N-[3-methoxyphenyl)ethyl-substituted] 1-arylpiperazine, 4-arylpiperidine and 1-aryl-4-aminopiperidine was synthesized and tested in radioligand binding assays at human cloned 5-HT7 and 5-HT1A receptors. In vitro metabolic stability of the target compounds was assessed after incubation with rat hepatic S9 microsomal fraction. Among the new compounds, 1-(2-biphenyl)-4-[2-(3-methoxyphenyl)ethyl]piperazine (1d) and 4-(2-biphenyl)-1-[2-(3-methoxyphenyl)ethyl]piperidine (2d) showed a good compromise between affinity at 5-HT7 receptor (K i = 7.5 nM and 13 nM, respectively) and in vitro metabolic stability (26 and 65 % recovery of parent compound, respectively) but were poorly selective over 5-HT1A receptor.

Naphthalenyl derivatives for hitting P-gp/MRP1/BCRP transporters.

Substituted naphthalenyl derivatives bearing oxazole, or thiazole or furyl heteronuclei have been carried out as bioisosters of aryl-oxazoles and -thiazoles derivatives previously reported in order to investigate the role of the hindrance on the activity towards P-gp/BCRP/and MRP1 transporters. In addition, the role of naphthalenyl group to modulate P-gp intrinsic activity of these compounds was ascertained. The results demonstrated that all naphthalenyl derivatives displayed comparable P-gp activity with respect to lead compounds previously characterized in our SAR studies but were less active towards BCRP and MRP1 pumps. In terms of intrinsic activity, the replacement of aryl with naphthalenyl moiety led to P-gp inhibitors, unambiguous or ambiguous substrates on the base of the heteronucleus and the substituent on the naphthalenyl fragment. Indeed, oxazole derivatives were: inhibitors (R=H, F, OH), unambiguous substrates (R=OCH(3)), or ambiguous substrate (R=Br); thiazole derivatives were: unambiguous substrates (R=OCH(3), Br), or ambiguous substrates (R=H, F). Finally furyl derivatives were ambiguous substrates.

LC-MS/MS Analysis on Infusion Bags and Filled Syringes of Decitabine: New Data on Physicochemical Stability of an Unstable Molecule.

Many anticancer drugs are reported to have low physicochemical stability after dilution; therefore, producers impose short times from reconstitution, dilution, and the end of administration. The precariousness of cancer patients' health in real-life experience within cancer hospitals often forces delays in the drug administration with respect to the standard treatment schedule timing, because of acute toxicities or the need to postpone a control analysis before administration. The public health costs for discarded anticancer drugs due to administration interruptions can be avoided, thanks to independent analytical studies, which integrate the producer's data reported in the technical sheet, referring to the real conditions of preparation in a sterile atmosphere under a cabin in a laboratory dedicated to handling cytotoxic drugs in controlled conditions of temperature, pressure, and particulate contamination. Decitabine is apparently an unstable molecule, whose reported stability is only 3 h at 2-8 °C when diluted, while the mother solution must be immediately used or, otherwise, discarded. This study has investigated the physicochemical stability of decitabine both in diluted infusion bags and in sterile water reconstituted syringes at 4 °C for 0, 24, 48, and 72 h. In all performed studies, the stability-indicating method involves, for the first time, the use of liquid chromatography-tandem mass spectrometry analysis. Unexpectedly, both diluted and reconstituted solutions of decitabine are more stable than previously reported data, with a 48 h-long physicochemical stability at 2-8 °C and protected from light.

Dose-response assessment of cerebral P-glycoprotein inhibition in vivo with [18F]MC225 and PET.

The Blood-Brain Barrier P-glycoprotein (P-gp) function can be altered in several neurodegenerative diseases and due to the administration of different drugs which may cause alterations in drug concentrations and consequently lead to a reduced effectiveness or increased side-effects. The novel PET radiotracer [18F]MC225 is a weak P-gp substrate that may show higher sensitivity to detect small changes in P-gp function than previously developed radiotracers. This study explores the sensitivity of [18F]MC225 to measure the dose-dependent effect of P-gp inhibitor tariquidar. Twenty-three rats were intravenously injected with different doses of tariquidar ranging from 0.75 to 12 mg/kg, 30-min before the dynamic [18F]MC225-PET acquisition with arterial sampling. Tissue and blood data were fitted to a 1-Tissue-Compartment-Model to obtain influx constant K1 and distribution volume VT, which allow the estimation of P-gp function. ANOVA and post-hoc analyses of K1 values showed significant differences between controls and groups with tariquidar doses >3 mg/kg; while applying VT the analyses showed significant differences between controls and groups with tariquidar doses >6 mg/kg. Dose-response curves were fitted using different models. The four-parameter logistic sigmoidal curve provided the best fit for K1 and VT data. Half-maximal inhibitory doses (ID50) were 2.23 mg/kg (95%CI: 1.669-2.783) and 2.93 mg/kg (95%CI: 1.135-3.651), calculated with K1 or VT values respectively. According to the dose-response fit, differences in [18F]MC225-K1 values could be detected at tariquidar doses ranging from 1.37 to 3.25 mg/kg. Our findings showed that small changes in the P-gp function, caused by low doses of tariquidar, could be detected by [18F]MC225-K1 values, which confirms the high sensitivity of the radiotracer. The results suggest that [18F]MC225 may allow the quantification of moderate P-gp impairments, which may allow the detection of P-gp dysfunctions at the early stages of a disease and potential transporter-mediated drug-drug interactions.