Effect of Permeation Enhancers on the Buccal Permeability of Nicotine: <Emphasis Type="BoldItalic">Ex vivo</Emphasis> Transport Studies Complemented by MALDI MS Imaging

Purpose

The purpose of this study was to assess the effect of several chemical permeation enhancers on the buccal permeability of nicotine and to image the spatial distribution of nicotine in buccal mucosa with and without buccal permeation enhancers.

Methods

The impact of sodium taurodeoxycholate (STDC), sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and Azone® on the permeability of [³H]-nicotine and [¹⁴C]-mannitol (a paracellular marker) across porcine buccal mucosa was studied ex vivo in modified Ussing chambers. The distribution of nicotine, mannitol and permeation enhancers was imaged using using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI).

Results

Despite STDC significantly increasing permeability of [¹⁴C]-mannitol, no enhancing effect was seen on [³H]-nicotine permeability with any of the permeation enhancers. Rather, SDS and DMSO retarded nicotine permeability, likely due to nicotine being retained in the donor compartment. The permeability results were complemented by the spatial distribution of nicotine and mannitol determined with MALDI MSI.

Conclusions

The buccal permeability of nicotine was affected in an enhancer specific manner, suggesting that nicotine primarily diffuses via the transcellular pathway. MALDI MSI was shown to complement ex vivo permeability studies and to be a useful qualitative tool for visualizing drug and penetration enhancer distribution in buccal mucosa.

     

Effects of <Emphasis Type="Italic">CYP2C19</Emphasis> and <Emphasis Type="Italic">CYP2C9</Emphasis> genotypes on pharmacokinetic variability of valproic acid in Chinese epileptic patients: nonlinear mixed-effect modeling

Purpose

To evaluate the effects of CYP2C19 and CYP2C9 genotypes on the pharmacokinetic variability of valproic acid (VPA) in epileptic patients using a population pharmacokinetic (PPK) approach.

Methods

VPA concentrations were measured in 287 epileptic patients, who were genotyped for CYP2C19*2/*3 and CYP2C9*3. Patients who were on monotherapy with VPA were divided into two groups, a PPK-model group (n?=?177) and a PPK-valid group (n?=?110). The PPK parameter values for VPA were calculated in the PPK-model group by using the NONMEM software. Ultimately, a biological model and a final model were established. Each model was then used to independently predict the concentrations of the PPK-valid group to validate the two models.

Results

There was a significant effect of the CYP2C19 and CYP2C9 genotypes on the pharmacokinetic (PK) variability (P?<?0.01) in the final PPK model of CL/F. The interindividual CL was calculated according to the final model: CL/F?=?0.0951?×?(1?+?e^{0.0267?×?(3???genotype)})?+?0.0071?×?age (L/h). The coefficient of variation (CV) (omega CL/F) of the final model was 29.3%, while that of the biological model was 31.7%. Based on the genotype, the individual PK parameters can be calculated more accurately than before.

Conclusion

The CYP2C19 and CYP2C9 genotypes significantly influenced the PK variability of VPA, as quantified by NONMEM software.

     

Supramolecular Chitosan Micro-Platelets Synergistically Enhance Anti-<Emphasis Type="BoldItalic">Candida albicans</Emphasis> Activity of Amphotericin B Using an Immunocompetent Murine Model

Purpose

The aim of this work is to design new chitosan conjugates able to self-organize in aqueous solution in the form of micrometer-size platelets. When mixed with amphotericin B deoxycholate (AmB-DOC), micro-platelets act as a drug booster allowing further improvement in AmB-DOC anti-Candida albicans activity.

Methods

Micro-platelets were obtained by mixing oleoyl chitosan and α-cyclodextrin in water. The formulation is specifically-engineered for mucosal application by dispersing chitosan micro-platelets into thermosensitive pluronic^® F127 20 wt% hydrogel.

Results

The formulation completely cured C. albicans vaginal infection in mice and had a superior activity in comparison with AmB-DOC without addition of chitosan micro-platelets. In vitro studies showed that the platelets significantly enhance AmB-DOC antifungal activity since the IC₅₀ and the MIC₉₀ decrease 4.5 and 4.8-times. Calculation of fractional inhibitory concentration index (FICI?=?0.198) showed that chitosan micro-platelets act in a synergistic way with AmB-DOC against C. albicans. No synergy is found between spherical nanoparticles composed poly(isobutylcyanoacrylate)/chitosan and AmB-DOC.

Conclusion

These results demonstrate for the first time the ability of flattened chitosan micro-platelets to have synergistic activity with AmB-DOC against C. albicans candidiasis and highlight the importance of rheological and mucoadhesive behaviors of hydrogels in the efficacy of the treatment.

     

Design and Modular Construction of a Polymeric Nanoparticle for Targeted Atherosclerosis Positron Emission Tomography Imaging: A Story of 25% <Superscript>64</Superscript>Cu-CANF-Comb

Purpose

To assess the physicochemical properties, pharmacokinetic profiles, and in vivo positron emission tomography (PET) imaging of natriuretic peptide clearance receptors (NPRC) expressed on atherosclerotic plaque of a series of targeted, polymeric nanoparticles.

Methods

To control their structure, non-targeted and targeted polymeric (comb) nanoparticles, conjugated with various amounts of c-atrial natriuretic peptide (CANF, 0, 5, 10 and 25%), were synthesized by controlled and modular chemistry. In vivo pharmacokinetic evaluation of these nanoparticles was performed in wildtype (WT) C57BL/6 mice after ⁶⁴Cu radiolabeling. PET imaging was performed on an apolipoprotein E–deficient (ApoE^?/?) mouse atherosclerosis model to assess the NPRC targeting efficiency. For comparison, an in vivo blood metabolism study was carried out in WT mice.

Results

All three ⁶⁴Cu-CANF-comb nanoparticles showed improved biodistribution profiles, including significantly reduced accumulation in both liver and spleen, compared to the non-targeted ⁶⁴Cu-comb. Of the three nanoparticles, the 25% ⁶⁴Cu-CANF-comb demonstrated the best NPRC targeting specificity and sensitivity in ApoE^?/? mice. Metabolism studies showed that the radiolabeled CANF-comb was stable in blood up to 9 days. Histopathological analyses confirmed the up-regulation of NPRC along the progression of atherosclerosis.

Conclusion

The 25% ⁶⁴Cu-CANF-comb demonstrated its potential as a PET imaging agent to detect atherosclerosis progression and status.

     

Bioactive flavanoids from <Emphasis Type="Italic">Glycosmis arborea</Emphasis>

Background

Glycosmis is a genus of evergreen glabrous shrub and distributed all over India. It possesses various medicinal properties and is used in indigenous medicine for cough, rheumatism, anemia, and jaundice. Glycosmis arborea is a rich source of alkaloids, terpenoids, coumarins, as well as flavonoids.

Results

The chemical investigation of methanol fraction of the leaves of G. arborea led to the isolation of one new flavone C-glycoside along with three known flavanoids, named as 5,7-dihydroxy-2-[4-hydroxy-3-(methoxy methyl) phenyl]-6-C-β-d-glucopyranosyl flavone (4), 5,7,4^′-trihydroxy-3^′-methoxy flavone (1), 5,4^′-dihydroxy-3^′-methoxy-7-O-β-d-glucupyranosyl flavanone (2), and 5,4^′-dihydroxy-3^′-methoxy-7-O-(α-l-rhamnosyl-(1?→6?)-β-d-glucopyranosyl) flavanone (3), respectively. The structures of all compounds were elucidated with the help of nuclear magnetic resonance spectrometry. Pure compounds and fractions were evaluated for pest antifeedant and antimicrobial activity.

Conclusion

Four compounds were isolated from the leaves of G. arborea. Among them, compound 4 showed significant antimicrobial activity.

     

Combination Metabolomics Approach for Identifying Endogenous Substrates of Carnitine/Organic Cation Transporter OCTN1

Purpose

Solute carrier SLC22A4 encodes the carnitine/organic cation transporter OCTN1 and is associated with inflammatory bowel disease, although little is known about how this gene is linked to pathogenesis. The aim of the present study was to identify endogenous substrates that are associated with gastrointestinal inflammation.

Methods

HEK293/OCTN1 and mock cells were incubated with colon extracts isolated from dextran sodium sulfate-induced colitis mice; the subsequent cell lysates were mixed with the amino group selective reagent 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS), to selectively label OCTN1 substrates. Precursor ion scanning against the fragment ion of APDS was then used to identify candidate OCTN1 substrates.

Results

Over 10,000 peaks were detected by precursor ion scanning; m/z 342 had a higher signal in HEK293/OCTN1 compared to mock cells. This peak was detected as a divalent ion that contained four APDS-derived fragments and was identified as spermine. Spermine concentration in peripheral blood mononuclear cells from octn1 gene knockout mice (octn1^?/?) was significantly lower than in wild-type mice. Lipopolysaccharide-induced gene expression of inflammatory cytokines in peritoneal macrophages from octn1^?/? mice was lower than in wild-type mice.

Conclusions

The combination metabolomics approach can provide a novel tool to identify endogenous substrates of OCTN1.

     

Lower striatal dopamine transporter binding in neuroleptic-naive schizophrenic patients is not related to antipsychotic treatment but it suggests an illness trait

Introduction

Drug induced parkinsonism (DIP) is directly related to dopamine D2 receptor blockade. However, there are many references describing parkinsonian signs (PS) in naive-patients. In our previous study, we observed lower DAT binding in a group of first-episode schizophrenic patients after short-term treatment with risperidone, compared with age-matched healthy controls.

Aim

To clarify if DAT decrease could be an illness trait, excluding the effect of antipsychotics on DAT availability, and to determine whether DAT availability before treatment with antipsychotics may predict subsequent development of PS.

Materials and methods

A new series of 20 neuroleptic-naive schizophrenic patients and 15 healthy subjects was recruited. SPECT with [¹²³I] FP-CIT (DaTSCAN®) was performed before starting antipsychotics and after 4 weeks of treatment. PS and psychopathological status were assessed by the Simpson–Angus (SAS), CGI and PANSS scales. Quantitative analyses of SPECTs were performed using ROIs placed in the caudate, putamen and occipital cortex.

Results

Schizophrenic patients showed lower DAT binding compared with the healthy subjects at baseline (p<0.001) and after a 4-week-treatment period (p=0.001). Six out of eight schizophrenic patients of the DIP group were symptomatic for PS at baseline, in comparison to two out of 12 in the NoDIP group. Nonetheless, no differences were observed on DAT between DIP and NoDIP, neither at baseline (p=0.360) nor at endpoint (p=0.984). Finally, no differences between baseline–endpoint DAT binding were observed, neither in the DIP group (p=0.767) nor in the NoDIP group (p=0.093).

Conclusion

Our new series of first-episode naive-schizophrenic patients (1) points out DAT dysfunction as an illness trait due to the significantly lower DAT binding in schizophrenic patients in comparison to healthy subjects; (2) supports the results of other authors who describe PS in never-treated patients; (3) confirms that [¹²³I] FP-CIT does not allow us to predict which patients will develop parkinsonism due to the lack of differences between DIP and NoDIP patients; and (4) confirms a null effect of antipsychotics on DAT due to the lack of differences in [¹²³I] FP-CIT before and after a 4-week-treatment period.

     

Predicting Pulmonary Pharmacokinetics from <Emphasis Type="Italic">In Vitro</Emphasis> Properties of Dry Powder Inhalers

Purpose

The ability of two semi-mechanistic simulation approaches to predict the systemic pharmacokinetics (PK) of inhaled corticosteroids (ICSs) delivered via dry powder inhalers (DPIs) was assessed for mometasone furoate, budesonide and fluticasone propionate.

Methods

Both approaches derived the total lung doses and the central to peripheral lung deposition ratios from clinically relevant cascade impactor studies, but differed in the way the pulmonary absorption rate was derived. In approach 1, the rate of in vivo drug dissolution/absorption was predicted for the included ICSs from in vitro aerodynamic particle size distribution and in vitro drug solubility estimates measured in an in vivo predictive dissolution medium. Approach 2 derived a first order absorption rate from the mean dissolution time (MDT), determined for the test formulations in an in vitro Transwell^® based dissolution system.

Results

Approach 1 suggested PK profiles which agreed well with the published pharmacokinetic profiles. Similarly, within approach 2, input parameters for the pulmonary absorption rate constant derived from dissolution rate experiments were able to reasonably predict the pharmacokinetic profiles published in literature.

Conclusion

Approach 1 utilizes more complex strategies for predicting the dissolution/absorption process without providing a significant advantage over approach 2 with regard to accuracy of in vivo predictions.

     

<Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">In Vitro</Emphasis> Evidence for Brain Uptake of 4-Phenylbutyrate by the Monocarboxylate Transporter 1 (MCT1)

Purpose

4-Phenylbutyrate (4-PBA) is expected to be a potential therapeutic for several neurodegenerative diseases. These activities require 4-PBA transport into the brain across the blood-brain barrier (BBB). The objective of the present study was to characterize the brain transport mechanism of 4-PBA through the BBB.

Methods

The brain transport of 4-PBA across the BBB was investigated following intravenous (IV) injection and internal carotid artery perfusion (ICAP) in vivo. The mechanism of transport was examined using TR-BBB cells, an in vitro model of the BBB.

Results

The volume of distribution (V_D) of 4-PBA by rat brain was about 7-fold greater than that of sucrose, a BBB impermeable vascular space marker, suggesting the blood-to-brain transport of 4-PBA through the BBB in the physiological state. [¹⁴C]4-PBA uptake by TR-BBB cells showed time-, pH- and concentration-dependence with a K _m of 13.4 mM at pH 7.4 and 3.22 mM at pH 6.0. The uptake was Na⁺ independent, and was significantly inhibited by alpha-cyano-4-hydroxycinnamate (a typical inhibitor for monocarboxylate transport), endogenous monocarboxylate compounds and monocarboxylic drugs. Lactate and valproate competitively inhibited [¹⁴C]4-PBA uptake with K _i value of 13.5 mM and 7.47 mM, respectively. These results indicate the role of monocarboxylate transporters (MCTs) in 4-PBA transport into the brain at the BBB. TR-BBB cells expressed mRNA of rMCT1, 2, and 4, especially, rMCT1 showed high mRNA expression level. In addition, [¹⁴C]4-PBA uptake was inhibited by rMCT1 specific small interfering RNA.

Conclusion

The transport mechanism of 4-PBA from blood to brain across the BBB likely involves MCT1.

     

Formulation and Evaluation of Chitosan-Based Buccal Bioadhesive Films of Zolmitriptan

Purpose

Therapeutic efficacy of zolmitriptan in oral therapy is primarily limited by the biopharmaceutical issues. The objective of this study is to design and optimize chitosan-based buccal bioadhesive system for the effective delivery of zolmitriptan in the treatment of migraine.

Methods

Factorial design (3²) is constructed and conducted in a fully randomized manner to study all nine possible experimental runs. The films were prepared by solvent casting method by varying the content of chitosan (X₁) and polyvinyl alcohol (X₂). The effect of these two independent variables on swelling index (Y1), percent drug release in 15 min (Y2) and 5 h (Y3), and mucoadhesive strength (Y4) of prepared films was evaluated.

Results

The physical and chemical characteristics displayed by the prepared films (F1–F9) were found to be optimal. It was observed that the factor X₁ has positive and X₂ has negative effect on response Y1. In contrast, factor X₁ showed negative effects on drug release at both time intervals (15 min and 5 h) while X₂ displayed positive responses for these variables (Y2 and Y3). However, the mucoadhesion increased with an increase in factor X₁ and decreased when the factor X₂ was increased. Indeed, the desirable characteristics exhibited by the film F7 are ideal for buccal application. Greater flux (63.93?±?12.51 μg/cm²/h) demonstrated in ex vivo studies substantiated the potential of optimized film to effectively deliver zolmitriptan across the buccal membrane.

Conclusions

This study concludes that the chitosan-based buccal film (F7) could be used in both prophylaxis and acute treatment of migraine, although need to be proved in vivo.

     

Pharmacokinetic interaction between HCV protease inhibitor boceprevir and methadone or buprenorphine in subjects on stable maintenance therapy

Purpose

Intravenous opioid use is a common route of hepatitis C virus (HCV) infection; consequently, the prevalence of HCV is high among patients on methadone or buprenorphine/naloxone. The authors evaluated the pharmacokinetic interaction of boceprevir with methadone or buprenorphine/naloxone in patients on stable maintenance therapy.

Methods

This was a two-center, open-label, fixed-sequence study in 21 adult volunteers on stable maintenance therapy. Oral methadone (20–150 mg once daily) or sublingual buprenorphine/naloxone (8/2–24/6 mg once daily) was administered alone or in combination with boceprevir (800 mg every 8 h) on days 2–7. Pharmacokinetic sampling occurred before and up to 24 h after the dose on days 1 and 7.

Results

Coadministration of boceprevir reduced the area under the concentration-time curve during a dosing interval τ (AUC _τ ) and maximum observed plasma (or serum) concentration (C _max) of R-methadone (geometric mean ratios (GMRs) [90 % confidence intervals (CIs)], 0.85 [0.74, 0.96] and 0.90 [0.71, 1.13]) and S-methadone (GMRs [90 % CIs], 0.78 [0.66, 0.93] and 0.83 [0.64, 1.09]). Boceprevir increased the AUC _τ and C _max of buprenorphine (GMRs [90 % CIs], 1.19 [0.91, 1.58] and 1.18 [0.93, 1.50]) and naloxone (GMRs [90 % CIs], 1.33 [0.90, 1.93] and 1.09 [0.79, 1.51]). Boceprevir exposure upon methadone or buprenorphine/naloxone coadministration was not clinically different from historical controls and there was no evidence of opioid withdrawal or excess.

Conclusions

There was no clinically meaningful impact of boceprevir on methadone or buprenorphine pharmacokinetics, suggesting that methadone/buprenorphine dose adjustments are not required upon coadministration with boceprevir. Individual patients may differ in their clinical experience and clinicians should maintain vigilance when coadministering these medications.

     

Radiofluorination and biological evaluation of <Emphasis Type="Italic">N</Emphasis>-aryl-oxadiazolyl-propionamides as potential radioligands for PET imaging of cannabinoid CB<Subscript>2</Subscript> receptors

Background

The level of expression of cannabinoid receptor type 2 (CB₂R) in healthy and diseased brain has not been fully elucidated. Therefore, there is a growing interest to assess the regional expression of CB₂R in the brain. Positron emission tomography (PET) is an imaging technique, which allows quantitative monitoring of very low amounts of radiolabelled compounds in living organisms at high temporal and spatial resolution and, thus, has been widely used as a diagnostic tool in nuclear medicine. Here, we report on the radiofluorination of N-aryl-oxadiazolyl-propionamides at two different positions in the lead structure and on the biological evaluation of the potential of the two tracers [¹⁸F]1 and [¹⁸F]2 as CB₂ receptor PET imaging agents.

Results

High binding affinity and specificity towards CB₂ receptors of the lead structure remained unaffected by the structural changes such as the insertion of the aliphatic and aromatic fluorine in the selected labelling sites of 1 and 2. Aliphatic and aromatic radiofluorinations were optimized, and [¹⁸F]1 and [¹⁸F]2 were achieved in radiochemical yields of ≥30% with radiochemical purities of ≥98% and specific activities of 250 to 450 GBq/μmol. Organ distribution studies in female CD1 mice revealed that both radiotracers cross the blood–brain barrier (BBB) but undergo strong peripheral metabolism. At 30 min after injection, unmetabolized [¹⁸F]1 and [¹⁸F]2 accounted for 60% and 2% as well as 68% and 88% of the total activity in the plasma and brain, respectively. The main radiometabolite of [¹⁸F]2 could be identified as the free acid [¹⁸F]10, which has no affinity towards the CB₁ and CB₂ receptors but can cross the BBB.

Conclusions

N-aryl-oxadiazolyl-propionamides can successfully be radiolabelled with ¹⁸F at different positions. Fluorine substitution at these positions did not affect affinity and specificity towards CB₂R. Despite a promising in vitro behavior, a rather rapid peripheral metabolism of [¹⁸F]1 and [¹⁸F]2 in mice and the generation of brain permeable radiometabolites hamper the application of these radiotracers in vivo. However, it is expected that future synthetic modification aiming at a replacement of metabolically susceptible structural elements of [¹⁸F]1 and [¹⁸F]2 will help to elucidate the potential of this class of compounds for CB₂R PET studies.

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Removing of Benzo[<Emphasis Type="Italic">a</Emphasis>]pyrene using the Isolated Fungi from Petroleum-polluted Soils

Objective

Benzo[a]pyrene, belonging to polycyclic aromatic hydrocarbons, is one of the most important industrial pollutants. This research was aimed to evaluate some fungal strains, with petroleum removing potency, for degradation of BaP from BaP-polluted media and also evaluation of Enzyme activity and protein content in the fungi growing in BaP-polluted media.

Methods

In a field study seven fungal specie were isolated from Tehran oil refinery and cultured in potato dextrose agar (PDA) media containing 30, 60 and 90 (mg/kg) BaP for adaptation of the fungal strains. Removing of BaP was measured after 45 days growth of the fungal colonies under different concentrations of BaP pollution in PDA media and soil. Peroxidase and catalase activity, and protein content were compared in the fungi growing in BaP-polluted media and control ones.

Results

The results showed that all the isolated fungi were able to growth in the BaP containing media and could remove BaP from the media. The highest removal efficiency was determined for Fusarium acuminatum (93%). Similar data obtained when the fungus used for bioremediation of BaP-polluted soil (91%). Total protein content and enzymatic activity (Peroxidase and Catalase) were increased with increasing of BaP pollution. The highest catalase activity was measured in F. acuminatum growing in the media containing 90 mg/kg BaP (2.2×10^-2 unit/mg protein) and the highest (5× 10^-3 unit/mg protein) peroxidase activity for Alternaria alternata. Protein content in the fungi was increased with increasing of BaP pollution. In F. acuminatum, the lowest amount of total protein was observed in the control sample (1×10^-3 mg/g FW) and the highest amount was belonging to the group treated by 90 mg/kg BaP (7.5×10^-3 mg/g FW).

Conclusion

It concluded that F. acuminatum showed the highest catalase activity, highest total protein content and also the highest BaP removal efficiency from both BaP-polluted media and soils.

     

Active cyamemazine metabolites in patients treated with cyamemazine (Tercian<Emphasis Type="Bold">®</Emphasis>): influence on cerebral dopamine D<Subscript>2</Subscript> and serotonin 5-HT<Subscript>2A</Subscript> receptor occupancy as measured by positron emission tomography (PET)

Rationale

Cyamemazine (Tercian®) is an antipsychotic agent blocking central dopamine D₂ receptors, which induces few extrapyramidal adverse effects, due to a potent antagonistic action at serotonin 5-HT_2A receptors. In vitro studies showed that the desmethyl metabolite of cyamemazine (N-desmethyl cyamemazine) has similar affinity for 5-HT_2A receptors as cyamemazine, whereas its D₂ receptor affinity is eight times lower (Benyamina et al. in Eur J Pharmacol 578(2–3):142–147, 2008). Moreover, cyamemazine sulfoxide showed modest affinity for 5-HT_2A receptors.

Objectives

The objective of this study is to measure steady-state plasma levels of N-desmethyl cyamemazine and cyamemazine sulfoxide in patients treated with clinically relevant doses of cyamemazine and correlate them with dopamine D₂ and serotonin 5-HT_2A receptor occupancies (RO) assessed by positron emission tomography (PET).

Methods

Eight patients received Tercian® 37.5, 75, 150, or 300 mg/day according to their symptoms. Dopamine D₂ and serotonin 5-HT_2A RO were assessed at steady-state cyamemazine plasma levels using [¹¹C]raclopride and [¹¹C]N-methyl-spiperone, respectively, for PET. Plasma levels of cyamemazine metabolites were determined using a validated high-performance liquid chromatography (PerkinElmer) associated with a mass spectrometry detection (API 365, PE SCIEX). The apparent equilibrium inhibition constant (K _i) was estimated by fitting RO with plasma levels of cyamemazine metabolites at the time of the PET scan.

Results

After 6 days of cyamemazine administration, plasma N-desmethyl cyamemazine reached steady-state levels at 2 to 12 times higher than those previously found for cyamemazine (Hode et al. in Psychopharmacology (Berl) 180:377–384, 2005). Plasma levels of N-desmethyl cyamemazine were closely related to striatal D₂ RO (r ²?=?0.942) and extrastriatal 5-HT_2A RO (r ²?=?0.901). The estimated K _i(app) value of N-desmethyl cyamemazine for striatal D₂ receptors was about fivefold higher than that for extrastriatal 5-HT_2A receptors (48.7 vs. 10.6 nM). Striatal D₂ RO increased with the plasma levels of N-desmethyl cyamemazine but remained below 75% even at its highest levels. At steady state, plasma cyamemazine sulfoxide levels were about double those of N-desmethyl cyamemazine. However, these cyamemazine sulfoxide levels should not contribute significantly to in vivo 5-HT_2A and D₂ receptor occupancy.

Conclusions

In patients orally given cyamemazine, N-desmethyl cyamemazine, but not cyamemazine sulfoxide, should significantly contribute to in vivo frontal 5-HT_2A and striatal D₂ receptor occupancy. The higher in vivo affinity of cyamemazine and its desmethyl metabolite for serotonin 5-HT_2A receptors compared with dopamine D₂ receptors should explain the low incidence of extrapyramidal adverse effects.

     

PEGylation of a High-Affinity Anti-(+)Methamphetamine Single Chain Antibody Fragment Extends Functional Half-Life by Reducing Clearance

Purpose

Methamphetamine (METH) abuse is a worldwide drug problem, yet no FDA-approved pharmacological treatments are available for METH abuse. Therefore, we produced an anti-METH single chain antibody fragment (scFv7F9Cys) as a pharmacological treatment for METH abuse. ScFv’s have a short half-life due to their small size, limiting their clinical use. Thus, we examined the pharmacokinetic effects of conjugating poly(ethylene) glycol (-PEG) to scFv7F9Cys to extend its functional half-life.

Methods

The affinity of scFv7F9Cys and PEG conjugates to METH was determined in vitro via equilibrium dialysis saturation binding. Pharmacokinetic and parameters of scFv7F9Cys and scFv7F9Cys-PEG20K (30 mg/kg i.v. each) and their ability to bind METH in vivo were determined in male Sprague-Dawley rats receiving a subcutaneous infusion of METH (3.2 mg/kg/day).

Results

Of three PEGylated conjugates, scFv7F9Cys-PEG20K was determined the most viable therapeutic candidate. PEGylation of scFv7F9Cys did not alter METH binding functionality in vitro, and produced a 27-fold increase in the in vivo half-life of the antibody fragment. Furthermore, total METH serum concentrations increased following scFv7F9Cys or scFv7F9Cys-PEG20K administration, with scFv7F9Cys-PEG20K producing significantly longer changes in METH distribution than scFv7F9Cys.

Conclusions

PEGylation of scFv7F9Cys significantly increase the functional half-life of scFv7F9Cys, suggesting it may be a long-lasting pharmacological treatment option for METH abuse.

     

Inter-Subject Variability in OCT1 Activity in 27 Batches of Cryopreserved Human Hepatocytes and Association with OCT1 mRNA Expression and Genotype

Purpose

OCT1/3 (Organic Cation Transporter-1 and -3; SLC22A1/3) are transmembrane proteins localized at the basolateral membrane of hepatocytes. They mediate the uptake of cationic endogenous compounds and/or xenobiotics. The present study was set up to verify whether the previously observed variability in OCT activity in hepatocytes may be explained by inter-individual differences in OCT1/3 mRNA levels or OCT1 genotype.

Methods

Twenty-seven batches of cryopreserved human hepatocytes (male and female, age 24–88 y) were characterized for OCT activity, normalized OCT1/3 mRNA expression, and OCT1 genetic mutation. ASP⁺ (4-[4-(dimethylamino)styryl]-N-methylpyridinium iodide) was used as probe substrate.

Results

ASP⁺ uptake ranged between 75 ± 61 and 2531 ± 202 pmol/(min × million cells). The relative OCT1 and OCT3 mRNA expression ranged between 0.007–0.46 and 0.0002–0.005, respectively. The presence of one or two nonfunctional SLC22A1 alleles was observed in 13 batches and these exhibited significant (p = 0.04) association with OCT1 and OCT3 mRNA expression. However, direct association between genotype and OCT activity could not be established.

Conclusion

mRNA levels and genotype of OCT only partially explain inter-individual variability in OCT-mediated transport. Our findings illustrate the necessity of in vitro transporter activity profiling for better understanding of inter-individual drug disposition behavior.

     

Regulatory Variants Modulate Protein Kinase C α (<Emphasis Type="Italic">PRKCA</Emphasis>) Gene Expression in Human Heart

Purpose

Protein kinase C α (PRKCA) is involved in multiple functions and has been implicated in heart failure risks and treatment outcomes. This study aims to identify regulatory variants affecting PRKCA expression in human heart, and evaluate attributable risk of heart disease.

Methods

mRNA expression quantitative trait loci (eQTLs) were extracted from the Genotype and Tissue Expression Project (GTEx). Allelic mRNA ratios were measured in 51 human heart tissues to identify cis-acting regulatory variants. Potential regulatory regions were tested with luciferase reporter gene assays and further evaluated in GTEx and genome-wide association studies.

Results

Located in a region with robust enhancer activity in luciferase reporter assays, rs9909004 (T?>?C, minor allele frequency =0.47) resides in a haplotype displaying strong eQTLs for PRKCA in heart (p?=?1.2?×?10^?23). The minor C allele is associated with both decreased PRKCA mRNA expression and decreased risk of phenotypes characteristic of heart failure in GWAS analyses (QT interval p?=?3.0?×?10^?14). While rs9909004 is the likely regulatory variant, other variants in high linkage disequilibrium cannot be excluded. Distinct regulatory variants appear to affect expression in other tissues.

Conclusions

The haplotype carrying rs9909004 influences PRKCA expression in the heart and is associated with traits linked to heart failure, potentially affecting therapy of heart failure.

     

BITC and <Emphasis Type="Italic">S</Emphasis>-Carvone Restrain High-Fat Diet-Induced Obesity and Ameliorate Hepatic Steatosis and Insulin Resistance

Purpose

To investigate the preventative activity of benzyl isothiocyante and S-carvone against high-fat diet-induced obesity and metabolic complications.

Methods

Ten-week-old C57BL/6 male mice were fed a high-fat diet and injected intraperitoneally twice per week with benzyl isothiocyante, S-carvone, or vehicle for 8 weeks. The body weight, food intake, and body composition were monitored, and glucose tolerance and insulin tolerance tests were performed at the end of the experiment. Serum and tissue samples were studied using serum biochemistry, histological, and gene expression analysis to define the effects of benzyl isothiocyante and S-carvone treatments on lipid and glucose metabolism and inflammatory responses.

Results

Benzyl isothiocyante and S-carvone blocked high-fat diet-induced weight gain, fat accumulation in the liver, and insulin resistance. The beneficial effects were found to be associated with an improvement of expression of macrophage marker genes in white adipose tissue, including F4/80, Cd11b, Cd11c, Cd206, and Tnf-α, and reduced expression of genes (Pparγ2, Scd1, Cd36) responsible for lipid synthesis and transport in the liver.

Conclusion

Benzyl isothiocyante and S-carvone block high-fat diet-induced obesity and metabolism disorders and can be considered for management of the obesity epidemic that affects approximately 36% of adults and 17% of children in the USA.

     

Enhanced Anti-Tumor Efficacy of Lipid-Modified Platinum Derivatives in Combination with <Emphasis Type="BoldItalic">Survivin</Emphasis> Silencing siRNA in Resistant Non-Small Cell Lung Cancer

Purpose

Cisplatin, is recognized as a first line therapeutic for the treatment of non-small cell lung cancer (NSCLC). Cisplatin resistance is identified as the most detrimental complication during treatment and has been associated with upregulation of several genes, such as the anti-apoptotic gene survivin. In this study, we have evaluated the cytotoxic activity of lipid (C6 and C8)-modified platinum compounds in combination with a survivin-silencing siRNA against cisplatin resistant tumors.

Methods

We synthesized and characterized several lipid-modified platinum compounds and evaluated their cytotoxic activity alone or in combination with survivin-silencing siRNA in vitro and in vivo against A549^DDP cells and in vivo in tumor xenograft model.

Results

The lipid-modified compounds exhibited significantly stronger cytotoxic activity in vitro compared to cisplatin, with CDDP-C6 and CDDP-C8 producing the most pronounced effect, in both A549 and A549^DDP cells. Pre-treatment of the A549^DDP cells with survivin-silencing siRNA enhanced the cytotoxic activity of these compounds. In vivo, the co-treatment of the survivin-silencing siRNA and CDDP-C8 produced the strongest tumor growth inhibition effect (64.5%, p?<?0.05) on a cancer mouse model of chemoresistant lung cancer. In contrast, cisplatin treatment exhibited no significant tumor growth inhibition (4.5%, no p).

Conclusions

Co-treatment of lipid-modified compounds and survivin-silencing siRNA can constitute a reliable alternative to cisplatin treatment for cisplatin-resistant lung tumors that merit further evaluation.