Jan L. Du Preez

Social isolation rearing induces mitochondrial, immunological, neurochemical and behavioural deficits in rats, and is reversed by clozapine or N–acetyl cysteine

Apart from altered dopamine (DA) function, schizophrenia displays mitochondrial and immune-inflammatory abnormalities, evidenced by oxidative stress, altered kynurenine metabolism and cytokine release. N-acetyl cysteine (NAC), an antioxidant and glutamate modulator, is effective in the adjunctive treatment of schizophrenia. Social isolation rearing (SIR) in rats is a valid neurodevelopmental animal model of schizophrenia. This study evaluated whether SIR-induced behavioural deficits may be explained by altered plasma pro- and anti-inflammatory cytokines, kynurenine metabolism, and cortico-striatal DA and mitochondrial function (via adenosine triphosphate (ATP) release), and if clozapine or NAC (alone and in combination) reverses these changes. SIR induced pronounced deficits in social interactive behaviours, object recognition memory, and prepulse inhibition (PPI), while simultaneously increasing striatal but reducing frontal cortical accumulation of ATP as well as DA. SIR increased pro- vs. anti-inflammatory cytokine balance and altered kynurenine metabolism with a decrease in neuroprotective ratio. Clozapine (5mg/kg/day×14days) as well as clozapine+NAC (5mg/kg/day and 150mg/kg/day×14days) reversed these changes, with NAC (150mg/kg/day) alone significantly but partially effective in some parameters. Clozapine+NAC was more effective than clozapine alone in reversing SIR-induced PPI, mitochondrial, immune and DA changes. In conclusion, SIR induces mitochondrial and immune-inflammatory changes that underlie cortico-striatal DA perturbations and subsequent behavioural deficits, and responds to treatment with clozapine or NAC, with an additive effect following combination treatment. The data provides insight into the mechanisms that might underlie the utility of NAC as an adjunctive treatment in schizophrenia.

Isolation rearing-induced deficits in sensorimotor gating and social interaction in rats are related to cortico-striatal oxidative stress, and reversed by sub-chronic clozapine administration

Social isolation rearing (SIR) in rats induces behavioral and glutamatergic changes akin to schizophrenia. We studied the effects of 8 weeks SIR on cortico-striatal redox and social and cognitive behaviors in rats. SIR increased superoxide dismutase activity, decreased oxidized:reduced glutathione ratio and increased lipid peroxidation in both brain regions, and induced deficits in prepulse inhibition and social and self-directed interactive behaviors. Both behavioral and cortico-striatal redox disturbances were corrected by clozapine (5 mg/kg/day×11days). Behavioral changes evoked by SIR are associated with cortico-striatal oxidative stress that is reversed by clozapine treatment, providing novel insight into the neurobiology and treatment of schizophrenia.

Development and validation of a single analytical method for the determination of tryptophan, and its kynurenine metabolites in rat plasma

It is highly beneficial to monitor the activity of the kynurenine pathway in a large series of samples with high accuracy and reliability in a single experimental protocol. We have developed a rapid specific solid-phase extraction (SPE)-liquid chromatography-electrospray ionization tandem mass spectrometry method for assaying tryptophan, kynurenine, kynurenic acid (KYNA), 3-hydroxyanthranilic acid (3OHAA), anthranilic acid and quinolinic acid (QA) in rat plasma. We also evaluated picolinic acid (PA) in this method, but it presented with unacceptable validation parameters. The assay involves pre-purification by SPE followed by chromatographic separation by C18 reversed phase chromatography. Mass spectrometric detection was performed using a mass spectrometer in positive and negative electrospray ionization; with a flow rate of 0.2 mL/min and an injection volume of 10 μL. Total run time including sample clean-up was 12 min. The assay method was found to be linear (R² > 0.95) and all the validation parameters were within acceptance range. The developed technique also demonstrated a significant elevation in plasma tryptophan, kynurenine, anthranilic acid and QA, and a significant decrease in KYNA, in rats subjected to post-weaning social isolation rearing, a putative animal model of relevance for depression and schizophrenia. This method can therefore be applied to measure metabolites of the kynurenine pathway in plasma accurately and precisely by LC-MS/MS, thereby helping to realize new opportunities in pharmacological and diagnostic research.

In vitro anti-cancer effects of artemisone nano-vesicular formulations on melanoma cells

UNLABELLED Artemisone is a 10-amino-artemisinin derivative that is markedly superior in vitro and in vivo to current artemisinins against malaria and also possesses antitumor activity. In seeking to capitalise on the last property, we have examined the encapsulation of artemisone in nano-vesicular niosomes and solid lipid nanoparticles, and have evaluated efficacies of the free and encapsulated artemisone against human melanoma A-375 cells and effects on human keratinocytes (HaCaT). Artemisone is successfully encapsulated into the nano-vesicles with encapsulation efficiencies of 67±6% and 79±5%, and with average particle sizes being 211±10nm and 295±18nm respectively. The formulations displayed highly selective cytotoxicity towards the melanoma cells with negligible toxicity towards the normal skin cells. The artemisone-loaded nano-vesicles almost completely inhibited the melanoma cells compared to the free drug. The results overall suggest a potentially more useful therapeutic strategy that needs to be evaluated for the treatment of melanoma and other cancers. FROM THE CLINICAL EDITOR Apart from being an effective anti-malarial drug, a surprising action of artemisone also has antitumor activity. Nonetheless, its low water solubility and bioavailability has limited its clinical use. In this article, the authors enacapsulated artemisone in nano- vesicles and solid lipid nano-particles (SLNs). In-vitro studies confirmed the selective cytotoxicity towards melanoma cells. Further in-vivo and pre-clinical studies are awaited.

Cortico-striatal oxidative status, dopamine turnover and relation with stereotypy in the deer mouse

The deer mouse presents with spontaneous stereotypic movements that resemble the repetitive behaviours of obsessive-compulsive disorder (OCD), and demonstrates a selective response to serotonin reuptake inhibitors. OCD has been linked to altered redox status and since increased dopamine signalling can promote stereotypies as well as oxidative stress, we investigated whether the severity of deer mouse stereotypy may be associated with altered dopamine turnover and cortico-striatal redox status. Deer mice were separated into high (HSB), low (LSB) and non-stereotypy (NS) groups. Frontal cortical and striatal dopamine, 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as superoxide dismutase (SOD) activity, reduced (GSH) and oxidised (GSSG) glutathione and glutathione redox index, were analysed as markers for regional dopamine turnover and oxidative stress, respectively. Dopamine and its metabolites and SOD activity did not differ across the stereotypy groups. Significantly reduced GSH and GSSG and increased glutathione redox index were only observed in the frontal cortex of HSB animals. Frontal cortical GSH and GSSG were inversely correlated while glutathione redox index was positively correlated with stereotypy. Deer mouse stereotypy is thus characterised by a deficient glutathione system in the frontal cortex but not striatum, and provides a therapeutic rationale for using glutathione-active antioxidants in OCD. The evidence for a primary frontal lesion has importance for future OCD research.

Dermal delivery of selected hydrophilic drugs from elastic liposomes: effect of phospholipid formulation and surfactants.

The effect of phospholipid formulation and choice of surfactant on skin permeation of selected hydrophilic drugs from elastic liposomes across human epidermal membrane has been studied. Sodium cholate and various concentrations of phosphatidylcholine were used for the preparation of liposomes namely hydrogenated phosphatidylcholine 90% (Phospholipon 90H), phosphatidylcholine 95% (Phospholipon 90G), phosphatidylcholine 78.6% (Phospholipon 80), and phosphatidylcholine 50% (Phosal PG). To investigate the effect of the surfactant, liposomes were prepared from 95% phosphatidylcholine (Phospholipon 90G) and various surfactants (sodium cholate, sodium deoxycholate, Span 20 (sorbitan monolaurate), Span 40 (sorbitan monopalmitate), Span 60 (sorbitan stearate) and Span 80 (sorbitan monooleate)). The vesicles were prepared by the conventional rotary evaporation technique. The film was hydrated with phosphate‐buffered saline (10 mL) containing 9, 2 and 2.5 mg mL−1 of methotrexate, idoxuridine and aciclovir, respectively. All formulations contained 7% ethanol. Homogenously‐sized liposomes were produced following extrusion through 100‐nm polycarbonate filters using Lipex Extruder. Particle size was characterized by transmission electron microscopy. Vertical Franz diffusion cells were used for the study of drug delivery through human epidermal membrane. For the three drugs, the highest transcutaneous fluxes were from elastic liposomes containing 95% phosphatidylcholine. In general, a higher flux value was obtained for liposomes containing sodium cholate compared with sodium deoxycholate. For the liposomes containing sorbitan monoesters, there was no clearly defined trend between alkyl chain length and flux values. Overall, transcutaneous fluxes of liposomal preparations of hydrophilic drugs were comparable with those from saturated aqueous solutions (P > 0.05).

Transport of aspalathin, a Rooibos tea flavonoid, across the skin and intestinal epithelium

Since Rooibos tea contains high levels of flavonoid antioxidants with potential health benefits when taken orally or applied topically, the quantity of the antioxidants crossing the physiological barriers is of scientific, clinical and commercial importance. This study investigated the in vitro transport of aspalathin, a unique flavonoid constituent of Rooibos tea, across intestinal epithelial cells and the human skin. The transport studies were conducted for both pure aspalathin solutions and extracts from unfermented (or green) Rooibos (Aspalathus linearis) aerial plant material across human abdominal skin in vertical Franz diffusion cells and Caco‐2 cell monolayers in Transwell 6‐well plates. The results obtained from the percutaneous permeation studies demonstrated that only 0.01% of the initial aspalathin dose from both the test solution and extract permeated through the skin, which was in accordance with the prediction from its log P value of −0.347. A portion of 0.07% of the initial aspalathin dose penetrated the different layers of the skin for the green Rooibos extract solution and 0.08% for the pure aspalathin solution. The transport of aspalathin across Caco‐2 cell monolayers was concentration dependent and reached almost 100% (Papp = 20.93 × 10−6 cm/s) of the initial dose in the highest concentration tested for the extract, while it was only 79.03% (Papp = 15.34 × 10−6 cm/s) of the initial dose for the highest concentration of the aspalathin solution. Copyright

Topical delivery of acyclovir and ketoconazole

Abstract Context: Viral and fungal cutaneous manifestations are regularly encountered in immunocompromised human immunodeficiency virus/acquired immunodeficiency syndrome individuals and can be treated by drugs such as acyclovir and ketoconazole, respectively. Objective: The aim of this study was to determine whether the novel Pheroid™ delivery system improved the transdermal delivery and/or dermal delivery of acyclovir and ketoconazole when incorporated into semi-solid formulations. Materials and methods: Semi-solid products (creams and emulgels) containing these drug compounds were formulated, either with or without (control) the Pheroid™ delivery system. The stability of the formulated semi-solid products was examined over a period of six months and included the assay of the actives, pH, viscosity, mass loss and particle size observation. Vertical Franz cell diffusion studies and tape stripping methods were used to determine the in vitro, stratum corneum (SC)-epidermis and epidermis-dermis delivery of these formulations. Results and discussion: Stability tests showed that none of the formulations were completely stable. Acyclovir showed a biphasic character during the in vitro skin diffusion studies for all the tested formulations. The Pheroid™ cream enhanced the transdermal, SC-epidermis and epidermis–dermis delivery of acyclovir the most. The average amount of ketoconazole diffused over 12 h showed improved delivery of ketoconazole, with the Pheroid™ emulgel exhibiting the best transdermal and epidermis–dermis delivery. Conclusion: The Pheroid™ formulae increased transdermal penetration as well as delivery to the dermal and epidermal skin layers. The Pheroid™ emulgel and the Pheroid™ cream increased the topical delivery of ketoconazole and acyclovir, respectively.

Polycyclic cage structures as carrier molecules for neuroprotective non-steroidal anti-inflammatory drugs

The blood-brain barrier is formed by the brain capillary endothelium and plays the predominant role in controlling the passage of substances between the blood and the brain. Recent studies on polycyclic structures, i.e. pentacyclo[5.4.0.0(2,6).0(3,10).0(5,9)]undecane and amantadine, indicated favourable distribution thereof to the brain and it was concluded that these polycyclic structures and their derivatives penetrate the blood-brain barrier readily. A series of novel polycyclic prodrugs incorporating the well known non-steroidal anti-inflammatory drugs (NSAIDs), acetylsalicylic acid and ibuprofen, were synthesised and screened for blood-brain barrier permeability and antioxidant activity. Increased levels of both NSAIDs were detected in the brain tissue of C57BL/6 mice after administration of the synthesised prodrugs, indicating favourable blood-brain barrier permeation. Results from a lipid peroxidation assay indicated that the ester and amide prodrugs significantly increased the ability of the drugs to attenuate lipid peroxidation. These novel prodrugs thus readily penetrate the blood-brain barrier and exhibit increased antioxidant activity when compared to the free NSAIDs.

In vitro wound healing and cytotoxic effects of sinigrin–phytosome complex

Sinigrin is a class of glucosinolates found naturally in plants of the Brassicaceae family. Lately, studies have shown that sinigrin possesses anticancer, antibacterial and anti-inflammatory activities. Since its efficacy has not been explored on wound healing, we examined the effect of sinigrin on HaCaT cells. We also aimed at formulating sinigrin into phytosome to form a sinigrin-phytosome complex and study the wound healing and cytotoxic activities on A-375 and HaCaT cells. Sinigrin was efficiently formulated into the phytosome with an average particle size of 153 ± 39 nm, zeta potential of 10.09 ± 0.98 mV and complex efficiency of 69.5 ± 5%. The formation of the sinigrin-phytosome complex was confirmed by DSC and FTIR analysis. The sinigrin-phytosome complex significantly exhibited wound healing effects when compared to sinigrin alone. After 42 h, the phytosome complex completely healed the wound, whereas sinigrin alone showed only 71% wound closure. The sinigrin-phytosome complex displayed minimal toxicity towards HaCaT cells and at higher concentrations, it showed potent activity towards A-375. The results indicated that sinigrin-phytosome complex augmented the therapeutic potential of sinigrin towards the wound healing activity and this approach should be explored further for cancerous wound treatment.