Influence of the Structure of Drug Moieties on the in Vitro Efficacy of HPMA Copolymer–Geldanamycin Derivative Conjugates

Purpose To optimize the structure of geldanamycin (GDM) derivative moieties attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via an enzymatically degradable spacer. Methods HPMA copolymers containing different AR-GDM (AR = 3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxy-propyl AP(OH)) were synthesized and characterized. Their cytotoxicity towards the A2780 human ovarian carcinoma cells was evaluated. Results The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM. Particularly, HPMA copolymer-bound AH-GDM, which possessed the longest substituent at the 17-position, demonstrated the highest efficacy among the polymer-bound GDM derivatives; however the activity of free AH-GDM was lower than that of the other free AR-GDMs. The relative increase of the activity of macromolecular AH-GDM when compared to AP-GDM or AP(OH)-GDM correlated with the enhanced recognition of AH-GDM terminated oligopeptide side-chains by the active site of the lysosomal enzyme, cathepsin B Drug stability and further stabilization upon binding to HPMA copolymer also contributed to the observed phenomena. Conclusion AH-GDM was found to be a suitable GDM derivative for the design of a drug delivery system based on HPMA copolymers and enzymatically-degradable spacers.     

Electro-Magnetic Nano-Particle Bound Beclin1 siRNA Crosses the Blood–Brain Barrier to Attenuate the Inflammatory Effects of HIV-1 Infection in Vitro

The purpose of this study was to evaluate a novel drug delivery system comprised of ferric-cobalt electro-magnetic nano-material (CoFe2O4@ BaTiO3; MENP) bound to siRNA targeting Beclin1 (MENP-siBeclin1) to cross the blood–brain barrier (BBB) and attenuate the neurotoxic effects of HIV-1 infection in the central nervous system following on-demand release of siRNA using an in vitro primary human BBB model. Beclin1 is a key protein in the regulation of the autophagy pathway and we have recently demonstrated the importance of Beclin1 in regulating viral replication and viral-induced inflammation in HIV-1-infected microglia. The MENP-siBeclin1 nano-formulation did not compromise the physiological function or integrity of the BBB model. Furthermore, the in vitro BBB data revealed that MENP-siBeclin1 could efficiently attenuate viral replication and viral-induced inflammation, likely due to STAT1/ NF-κB signaling pathways. MENP-siBeclin1 also silenced Beclin1 protein expression in HIV-1-infected microglial cells within the model system. In addition, the cytotoxic effects of direct treatment with siBeclin1 and MENP alone or in nano-formulation on primary human neuronal cells showed a minimal amount of cell death. Overall, the data shows that the nano-formulation can silence the BECN1 gene as an effective mechanism to attenuate HIV-1 replication and viral-induced inflammation in the context of the BBB.     

Pharmacokinetic–Pharmacodynamic–Efficacy Analysis of Efalizumab in Patients with Moderate to Severe Psoriasis

Purpose Efalizumab is a humanized anti-CD11a monoclonal antibody that demonstrated efficacy in the treatment of patients with psoriasis. The objective of this study was to perform a pharmacokinetic (PK)–pharmacodynamic (PD)–efficacy (E) modeling analysis with intersubject variability assessment to increase our understanding of the interaction of efalizumab with CD11a on T cells and consequent reduction in severity of disease in psoriasis patients.Methods A total of 6,329 samples from 240 patients in five Phase I and II clinical studies were used in the analysis. For the analysis, plasma efalizumab concentration was used as the PK measurement, the percent of predose CD11a was used as the PD measurement, and the psoriasis area and severity index was used as the measure of efficacy. A receptor-mediated PK/PD model was developed that describes the dynamic interaction of efalizumab binding with CD11a. In the efficacy model, the rate of psoriasis skin production is directly proportional to the amount of free surface CD11a on T cells, which is offset by the rate of skin healing. An additional CD11a-independent component to psoriasis skin production accounted for incomplete response to efalizumab therapy. A Monte Carlo parametric expectation maximization method implemented in the ADAPT II program was used to obtain the estimate of population parameters and inter- and intrasubject variability.Results and Conclusions The final model described the PK/PD/E data in psoriasis patients reasonably well. In addition, simulations using the final model suggested that efalizumab administered less frequently could possibly be more convenient with similar efficacy.     

The Protective Effect of Radicicol Against Renal Ischemia–Reperfusion Injury in Mice

Overexpression of heat shock protein 70 kDa (HSP70) is known to confer cellular protection against ischemia–reperfusion (I/R) injury. Radicicol, a HSP90 inhibitor, has been reported to induce the expression of HSP70 protein. Here we studied whether radicicol attenuated renal I/R injury in vivo. Treatment of mice with radicicol ameliorated renal I/R injury and increased renal HSP70 mRNA and protein. Administration of radicicol with quercetin, an inhibitor of HSP70 induction, eliminated the renoprotective effect of radicicol. Our results suggest that the up-regulation of renal HSP70 protein by radicicol leads to a novel drug therapy against renal I/R injury.     

Investigation of the Therapeutic Efficacy of Codelivery of psiRNA–Vascular Endothelial Growth Factor and pIL-4 into Chitosan Nanoparticles in the Breast Tumor Model

Angiogenesis has been known to increase tumor growth and for its metastatic potential in human tumors. Vascular endothelial growth factor (VEGF) plays an important role in tumor angiogenesis and is a promising therapeutic target for breast cancer. VEGF is an essential target for RNAi-based gene therapy of breast cancer. Interleukin-4 (IL-4) may act as an anti-angiogenic molecule that inhibits tumor growth and migration in rats. The purpose of the present study was to improve therapeutic efficacy in breast cancer with the codelivery of siRNA-expressing plasmid targeting VEGF and IL-4-expressing plasmid encapsulating into chitosan nanoparticles (NPs). The codelivery of psiVEGF and pIL-4 plasmids greatly enhanced in vitro and in vivo gene-silencing efficiency. For the in vitro study, when psiVEGF and pIL-4 into chitosan NPs were combined (81%), the gene-silencing effect was higher than psiVEGF and pIL-4 NPs alone. The in vivo study breast tumor model demonstrated that the administration of coencapsulation of psiVEGF and pIL-4 into chitosan NPs caused an additive effect on breast tumor growth inhibition (97%), compared with containing NPs psiVEGF or pIL-4 alone. These results indicate that chitosan NPs can be effectively used for the codelivery of pIL-4 and siVEGF-expressing plasmid in a combination therapy against breast cancer.     

Histological Protection by Donepezil Against Neurodegeneration Induced by Ischemia–Reperfusion in the Rat Retina

Although a blockade of acetylcholine esterase has been reported to suppress neuronal cell death induced by exogenous glutamate and β-amyloid, information is still limited regarding the neuroprotective effects of the acetylcholine esterase inhibitor donepezil. We histologically examined the effects of donepezil on neuronal injury induced by ischemia–reperfusion. Intravenous and intravitreous treatment with donepezil 15 min prior to ischemia dramatically reduced the retinal damage. The protective effect of donepezil in the ganglion cell layer was not affected by mecamylamine, a nicotinic acetylcholine-receptor antagonist, nor scopolamine, a muscarinic acetylcholine-receptor antagonist. The protective effect of donepezil in the inner plexiform layer was reduced not by mecamylamine, but by scopolamine. Neostigmine, a choline-esterase inhibitor, and pilocarpine, a muscarinic acetylcholine-receptor agonist, have protective effects in the inner plexiform layer and the inner nuclear layer. These results suggest that not only the activation of acetylcholine receptors but also a mechanism unrelated to acetylcholine-esterase inhibition contribute to the protective effect of donepezil on the ganglion cells in the ischemic–reperfused rat retina. Donepezil may be useful as a therapeutic drug against retinal diseases that cause neuronal cell death such as glaucoma with high intraocular pressure.     

Efficacy of the combination of an α1-blocker with an anticholinergic agent in the treatment of lower urinary tract symptoms associated with bladder outlet obstruction

The combination of an α1-blocker with an anticholinergic is a new and promising therapeutic approach for bladder outlet obstruction and detrusor overactivity. Both placebo-controlled and comparative studies have demonstrated that the addition of an anticholinergic in the conventional treatment of patients with bladder outlet obstruction is safe, as the likelihood of acute urinary retention is low. Although the pathophysiology of detrusor overactivity is unknown and most probably multifactorial, it is not expected that the voiding phase is influenced by regular doses of anticholinergics, although high doses may affect detrusor contraction. However, safety issues must be studied further. The combination of tamsulosin with propiverine or tolterodine, and of doxasosin with tolterodine has been shown to cause a significant improvement of lower urinary tract symptoms when compared with α1-blocker monotherapy. Indisputably, the existing literature provides clear evidence that the combination of an α1-blocker with an anticholinergic extends physicians ability to manage lower urinary tract symptoms caused by bladder outlet obstruction and overactive bladder syndrome.     

Bias in the Wagner–Nelson Estimate of the Fraction of Drug Absorbed

Purpose. To examine and quantify bias in the Wagner-Nelson estimate of the fraction of drug absorbed resulting from the estimation error of the elimination rate constant (k), measurement error of the drug concentration, and the truncation error in the area under the curve. Methods. Bias in the Wagner-Nelson estimate was derived as a function of post-dosing time (t), k, ratio of absorption rate constant to k (r), and the coefficient of variation for estimates of k (CV _k), or CV _c for the observed concentration, by assuming a one-compartment model and using an independent estimate of k. The derived functions were used for evaluating the bias with r= 0.5, 3, or 6; k= 0.1 or 0.2; CV _c = 0.2 or 0.4; and CV _k =0.2 or 0.4; for t= 0 to 30 or 60. Results. Estimation error of k resulted in an upward bias in the Wagner-Nelson estimate that could lead to the estimate of the fraction absorbed being greater than unity. The bias resulting from the estimation error of k inflates the fraction of absorption vs. time profiles mainly in the early post-dosing period. The magnitude of the bias in the Wagner-Nelson estimate resulting from estimation error of k was mainly determined by CV _k. The bias in the Wagner-Nelson estimate resulting from to estimation error in k can be dramatically reduced by use of the mean of several independent estimates of k, as in studies for development of an in vivo-in vitro correlation. The truncation error in the area under the curve can introduce a negative bias in the Wagner-Nelson estimate. This can partially offset the bias resulting from estimation error of k in the early post-dosing period. Measurement error of concentration does not introduce bias in the Wagner-Nelson estimate. Conclusions. Estimation error of k results in an upward bias in the Wagner-Nelson estimate, mainly in the early drug absorption phase. The truncation error in AUC can result in a downward bias, which may partially offset the upward bias due to estimation error of k in the early absorption phase. Measurement error of concentration does not introduce bias. The joint effect of estimation error of k and truncation error in AUC can result in a non-monotonic fraction-of-drug-absorbed-vs-time profile. However, only estimation error of k can lead to the Wagner-Nelson estimate of fraction of drug absorbed greater than unity.     

Efficacy and Tolerability of Low-dose Transdermal Estrogen (Oesclim®) in the Treatment of Menopausal Symptoms

Summary

Objective:To establish the proportion of symptomatic postmenopausal women who can be satisfactorily maintained on a low HRT dose of 25?μg/day 17-β-estradiol (Oesclim® 25 transdermal patches), after 8 weeks of treatment.

Study design and patients: This was a multicenter open label non-comparative trial. Treatment was initiated with 25?μg/day dosage, which could be increased to 50?μg/day if required after 8 weeks, according to clinical evaluation. Sequential treatment with an oral progestogen was also given for >12 days/month in all non-hysterectomized women. The primary criterion for evaluation of efficacy was the proportion of patients who remained on Oesclim® 25 after 8 weeks of treatment in comparison to patients requiring Oesclim® 50.

Results: Sixty-two patients were included in the study and 60 were treated. 88.3% of treated patients [CI: 78.7–94.9] fulfilled the primary criterion, remaining with the Oesclim® 25 dosage after 8 weeks of treatment. All clinical menopausal symptoms showed a decrease from baseline to the end of the study. The mean daily number of vasomotor symptoms decreased from

8.2 (±5.6) at baseline, for the entire treated population, to 1.0 (±2.2) and 1.0 (±1.2) at the end of the study in patients remaining with Oesclim® 25 and in those requiring Oesclim® 50, respectively. At the interim visit, patients in the Oesclim® 50 group had a higher number of symptoms than those maintained on Oesclim® 25. The global efficacy of the treatment was evaluated as very effective/effective by 93% of all patients and very good/good by investigators for 91% of their patients. Overall 91% of all patients evaluated the global tolerability as very well/well, while investigators rated it very good/good for 97% of their patients. The vast majority of all patients (93%) were very satisfied/satisfied with the trial treatment, and 90% of them were willing to continue the study drug.

Conclusion: Oesclim® low dose (25?μg) hormonal transdermal therapy was efficient in management of climacteric symptoms in this 16-week study. The good acceptance of the treatment was associated with its high efficiency and tolerability.     

Evaluation of the polyethylene glycol–KF–water system in the context of purifying PEG–protein adducts

Covalent binding of PEG to proteins leads to conjugates widely investigated in several biotechnological processes. Their use as pharmaceuticals requires both careful purification and proper characterization. In this context, this paper examines the potentialities offered by hydrophobic interaction chromatography and compares aqueous potassium fluoride and ammonium sulfate as the eluents. Relative contribution of the various forces which dictate the chromatographic behaviour of PEG–protein adducts on Fractogel TSK–Butyl 650 is discussed.     

Neuroprotective Effect of Curcuminoids Against Inflammation-Mediated Dopaminergic Neurodegeneration in the MPTP Model of Parkinson’s Disease

The present study investigated the neuroprotective effect of curcuminoids, the active polyphenols of Curcuma longa (L.) rhizomes against inflammation-mediated dopaminergic neurodegeneration in the 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP) model of Parkinson's disease (PD). Male C57BL/6 mice were pre-treated with curcuminoids (150?mg/kg/day) for 1?week, followed by four intra-peritoneal (i.p.) injections of MPTP (20?mg/kg) at 2?h intervals with further administration of curcuminoids or deprenyl (3?mg/kg/day) for 2?weeks. Our results show that oral administration of curcuminoids significantly prevented MPTP-mediated depletion of dopamine and tyrosine hydroxylase (TH) immunoreactivity. In-addition, pre-treatment with curcuminoids reversed glial fibrillary acidic protein (GFAP) and inducible nitric oxide synthase (iNOS) protein expression, as well as, reduced pro-inflammatory cytokine and total nitrite generation in the striatum of MPTP-intoxicated mice. Significant improvement in motor performance and gross behavioural activity, as determined by rota-rod and open field tests were also observed. Taken together, our findings suggest that curcuminoids exert a neuroprotective effect against MPTP-induced dopaminergic neurodegeneration through its anti-inflammatory action and thus holds immense potential as a therapeutic candidate for the prevention and management of PD.     

Assessing the Efficacy of an Aroclor 1254–Induced Exogenous Metabolic Activation System for Fetax

ABSTRACT

The developmental toxicity of N-nitrosodimethyl-amine (NDMA) and trichloroethylene (TCE) was assessed with Frog Embryo Teratogenesis Assay: Xenopus (FETAX). Late Xenopus laevis blastulae were exposed to NDMA and TCE for 96–h in two separate static-renewal tests with and without the presence of three differently induced exogenous metabolic activation systems (MAS). The MAS consisted of Aroclor 1254–induced (Aroclor 1254 MAS), isoniazid-induced (INH MAS), and a post-isolation mixture (mixed MAS) of Aroclor 1254– and isoniazid-induced rat liver microsomes. Addition of the INH MAS and the mixed MAS increased the Teratogenic Index [TI=LC50/EC50 (malformation)] of NDMA and TCE nearly 2.0– and 2.1–fold and 2.1– and 1.7–fold, respectively. Inclusion of the Aroclor 1254 MAS did not alter the developmental toxicity of either compound. Based on TI values, embryo growth, and types and severity of induced malformations, both NDMA and TCE were developmentally toxic. Use of post-microsome isolation mixtures from differentially induced rat livers increased the efficacy of the exogenous MAS routinely used by FETAX.     

Efficacy of 5-Vinyl-l-β-D-arabinofuranosyluracil (VaraU) Against Herpes Simplex Virus Type 2 Strains in Cell Cultures and Against Experimental Herpes Encephalitis in Mice: Comparison with Acyclovir and Foscarnet

The sensitivity of different herpes simplex virus type 2 (HSV-2) strains to inhibition by 5-vinyl-l--D-arabinofuranosyluracil (VaraU) was evaluated in comparison to 9-(2-hydroxyethoxymethyl)guanine (ACV; acyclovir) and trisodiumphosphonoformate (Na₃PFA; foscarnet), using a plaque inhibition assay in primary rabbit testes (PRT) cells as well as in human embryonic lung fibroblast (HELF) cell cultures. The order of decreasing activity found was ACV VaraU > Na₃PFA in PRT cells and ACV > VaraU Na₃PFA in HELF cells, with 50% inhibition doses (ID₅₀) of 1.8, 8.8, and >110 µM for the three drugs in HELF cells, respectively. After 72hr of drug treatment, inhibition of HELF cell proliferation by VaraU (ID₅₀, >1000 µM) was less than that by ACV and Na₃PFA, resulting in high selectivity indexes of >100 against HSV-2 for VaraU and ACV. Their in vivo efficacy was assessed in a mouse encephalitis model. Using a treatment schedule of three daily intraperitoneal (ip) doses over a period of 5 days, only the survival times of mice were considerably prolonged by VaraU (150 or 300 mg/kg per day; P < 0.05 or P < 0.001, respectively). In contrast, ACV treatment (150 mg/kg per day) led to a nearly complete prevention of encephalitis and death (P < 0.001). Similar therapy results with VaraU application through the drinking water were obtained using only one-sixth of the high ip dose (50 mg/kg per day) but over a prolonged period of treatment. Under similar conditions no therapeutic effect of oral Na₃PFA was observed.     

Involvement of the dopamine D1 receptor system in the anxiolytic effect of cedrol in the elevated plus maze and light–dark box tests

Cedrol, mainly derived from Juniperus virginiana L. essential oil, has been demonstrated the anxiolytic effect, although its mechanism of action is still not fully established. In the present study, male ICR mice were submitted to the elevated plus maze (EPM) and light–dark box (LDB) tests to investigate the putative mechanism of anxiolytic effect. WAY100635 (5-HT_1A receptor antagonist), flumazenil (benzodiazepine receptor antagonist), SCH23390 (dopamine D₁ receptor antagonist) or sulpiride (dopamine D₂/D₃ receptor antagonist) were used in the behavioral experiment to determine the mechanism of action of cedrol. Subsequently, the monoamine neurotransmitter levels were evaluated after behavioral tests. The data suggest that no significant effect in behavioral parameters were observed after sole intraperitoneal (i.p.) injection of antagonists compared to saline group. The anxiolytic effect of cedrol in behavioral procedures was blocked by either WAY100635 or flumazenil. The anxiolytic effect of cedrol (1200 mg/kg) was effectively antagonized by SCH23390 (0.125 mg/kg). Furthermore, cedrol decreased the DA and NE levels in hippocampus, striatum and hypothalamus. The present findings suggest that the dopaminergic system (D₁ receptor) rather than serotoninergic or GABAergic system may potentially be involved in the modulation of cedrol-induced anxiolytic-like behaviors in mice.     

Involvement of the cytokine–IDO1–AhR loop in zinc oxide nanoparticle-induced acute pulmonary inflammation

Zinc oxide nanoparticles (ZnONPs) are widely used in our daily life, such as in sunscreens and electronic nanodevices. However, pulmonary exposure to ZnONPs causes acute pulmonary inflammation, which is considered as an initial event for various respiratory diseases. Thus, elucidation of the underlying cellular mechanisms of ZnONPs can help us in predicting their potential effects in respiratory diseases. In this study, we observed that ZnONPs increased proinflammatory cytokines, accompanied with an increased expression of aryl hydrocarbon receptor (AhR) and its downstream target cytochrome P450 1A1 (CYP1A1) in macrophages in vitro and in mouse lung epithelia in vivo. Moreover, zinc nitrate, but not silica or titanium dioxide nanoparticles (NPs), had similar effects on macrophages, indicating that the zinc element or ion released from ZnONPs is likely responsible for the activation of the AhR pathway. Cotreatment with an AhR antagonist or AhR knockout reduced ZnONPs-induced cytokine secretion in macrophages or mice, respectively. Furthermore, kynurenine (KYN), an endogenous AhR agonist and a tryptophan metabolite catalyzed by indoleamine 2,3-dioxygenase (IDO), was increased in the serums of mice that aspirated ZnONPs. Consistently, ZnONPs increased IDO1 expression in lung cells in vitro and in vivo. Finally, AhR knockout reduced ZnONPs-induced pulmonary inflammation, cytokine secretion and KYN production in mice, suggesting that AhR activation is involved in ZnONPs-induced cytokine secretion and pulmonary inflammation. In summary, we demonstrated that the pulmonary exposure of ZnONPs stimulated the cytokine–IDO1–AhR loop in the lungs, which has been implied to play roles in immune dysfunctions.     

Improvement in the handling of drug–drug interactions

Approximately one in 200 hospitalised patients has a serious adverse drug effect caused by drug–drug interactions (DDIs). Such adverse effects should be avoidable, but current information provided on DDIs is often incomplete and difficult or even impossible to translate into true risk and appropriate tangible action. Clinicians need to know the mean and maximal expected effect of a DDI on clinical endpoints, any dose adjustments required, and how to monitor tolerability and efficacy in patients subject to a DDI. To this end, improved study designs should take the objective of improving treatment explicitly into account, and any existing DDI data should be publicly accessible. Modelling needs to be used more extensively in order to quantitatively predict the effects of DDIs on clinical endpoints in patients and to relate clinical endpoint effects considered as acceptable to respective changes in experimental and clinical studies. Computer-based expert systems will be required to convert such DDI data into recommendations applicable to the individual patient. Therefore, the incorporation of DDIs in a more general procedure for personalisation of drug therapy is desirable.     

Role of Drug – Drug Interactions in the Efficacy and Safety of Clarithromycin Treatment for Helicobacter Pylori Eradication

Pharmaceutical Chemistry Journal - Current risks from resistance, adverse drug reactions, and drug – drug interactions of clarithromycin used as a component of Helicobacter pylori eradication...