Mapping Ion-Induced Mesophasic Transformation in Lyotropic In Situ Gelling System and its Correlation with Pharmaceutical Performance

Purpose

To investigate influence of ion induced mesophasic transformation on pharmaceutical performance of in situ gelling system consisting of glyceryl monooleate.

Methods

The prepared system showed mesophasic transformation during its conversion from sol to gel upon controlled hydration. The process of mesophasic transformation was studied by SAXS, DSC, rheology and plane polarized light microscopy. Further the influence of additives i.e. naproxen salts (sodium and potassium) and naproxen (base) on the process of mesophasic transformation was also elucidated.

Results

It was observed that addition of salt form of naproxen transformed W/O emulsions into cubic mesophase whereas addition of base form of naproxen formed reverse hexagonal (H_II) phase upon controlled hydration. The cubic mesophase formed by naproxen salts retarded the drug release for initial 3 h whereas H_II phase showed sustained drug release characteristics for naproxen base following Higuchi drug release kinetics.

Conclusion

The current work suggests that formulations with tailor made pharmaceutical performance can be developed by selecting proper additives in the system so as to obtain the desired mesophase ‘on demand’ thereby controlling drug release characteristics.

Tanshinones from Chinese Medicinal Herb Danshen (Salvia miltiorrhiza Bunge) Suppress Prostate Cancer Growth and Androgen Receptor Signaling

Purpose

To test whether tanshinones inhibit prostate cancer (PCa) growth at least in part through inhibiting androgen receptor (AR) signaling.

Methods

We evaluated cell growth, survival and AR signaling parameters of PCa cells after exposure to tanshinones in in vitro models. We also tested the in vivo inhibitory efficacy of tanshinone IIA (TIIA) against LNCaP xenograft model in athymic nude mice.

Results

For androgen-dependent LNCaP cells, a colony growth assay showed strong inhibitory potency following the order of TIIA??cryptotanshinone>tanshinone I, being 10?C30 folds higher than Casodex (racemic). TIIA inhibited growth of LNCaP cells more than several androgen-independent PCa cell lines. All 3 tested tanshinones were devoid of AR agonist activity under castrate condition. Mechanistically, tanshinones inhibited AR nuclear translocation within 2?h, decreased protein and mRNA abundance of AR and its target prostate-specific antigen within 12?h, and stimulated proteosomal degradation of AR. Oral administration of TIIA (25?mg/kg, once daily) retarded LNCaP xenograft growth and down-regulated tumor AR abundance in athymic nude mice.

Conclusion

AR targeting action of tanshinones was distinct from Casodex and contributed to prostate cancer growth suppression in vitro and in vivo.     

Effect of Compression on Non-isothermal Crystallization Behaviour of Amorphous Indomethacin

Purpose

To evaluate the effect of tablet compression on the physical stability of amorphous indomethacin.

Methods

The amorphous indomethacin generated by melt cooling, rapid (5°C/min) or slow (0.2°C/min) cooling, was evaluated by PXRD, mDSC and FTIR analysis. Non-isothermal crystallisation behaviour was assessed using mDSC and any structural changes with compression were monitored by FTIR. Amorphous indomethacin was compressed in a DSC pan using a custom made die cavity-punch setup and further analysed in the primary container to minimize stress due to sample transfer and preparation.

Results

Compression of amorphous indomethacin induced and increased the extent of crystallisation upon heating. DSC results revealed that amorphous indomethacin generated by rapid cooling is more prone to compression induced crystallisation than the slowly cooled one. Onset temperature for crystallisation (T _c ) of uncompressed slowly and rapidly cooled samples are 121.4 and 124°C and after compression T _c decreased to ca 109 and ca 113°C, respectively. Compression of non-aged samples led to higher extent of crystallisation predominantly into ??-form. Aging followed by compression led to crystallisation of mainly the ??-form.

Conclusions

Compression affects the physical stability of amorphous indomethacin. Structural changes originated from tablet compression should be duly investigated for the stable amorphous formulation development.     

Predicting Nonlinear Pharmacokinetics of Omeprazole Enantiomers and Racemic Drug Using Physiologically Based Pharmacokinetic Modeling and Simulation: Application to Predict Drug/Genetic Interactions

Purpose

The objective of this study is to develop a physiologically-based pharmacokinetic (PBPK) model for each omeprazole enantiomer that accounts for nonlinear PK of the two enantiomers as well as omeprazole racemic drug.

Methods

By integrating in vitro, in silico and human PK data, we first developed PBPK models for each enantiomer. Simulation of racemic omeprazole PK was accomplished by combining enantiomer models that allow mutual drug interactions to occur.

Results

The established PBPK models for the first time satisfactorily predicted the nonlinear PK of esomeprazole, R-omeprazole and the racemic drug. The modeling exercises revealed that the strong time-dependent inhibition of CYP2C19 by esomeprazole greatly altered the R-omeprazole PK following administration of racemic omeprazole as in contrast to R-omeprazole given alone. When PBPK models incorporated both autoinhibition of each enantiomer and mutual interactions, the ratios between predicted and observed AUC following single and multiple dosing of omeprazole were 0.97 and 0.94, respectively.

Conclusions

PBPK models of omeprazole enantiomers and racemic drug were developed. These models can be utilized to assess CYP2C19-mediated drug and genetic interaction potential for omeprazole and esomeprazole.     

Rapid Assessment of Homogeneity and Stability of Amorphous Solid Dispersions by Atomic Force Microscopy—From Bench to Batch

Purpose

To verify the robustness and fundamental value of Atomic Force Microscopy (AFM) and AFM-based assays to rapidly examine the molecular homogeneity and physical stability of amorphous solid dispersions on Hot-Melt-Extrudates.

Methods

Amorphous solid dispersions were prepared with a Hot-Melt Extruder (HME) and profiled by Raman Microscopy and AFM following a sequential analytical routine (Multi-Scale-Imaging-of-Miscibiliy (MIMix)). Extrudates were analyzed before and after incubation at elevated temperature and humidity. The data were compared with published results as collected on miniaturized melt models. The value of molecular phase separation rates for long term stability prediction was assessed.

Results

Data recorded on the extrudates are consistent with those published, and they can be compared side by side. Such direct data comparisons allow the identification of possible sources of extrudate heterogeneities. The surface roughness analysis of fracture-exposed interfaces is a novel quantitative way to trace on the nanometer scale the efficiencies of differently conducted HME-processes. Molecular phase separation rates are shown to be relevant for long term stability predictions.

Conclusions

The AFM-based assessment of API:excipient combinations is a robust method to rapidly identify miscible and stable solid dispersions in a routine manner. It provides a novel analytical tool for the optimization of HME processes.     

“MCC SANAQ®burst”—A New Type of Cellulose and its Suitability to Prepare Fast Disintegrating Pellets

Introduction

Microcrystalline cellulose (MCC) is the commonly used pelletization aid in wet extrusion-spheronization processes. MCC has the structure of cellulose I and is denoted as MCC I. Recently, MCC II, a different polymorphic type of MCC, became commercially available, known under the name MCC SANAQ®burst. Due to the fact, that MCC II can be used as a filler and a disintegrant in tableting, MCC SANAQ®burst was investigated as new pelletization aid with the goal to prepare disintegrating pellets.

Materials

MCC II pellets were compared to the corresponding conventional pellets, manufactured on the basis of MCC I, namely Avicel® PH 102. Formulations with 10%, 20%, and 50% of either MCC I or MCC II as pelletization aids were produced.

Methods

One series of binary mixtures, contained lactose monohydrate as filler and a second series chloramphenicol as model drug. All pellets were characterized by their yield, aspect ratio, equivalent diameter, water content, tensile strength, disintegration behavior and—if applicable—drug release.

Results and Discussion

The production of pellets with sufficient quality properties by addition of 10%, 20%, and 50% of MCC II as pelletization aid was possible. In contrast to MCC I pellets, MCC II-based pellets showed disintegration resulting in a much faster drug release.

Conclusion

MCC SANAQ®burst is a promising pelletization aid providing disintegrating and fast-dissolving pellets.     

A Bodyweight-Dependent Allometric Exponent for Scaling Clearance Across the Human Life-Span

Purpose

To explore different allometric equations for scaling clearance across the human life-span using propofol as a model drug.

Methods

Data from seven previously published propofol studies ((pre)term neonates, infants, toddlers, children, adolescents and adults) were analysed using NONMEM VI. To scale clearance, a bodyweight-based exponential equation with four different structures for the exponent was used: (I) 3/4 allometric scaling model; (II) mixture model; (III) bodyweight-cut-point separated model; (IV) bodyweight-dependent exponent model.

Results

Model I adequately described clearance in adults and older children, but overestimated clearance of neonates and underestimated clearance of infants. Use of two different exponents in Model II and Model III showed significantly improved performance, but yielded ambiguities on the boundaries of the two subpopulations. This discontinuity was overcome in Model IV, in which the exponent changed sigmoidally from 1.35 at a hypothetical bodyweight of 0?kg to a value of 0.56 from 10?kg onwards, thereby describing clearance of all individuals best.

Conclusions

A model was developed for scaling clearance over the entire human life-span with a single continuous equation, in which the exponent of the bodyweight-based exponential equation varied with bodyweight.     

Investigation of Follicular and Non-follicular Pathways for Polyarginine and Oleic Acid-Modified Nanoparticles

Purpose

To investigate the percutaneous permeation pathways of cell penetrating peptide modified lipid nanoparticles and oleic acid modified polymeric nanoparticles.

Methods

Confocal microscopy was performed on skin cultures (EpiDermFT?) for modified and un-modified nanoparticles. Differential stripping was performed following in vitro skin permeation of Ibuprofen (Ibu) encapsulated nanoparticles to estimate Ibu levels in different skin layers and receiver compartment. The hair follicles (HF) were blocked and in vitro skin permeation of nanoparticles was then compared with unblocked HF. The surface modified nanoparticles were investigated for response on allergic contact dermatitis (ACD).

Results

Surface modified nanoparticles showed a significant higher (p?<?0.05) in fluorescence in EpiDermFT? cultures compared to controls. The HF play less than 5% role in total nanoparticle permeation into the skin. The Ibu levels were significantly high (p?<?0.05) for surface modified nanoparticles compared to controls. The Ibu levels in skin and receiver compartment were not significantly different when HF were open or closed. Modified nanoparticles showed significant improvement in treatment of ACD compared to solution.

Conclusions

Our studies demonstrate that increased skin permeation of surface modified nanoparticles is not only dependent on a follicular pathway but also occur through non-follicular pathway(s).     

Curcumin,demethoxycurcumin and bisdemethoxycurcumin differentially inhibit morphine's rewarding effect in rats

Rationale

Recent animal studies reported that curcumin, the active constituent of Curcuma longa, has several central actions and may attenuate morphine tolerance.

Objectives

In the present study, we utilized the intracranial self-stimulation (ICSS) paradigm to examine the effects of the commercially available curcuminoid mixture and each one of its components, individually, on brain stimulation reward and on the reward-facilitating effect of morphine.

Methods

Male Sprague-Dawley rats were implanted with an electrode into the medial forebrain bundle and trained to respond for electrical stimulation using a rate-frequency paradigm. In the first study, rats were injected with graded doses either of the curcuminoid mixture, or curcumin I, or II, or III. In the second study, we examined whether a low dose of the curcuminoid mixture or each individual curcumin analogue composing it could counteract the reward-facilitating effect of morphine.

Results

At low doses, both the curcuminoid mixture and curcumin I did not affect brain stimulation reward, whereas, higher doses increased ICSS thresholds. Curcumin II and curcumin III did not affect brain stimulation reward at any doses. Subthreshold doses of the curcuminoid mixture and curcumin I inhibited the reward-facilitating effect of morphine.

Conclusion

Both the curcuminoid mixture and curcumin I lack hedonic properties and moderate the reward-facilitating effect of morphine. Our data suggest that curcumin interferes with brain reward mechanisms responsible for the expression of the acute reinforcing properties of opioids and provide evidence that curcumin may be a promising adjuvant for attenuating morphine’s rewarding effects in patients who are under long-term opioid therapy.     

A Rifapentine-Containing Inhaled Triple Antibiotic Formulation for Rapid Treatment of Tubercular Infection

Purpose

The potential for rifapentine-containing oral therapeutic regimens to significantly shorten the current six-month anti-tubercular treatment regimen is confounded by high plasma protein binding of rifapentine. Inhaled aerosol delivery of rifapentine, a more potent anti-tubercular antibiotic drug, in combination with other first-line antibiotics may overcome this limitation to deliver a high drug dose at the pulmonary site of infection.

Methods

A formulation consisting of rifapentine, moxifloxacin and pyrazinamide, with and without leucine, was prepared by spray-drying. This formulation was assessed for its physico-chemical properties, in vitro aerosol performance and antimicrobial activity.

Results

The antibiotic powders, with and without leucine, had similar median aerodynamic diameters of 2.58?±?0.08 μm and 2.51?±?0.06 μm, with a relatively high fine particle fraction of 55.5?±?1.9% and 63.6?±?2.0%, respectively. Although the powders were mostly amorphous, some crystalline peaks associated with the δ polymorph for the spray-dried crystalline pyrazinamide were identified.

Conclusions

Stabilisation of the powder with 10% w/w leucine and protection from moisture ingress was found to be necessary to prevent overt crystallisation of pyrazinamide after long-term storage. In vitro biological assays indicated antimicrobial activity was retained after spray-drying. Murine pharmacokinetic studies are currently underway.     

The Development of Thermal Nanoprobe Methods as a Means of Characterizing and Mapping Plasticizer Incorporation into Ethylcellulose Films

Purpose

The phase composition and distribution of ethylcellulose (EC) films containing varying amounts of the plasticizer fractionated coconut oil (FCO) were studied using a novel combination of thermal and mapping approaches.

Methods

The thermal and thermomechanical properties of films containing up to 30% FCO were characterized using modulated temperature differential scanning calorimetry (MTDSC) and dynamic mechanical analysis (DMA). Film surfaces were mapped using atomic force microscopy (AFM; topographic and pulsed force modes) and the composition of specific regions identified using nanothermal probes.

Results

Clear evidence of distinct conjugate phases was obtained for the 20?C30% FCO/EC film systems. We suggest a model whereby the composition of the distinct phases may be estimated via consideration of the glass transition temperatures observed using DSC and DMA. By combining pulsed force AFM and nano-thermal analysis we demonstrate that it is possible to map the two separated phases. In particular, the use of thermal probes allowed identification of the distinct regions via localized thermomechanical analysis, whereby nanoscale probe penetration is measured as a function of temperature.

Conclusion

The study has indicated that by using thermal and imaging techniques in conjunction it is possible to both identify and map distinct regions in binary films.     

The effects of perinatal fluoxetine treatment on the circadian system of the adult mouse

Rational

Depression is prevalent among women of childbearing age and is frequently treated with selective serotonin reuptake inhibitors (SSRIs). As some SSRIs, such as fluoxetine (Flx), can cross the placenta, it is possible that the neurodevelopment of the fetus may be affected, leading to altered behavior in adulthood.

Objectives

In this study, we examined the effects of perinatal Flx exposure on the subsequent expression of circadian rhythms in adult mice.

Methods

Dams were treated with 25 mg/kg/day Flx in their drinking water from embryonic day 15 to postnatal day 12. Circadian organization of wheel running rhythms and phase shifts to photic and non-photic stimuli were assessed in the offspring starting at 6 weeks of age.

Results

We found that perinatal Flx exposure led to larger light-induced phase advances (1.19?±?0.51 vs. 0.55?±?0.25 h), smaller phase advances to the serotonin agonist 8-OH-DPAT during the mid-subjective day (0.44?±?0.15 vs. 0.70?±?0.17 h), and a shorter free-running period in constant darkness (23.47?±?0.13 vs. 23.64?±?0.13 h).

Conclusions

These results suggest that perinatal exposure to SSRIs may have consequences for the functioning of the circadian system later in life.     

Effect of clarithromycin and fluconazole on the pharmacokinetics of montelukast in human volunteers

Objective

Montelukast, a leukotriene receptor antagonist, is used in the treatment of asthma. The objective of the study reported here was to determine whether multiple doses of clarithromycin or fluconazole affect the pharmacokinetics of montelukast.

Methods

This was a four-phase cross-over study with a washout period of 2 weeks between phases. In phase 1, 12 volunteers received a single oral dose of 10?mg montelukast. In phase 2, the volunteers received a single, oral dose of 1,000?mg clarithromycin once daily for 2 days, followed by, on day 3, a single oral dose of 10?mg montelukast co-administered with clarithromycin. In phase 3, a single oral dose of 50?mg fluconazole was given once daily for 6 days, followed by, on day 7, a single oral dose of 10?mg montelukast co-administered with 50?mg fluconazole. In the last phase (phase 4), a single oral dose of 150?mg fluconazole was given once daily for 6 days, followed by, on day 7, a single oral dose of 10?mg montelukast co-administered with 150?mg fluconazole. The plasma concentration of montelukast was measured by high performance liquid chromatography for 24?h.

Results

Following clarithromycin co-administration, the area under the concentration–time curve from zero to infinity ( AUC_0-∞) of montelukast increased by 144% [90% confidence interval (CI) 2.03–2.86]. The co-administration of a single oral dose of 150 and 50?mg fluconazole decreased the montelukast AUC_0–∞ by 30.7 (90% CI 0.53–0.81) and 38.8% (90% CI 0.57–0.69), respectively.

Conclusions

Clarithromycin increased the plasma concentrations of montelukast whereas fluconazole reduced the plasma concentrations of montelukast. The mechanism of the interaction is probably due to interference of the interacting drugs with transporters mediating the uptake of montelukast.     

Influence of Soluplus on Solid-State Properties and Physical Stability of Indomethacin-Saccharin Co-crystal Formation Prepared by Air-Drying Process

Purpose

The effect of Soluplus on the solid-state characteristics and physical transformation of indomethacin (IMC), saccharin (SAC), IMC-SAC physical mixture, and the previously prepared IMC-SAC co-crystal was investigated after solvent evaporation via air-drying process.

Methods

Soluplus with γ-IMC or SAC (weight ratio?=?1:1) was respectively prepared by completely dissolving both components in acetone by ultrasonication, and then evaporated via air-drying process. Soluplus was fully co-dissolved with the previously prepared IMC-SAC co-crystal (procedure I) or completely dissolved together with IMC-SAC physical mixture (procedure II) in acetone by ultrasonication, and then evaporated through air-drying process. The physical stability of both Soluplus/IMC-SAC systems was also carried out under accelerated storage conditions. All the samples were determined by thermoanalytical and FTIR spectroscopic studies with spectral curve-fitting technique.

Results

Soluplus caused the amorphous formation of IMC or SAC in the Soluplus solid dispersion, in which the intermolecular hydrogen bonding interaction between SAC and Soluplus occurred. Although Soluplus did not influence the co-crystal formation between IMC and SAC in the solid dispersion, the polymorphic transformation and physical stability of Soluplus/IMC-SAC solid dispersion were markedly influenced by procedures I and II after evaporation via air-drying process.

Conclusion

Soluplus influences the amorphous or crystalline IMC-SAC co-crystal formulated in the solid dispersion prepared by procedure I or II via air-drying process. The amorphous IMC-SAC co-crystal in the Soluplus solid dispersion prepared by procedure I gradually transformed to a crystalline IMC-SAC co-crystal after storage at 40?±?2 °C/75?±?5 % RH conditions for 28 days, whereas the crystalline IMC-SAC co-crystal in the Soluplus solid dispersion prepared by procedure II via air-drying process maintained a long-term storage stability.

     

Sensitization to cocaine is inhibited after intra-accumbal GR103691 or rimonabant, but it is enhanced after co-infusion indicating functional interaction between accumbens D3 and CB1 receptors

Rationale

Dopamine D₃ receptors and cannabinoid CB₁ receptors are both expressed in the nucleus accumbens, and they have been involved in motor sensitization to cocaine. The objectives were: (1) to study the effects of blockade of these receptors on sensitization to repeated cocaine, by using GR103691, D₃ receptor blocker, and rimonabant, CB₁ receptor ligand, and (2) to discern if both receptors interact by co-infusing them.

Materials and methods

Cocaine (10?mg/kg) was injected daily for 3?days (induction phase) and later on day?8 (expression phase), and locomotor activity was measured during 2?h after cocaine. GR103691 and rimonabant were bilaterally injected (0.5???l volume of each infusion) in the nucleus accumbens through cannulae (GR103691, 0, 4.85, and 9.7???g/??l; rimonabant, 0, 0.5, and 1.5???g/??l), before cocaine, during either induction or expression phases of sensitization.

Results

The findings indicated that sensitizing effects of cocaine were abolished after D₃ receptor blocking during both induction and expression phases, as well as rimonabant infusion during the expression (not induction) phase. A functional interaction between both receptors was also observed, because if GR103691 was injected during induction and rimonabant during expression, sensitizing effects of cocaine were observed to be normal or further enhanced.

Conclusion

Dopamine D₃ receptors within the nucleus accumbens are critical for the development and consolidation of sensitization, and cannabinoid CB₁ receptors are critical for the expression of sensitization. Co-blockade of D₃ and CB₁ receptors exert opposite effects to blockade of these receptors separately, revealing the existence of a functional interaction between them.     

Amphetamine challenge decreases yohimbine binding to α2 adrenoceptors in Landrace pig brain

Rationale

The noradrenaline (NA) system is implicated in neurodegenerative and psychiatric disorders; however, our understanding is impaired by the lack of well-validated radioligands to assess NA function and release. Yohimbine, an ??2 adrenoceptor antagonist, has recently been developed as a carbon-11 [11C]-labeled radioligand for positron emission tomography (PET) imaging studies.

Objectives

Here we explore the hypothesis that yohimbine can be used as an in vivo tracer of NA receptor binding and release during amphetamine challenges in Landrace pigs.

Methods

Pigs underwent baseline PET scans with [11C]yohimbine and were then challenged with 10?mg/kg?d-amphetamine 20?min prior to a second [11C]yohimbine scan. Using the Logan analysis model, volumes of distribution were calculated from fits of the kinetic data 25?C90?min post-yohimbine injection.

Results

Amphetamine decreased [11C]yohimbine volume of distribution in the brain regions under investigation, including the thalamus, caudate nucleus, and cortical regions.

Conclusion

These data suggest that the binding of [11C]yohimbine to ??2 adrenoceptors may be displaceable by increases in synaptic concentrations of the endogenous ligand, NA, and possibly dopamine, suggesting the possibility that [11C]yohimbine may be used as a surrogate marker of NA release in vivo.     

Effects of one-time apple juice ingestion on the pharmacokinetics of fexofenadine enantiomers

Purpose

We examined the effect of a single apple juice intake on the pharmacokinetics of fexofenadine enantiomers in healthy Japanese subjects.

Methods

In a randomized two phase, open-label crossover study, 14 subjects received 60 mg of racemic fexofenadine simultaneously with water or apple juice. For the uptake studies, oocytes expressing organic anion-transporting polypeptide 2B1 (OATP2B1) were incubated with 100 μM (R)- and (S)-fexofenadine in the presence or absence of 10 % apple juice.

Results

One-time ingestion of apple juice significantly decreased the area under the plasma concentration–time curve (AUC_0–24) for (R)- and (S)-fexofenadine by 49 and 59 %, respectively, and prolonged the time to reach the maximum plasma concentration (t _max) of both enantiomers (P?R)- and (S)-fexofenadine excretion into urine (Ae_0–24) by 54 and 58 %, respectively, the renal clearances of both enantiomers were unchanged between the control and apple juice phases. For in vitro uptake studies, the uptake of both fexofenadine enantiomers into OATP2B1 complementary RNA (cRNA)-injected oocytes was significantly higher than that into water-injected oocytes, and this effect was greater for (R)-fexofenadine. In addition, apple juice significantly decreased the uptake of both enantiomers into OATP2B1 cRNA-injected oocytes.

Conclusions

These results suggest that OATP2B1 plays an important role in the stereoselective pharmacokinetics of fexofenadine and that one-time apple juice ingestion probably inhibits intestinal OATP2B1-mediated transport of both enantiomers. In addition, this study demonstrates that the OATP2B1 inhibition effect does not require repeated ingestion or a large volume of apple juice.     

Lithium reduces the effects of rotenone-induced complex I dysfunction on DNA methylation and hydroxymethylation in rat cortical primary neurons

Rationale

Mitochondrial complex I dysfunction and alterations in DNA methylation levels are consistently reported in bipolar disorder (BD) and are regulated by lithium. One of the mechanisms by which lithium may exert its effects in BD is by improving mitochondrial complex I function. Therefore, we examined whether complex I dysfunction induces methylation and hydroxymethylation of DNA and whether lithium alters these effects in rat primary cortical neurons.

Methods

Rotenone was used to induce mitochondrial complex I dysfunction. Cell viability was measured by MTT assay, and ATP levels were assessed by Cell-Titer-Glo®. Complex I activity was measured using an ELISA-based assay. Apoptosis, DNA methylation, and hydroxymethylation levels were measured by immunocytochemistry.

Results

Rotenone decreased complex I activity and ATP production, but increased cell death and apoptosis. Rotenone treatment increased levels of 5-methylcytosine (5mc) and hydroxymethylcytosine (5hmc), suggesting a possible association between complex I dysfunction and DNA alterations. Lithium prevented rotenone-induced changes in mitochondrial complex I function, cell death and changes to DNA methylation and hydroxymethylation.

Conclusions

These findings suggest that decreased mitochondrial complex I activity may increase DNA methylation and hydroxymethylation in rat primary cortical neurons and that lithium may prevent these effects.