Homocysteine levels in adolescent schizophrenia patients

Homocysteine is a sulfur containing amino acid that has been widely investigated for its putative role in cardiovascular and neuropsychiatric disorders. It has been suggested that homocysteine has implications especially in young, male schizophrenia patients. In this prospective case-control study, we compared plasma homocysteine levels in a group of adolescent schizophrenia inpatients (aged 14–21 years; n = 23) to normal healthy controls (n = 51). Mean plasma homocysteine levels were significantly higher in the patient group than in the control group (15.40 ± 2.00 and 9.78 ± 0.33 μmol/L, respectively, p < 0.032). The difference was almost entirely attributable to the male schizophrenia subgroup (18.18 ± 5.65 in male patients vs. 10.31 ± 5.33 μmol/L in female patients). The group × sex interaction was statistically significant (p = 0.0035). These data indicate that a subgroup of male adolescent schizophrenia patients has high homocysteine blood levels. The role of homocysteine in the pathophysiology of adolescent-onset schizophrenia merits further investigation.     

Further evidence of an association between adolescent bipolar disorder with smoking and substance use disorders: a controlled study

Although previous work suggests that juvenile onset bipolar disorder increases risk for substance use disorders and cigarette smoking, the literature on the subject is limited. We evaluated the association of risk for substance use disorders and cigarette smoking with bipolar disorder in adolescents in a case–control study of adolescents with bipolar disorder (n = 105, age 13.6 ± 2.5 years [mean]; 70% male) and without bipolar disorder (“controls”; n = 98, age 13.7 ± 2.1 years; 60% male). Rates of substance use and other disorders were assessed with structured interviews (KSADS-E for subjects younger than 18, SCID for 18-year-old subjects). Bipolar disorder was associated with a significant age-adjusted risk for any substance use disorder (hazard ratio[95% confidence interval] = 8.68[3.02 25.0], χ² = 16.06, p < 0.001, df = 1), alcohol abuse (7.66 [2.20 26.7], χ² = 10.2, p = 0.001, df = 1), drug abuse (18.5 [2.46 139.10], χ² = 8.03, p = 0.005, df = 1) and dependence (12.1 [1.54 95.50], χ² = 5.61, p = 0.02, df = 1), and cigarette smoking (12.3 [2.83 53.69], χ² = 11.2, p < 0.001, df = 1), independently of attention deficit/hyperactivity disorder, multiple anxiety, and conduct disorder (CD). The primary predictor of substance use disorders in bipolar youth was older age (BPD − SUD versus BPD + SUD, logistic regression: χ² = 89.37, p < 0.001). Adolescent bipolar disorder is a significant risk factor for substance use disorders and cigarette smoking, independent of psychiatric comorbidity. Clinicians should carefully screen adolescents with bipolar disorder for substance and cigarette use.     

NCX 6560, a nitric oxide-releasing derivative of atorvastatin, inhibits cholesterol biosynthesis and shows anti-inflammatory and anti-thrombotic properties

We compared the lipid-lowering, vasodilating, anti-thrombotic and anti-inflammatory properties of NCX 6560, a novel NO-releasing derivative of atorvastatin, with those of atorvastatin. NCX 6560 and atorvastatin induced similar inhibition of cholesterol biosynthesis in rat smooth muscle cells (IC₅₀ = 1.9 ± 0.4 and 3.9 ± 1.0 μM, respectively). However, in hyperlipidemic mice, a 5-week oral treatment with NCX 6560 (46.8 mg/kg/day, p.o.) was more effective than equivalent atorvastatin (40 mg/kg/day, p.o.) at lowering serum cholesterol (NCX 6560: − 21% vs controls, P < 0.05; atorvastatin: − 14% vs control, P = NS). In norepinephrine-precontracted rabbit aortic rings, NCX 6560-induced vasodilation (EC₅₀ = 53.5 ± 8.3 μM) and in PC12 cells it stimulated cGMP formation (EC₅₀ = 1.8 ± 0.7 μM), while atorvastatin was inactive. In lipopolysaccharide from Escherichia coli (LPS)-treated RAW 264.7 macrophages, NCX 6560 reduced iNOS expression and dimer assembly more efficiently than atorvastatin and inhibited nitrite accumulation (IC₅₀ = 6.7 ± 1.6 μM) and TNF release. U46619- or collagen plus epinephrine-induced platelet pulmonary thromboembolism in mice was reduced by NCX 6560 at 46.8 mg/kg p.o. (mortality: − 44% and − 56% vs vehicle, respectively; P < 0.05), but not by atorvastatin 40 mg/kg, p.o. In the U46619-induced mortality model, isosorbide mononitrate (ISMN) (20 mg/kg, p.o.), a pure NO-donor, was also active (mortality: − 40%, P < 0.05). NCX 6560 significantly reduced ex vivo platelet adhesion to collagen at high shear (− 31 ± 1.3% vs vehicle), and so did ISMN (− 33.3 ± 1.7% vs vehicle). Atorvastatin was ineffective. NCX 6560, but not atorvastatin, reduced blood pressure in eNOS knockout mice (− 16%, P < 0.001 vs vehicle), an effect not observed in wild type mice. On the contrary, ISMN provoked a significant drop of blood pressure both in wild type (− 20%, P < 0.05 vs vehicle) and in eNOS−/− mice (− 21%, P < 0.05 vs vehicle). In conclusion, NCX 6560 exerts greater lipid-lowering, anti-thrombotic and anti-inflammatory effects than atorvastatin, due to a large extent to NO release.     

Linezolid reduces length of stay and duration of intravenous treatment compared with vancomycin for complicated skin and soft tissue infections due to suspected or proven methicillin-resistant Staphylococcus aureus (MRSA)

We compared the health outcomes in patients treated with linezolid or vancomycin for complicated skin and soft tissue infections (cSSTIs). This analysis is part of a randomised, open-label, multinational trial involving 1200 adult patients hospitalised with cSSTIs due to suspected or proven methicillin-resistant Staphylococcus aureus (MRSA). Subjects received linezolid 600 mg intravenous (i.v.) or oral, or vancomycin 1 g i.v. every 12 h. A test-of-cure was assessed at 7 days post therapy. Compared with vancomycin, linezolid treatment was associated with significantly shorter length of stay (all P < 0.01), decreased i.v. antibiotic treatment duration (all P < 0.0001) and higher discharge rates (all P < 0.05). Thus, linezolid has the potential to reduce medical resource use for the treatment of cSSTIs.     

Differential expression of alpha1-adrenergic receptor subtypes in coronary microvascular endothelial cells in culture

Nicorandil has an anti-apoptotic effect on ischemic myocardium through the activation of ATP-sensitive potassium (K_ATP) channel. We tested the hypothesis that oral administration of nicorandil had a protective effect on ischemic skin flaps. A cranially based skin flap measuring 3 × 7 cm in full thickness was made on the back of rats. The rats were divided into a control group and 8 nicorandil groups (group 1–8) according to different doses and timings of administration. On day 7 at 5 cm, groups 1 to 6 (10 or 30 mg/kg twice per day for 3 days starting at 24 h before, 0.5 h before or 0.5 h after the operation) showed significantly higher blood perfusion change rate (73.3 ± 2.9%–79.1 ± 4.1% vs. 25.9 ± 8.6%, P < 0.01), and significantly higher survival rate (68.8 ± 4.8–75.2 ± 8.2% vs. 47.0 ± 2.8%, P < 0.05) than the control group. Many more surviving blood vessels were also observed in these groups. In contrast, no significant effects were found either in group 7 (30 mg/kg twice per day for 3 days starting 24 h after the operation) or group 8 (30 mg/kg once at 0.5 h after the operation). We did not find an angiogenic effect of nicorandil in vitro. Therefore, our results confirmed that the oral administration of nicorandil could protect tissues from necrosis in ischemic skin flaps. In addition, its protective effect depends on the time of first administration and the duration.     

Lactobacillus rhamnosus strain GG restores alkaline phosphatase activity in differentiating Caco-2 cells dosed with the potent mycotoxin deoxynivalenol

Deoxynivalenol (DON) contamination of cereal crops occurs frequently, and may cause acute exposure at high levels or chronic more moderate exposure. DON has proven toxicity including restriction of enterocyte differentiation, which may play a part in DON induced gastroenteritis. The probiotic bacteria Lactobacillus rhamnosus strain GG (GG) can bind DON, and therefore potentially restrict bioavailability of this toxin. Binding efficacy is not significantly altered by heat treatment, and therefore this in vitro study evaluated whether heat inactivated GG could restore the differentiation process in Caco-2 cells, using alkaline phosphatase (ALP) activity as a marker of differentiation. DON (200 ng/mL) caused a significant (p < 0.001) 36% reduction in ALP activity (1598 ± 137 U/mg protein) compared to untreated cells (2502 ± 80 U/mg). A dose dependant restoration of ALP activity was observed where DON treated cells were co-incubated with heat inactivated GG (1719 ± 84; 2007 ± 142; 2272 ± 160 U/mg for GG at 1 × 10⁴ (p > 0.9), 1 × 10⁷ (p < 0.001), and 1 × 10¹⁰ CFU/mL (p < 0.001), respectively). Co-incubation of the non-binding strain, LC-705 (1 × 10¹⁰ CFU/mL), with DON did not significantly restore the ALP (1841 ± 97 U/mg, p < 0.077) compared to DON only treated cells. When viable GG were co-incubated with DON a similar restoration of ALP activity was observed as seen for heat inactivated GG. These combined data suggest that the major effect of GG on restoring ALP activity, and therefore Caco-2 cell differentiation, was due to specific binding of DON, with possibly a more minor role of non-specific bacterial interference.     

Microcalorimetric investigation of the toxic action of ammonium ferric(III) sulfate on the metabolic activity of pure microbes

A series of calorimetric experiments were performed to investigate toxic action of ammonium ferric sulfate (AFS) on Bacillus subtilis, Pseudomonas putida and Candida humicola. The power–time curves of micro-organism metabolism were obtained, and the action of them by addition of AFS was studied. C. humicola, B. subtilis and P. putida were inhibited completely when the concentrations were up to 320.0, 160.0 and 160.0 μg mL⁻¹, respectively. The relationships between growth rate constant (k) and doses of AFS were approximately linear for three microbes, P. putida for 10.0–160.0 μg mL⁻¹ (R = −0.9746), B. subtilis for 0–160.0 μg mL⁻¹ (R = −0.9868) and C. humicola for 10.0–320.0 μg mL⁻¹ (R = −0.9955). The total heat dissipated per milliliter (Q_T) for three microbes remained balance approximately during the lower doses, P. putida and B. subtilis less than the dose of 20.0 μg mL⁻¹, 0.56 ± 0.01 and 0.26 ± 0.01 J mL⁻¹, respectively, C. humicola less than the dose of 40.0 μg mL⁻¹, 0.58 ± 0.03 J mL⁻¹. The biomass and OD₆₀₀ of three micro-organisms growth in the absence of AFS also were obtained. The power–time curve of C. humicola growth coincided with its turbidity curve. It elucidates that microcalorimetric method agreed with the routine microbiology method.     

Multidrug resistance to antimicrobials as a predominant factor influencing patient survival

The impact of multidrug resistance to antimicrobials was assessed in a cohort of 243 patients with microbiologically documented infections by a variety of susceptible and multidrug-resistant (MDR) species. Multidrug resistance was defined as resistance to more than two antimicrobial agents of different chemical structure. Cox regression analysis was performed to define differences and the significance of any predisposing factors. Overall survival of patients infected by susceptible isolates was prolonged compared with patients infected by MDR isolates (P = 0.013). Mortality rates of infections caused by susceptible and MDR isolates were 4.87% and 16.15%, respectively (P = 0.013); the higher mortality rate for MDR isolates was more pronounced for infections by Klebsiella pneumoniae and Pseudomonas aeruginosa. Mean (± standard error (S.E.)) survival of patients infected by susceptible and MDR isolates in patients without signs of severe sepsis was 28 days and 27.29 ± 0.35 days, respectively (P = not significant). Mean (± S.E.) survival of patients with severe sepsis caused by susceptible and MDR isolates was 7.70 ± 4.62 days and 10.45 ± 2.18 days, respectively (P = 0.048). Diabetes mellitus type 2, the presence of severe sepsis and any underlying malignancy were the most important risk factors affecting survival. It is concluded that infections by MDR isolates were accompanied by higher mortality rates and decreased survival compared with infections by susceptible isolates. Diabetes mellitus type 2 and underlying malignancies were significant co-morbid conditions, whereas survival after infection by susceptible isolates was particularly decreased in the event of severe sepsis.     

Evaluation of gatifloxacin pharmacokinetics and pharmacodynamics in severely ill adults in a medical Intensive Care Unit

A prospective, open-label study investigated the steady-state pharmacokinetics of gatifloxacin in 20 adult patients in a medical Intensive Care Unit (ICU). Twelve patients had normal or moderately impaired renal function (creatinine clearance (CrCL) ≥40 mL/min) and received gatifloxacin 400 mg intravenously once daily. Eight patients had CrCL < 40 mL/min and received 200 mg doses. Gatifloxacin plasma and urine concentrations were determined by validated high-performance liquid chromatography. Mean ± standard deviation gatifloxacin elimination half-life (t_1/2), systemic clearance and volume of distribution in patients with CrCL ≥ 40 mL/min were 10.8 ± 1.5 h, 156 ± 29 mL/min and 1.8 ± 0.2 L/kg, respectively. Maximum and minimum serum concentrations (C_max and C_min) and area under the serum concentration–time curve from 0–24 h (AUC_0–24) in these patients were 4.77 ± 0.76 mg/L, 1.08 ± 0.28 mg/L and 44.4 ± 9.2 mg h/L, respectively. Observed t_1/2, C_max and AUC_0–24 following 200 mg doses in patients with poor renal function (CrCL < 40 mL/min) were 18.2 ± 3.3 h, 2.85 ± 0.76 mg/L and 36.6 ± 3.4 mg h/L, respectively. Statistically significant (P < 0.05) increase in AUC_0–24 and decreases in t_1/2 and clearance (total and renal) were observed in ICU patients administered intravenous gatifloxacin compared with previous data in healthy volunteers. Pharmacodynamic evaluation by Monte Carlo simulation indicated that approved gatifloxacin dosage regimens appear to be adequate for most pathogens (minimum inhibitory concentration (MIC) ≤0.5 μg/mL) associated with community-acquired infections in severely ill ICU patients; less susceptible pathogens (MIC ≥ 1 μg/mL) do not appear to be optimally treated with currently approved doses.     

Alpha1L-adrenoceptors mediate contractions of the isolated mouse prostate

The subtype of ₁-adrenoceptor mediating noradrenaline-induced contractile responses in isolated mouse prostate glands was investigated. Adrenoceptor agonists were able to produce concentration-dependent contractions with the following rank order of potency: adrenaline ≥ noradrenaline ≥ clonidine = phenylephrine > dopamine ≥ isoprenaline. Concentration–response curves to noradrenaline of the prostatic smooth muscle were antagonised by prazosin, N-[2-(2-cyclopropylmethoxyphenoxy)ethyl]-5-chloro-, -dimethyl-1H-indole-3-ethanamine (RS-17053), 2-(2,6-dimethoxyphenoxyethyl)aminomethyl-1,4-benzodioxane (WB 4101), tamsulosin and yohimbine with mean antagonist affinity estimates (pA₂ or apparent pK_B) of 8.12 ± 0.10, 6.56 ± 0.11, 8.38 ± 0.06, 10.14 ± 0.19 and 7.38 ± 1.36 respectively. Propranolol (1 μM) had no antagonist activity (P = 0.994, n = 6). Yohimbine (0.01, 0.1, 1 μM) had no antagonist activity in the presence of prazosin (0.1 μM) (P ≥ 0.059). The results obtained indicate that ₁-adrenoceptors mediate the contractile response in isolated preparations of the mouse prostate. Furthermore, the particular subtype of ₁-adrenoceptor mediating the response to exogenously administered noradrenaline corresponds to the _1L-subtype, the same subtype as that which has been shown to mediate noradrenaline-induced contractile activity in the human prostate.     

A new hydrogel for the extended and complete prednisolone release in the GI tract

The issue of incomplete release of poorly soluble drugs from sustained-release oral formulations is addressed using prednisolone (PDS) as the model drug and a novel highly swelling hydrogel as the rate-controlling material. The hydrogel was formed by heating N-carboxymethylchitosan (CMC) to 80 °C for 24 h. Swelling, alkalimetry, FTIR, DSC, and solid-state NMR studies showed that the treatment produced physical crosslinking, i.e., polymer chain entanglement. A controlled-release system was prepared by coating an inert compacted support of ethylcellulose (50 mg; diameter, 6 mm) with a CMC layer containing dispersed PDS powder (10–50 μm). The system was heated to crosslink the CMC coating, then drug release to simulated GI fluids was studied in vitro. The drug release pattern and term were modulated via the layer mass (LM) (10 or 14 mg cm⁻²) and/or the drug–polymer wt ratio (D/P) (1:5 or 2:5). The rate parameter, K, and the time exponent, n, of the Peppas equation were: K = 26.6 ± 0.3 h⁻ⁿ, n = 0.78 ± 0.02 (LM, 10 mg cm⁻²; D/P, 1:5); K = 24.7 ± 0.7 h⁻ⁿ, n = 0.56 ± 0.02 (LM, 14 mg cm⁻²; D/P, 1:5); K = 20.7 ± 0.3 h⁻ⁿ, n = 0.76 ± 0.01 (LM, 10 mg cm⁻²; D/P, 2:5). Hydrogel swelling was faster than drug release. This was controlled, in a first stage, by drug dissolution–diffusion in the swollen gel, and subsequently, by diffusion. The drug release rate was unaffected by the GI pH variations, and slightly affected by the environmental hydrodynamics. The system promises an extended and complete release of poorly soluble drugs in the GI tract.     

Lymphocyte muscarinic receptors and platelet monoamine oxidase-B as biomarkers of CNS function: effects of age and gender in healthy humans

Lymphocyte cholinergic muscarinic receptors (MRs) and platelet monoamine oxidase-B (MAO-B) activity are considered surrogate markers of the same parameters in the central nervous system. Lymphocyte MR binding and platelet MAO-B activity were measured in a consistent number of healthy human adults and analysed according to gender and age. The mean value ± S.D. of MR binding neither differed between males (12.2 ± 10.0 fmol/10⁶ cells, range: 0.5–37.9, n = 86) and females (10.7 ± 9.7 fmol/10⁶ cells, range: 0.5–39.7, n = 69) nor among age groups. MAO-B activity was significantly higher in women (geometric mean: 11.3 nmol/mg protein/h, with 65% of values from 7.3 to 17.6; n = 43), than in men (7.7 nmol/mg protein/h, with 65% of values from 4.5 to 13; n = 95). Males aged 56–66 years displayed a higher, though not statistically significant, basal enzyme activity than younger subjects. Altogether these data indicate gender-related differences in MAO activity, but not in MR binding, and inter-individual differences in the basal values of both peripheral blood markers in healthy subjects.     

Effects of diazinon on acetylcholinesterase activity and lipid peroxidation in the brain of Oreochromis niloticus

The aim of this study was to investigate the effects of organophosphorus (OP) pesticide diazinon on acetylcholinesterase (AChE: EC 3.1.1.7) activity and its relationship to lipid peroxidation (LPO) in the brain of a freshwater fish, Oreochromis niloticus. Malondialdehyde (MDA) content was used as biomarker for LPO. Fish were exposed to 1 and 2 mg/L sublethal concentrations of diazinon for 1, 7, 15 and 30 days. In the entire experimental group, AChE activity in brain significantly decreased (up to 93% of control), whereas MDA content decreased after 1 day, and increased after 7 and 15 days of exposures. MDA was in similar level with the control group after diazinon exposure of 30 days. The findings of the present study show that diazinon inhibited AChE activity and it has LPO-inducing potential in fish. The inhibition of AChE activity in the brain of O. niloticus correlated with increased MDA levels after 7 and 15 days diazinon exposures (r = −0.661, P < 0.019; r = −0.652, P < 0.022, respectively).     

Blood manganese concentrations and intrauterine growth restriction

To assess the relationship between blood concentrations of manganese (Mn) and intrauterine growth restriction (IUGR), Mn levels in the umbilical cord blood (UCB) and the mother whole blood (MWB) samples were measured in apparently healthy mothers and their newborns. Measurement was conducted by an inductively coupled plasma mass spectrometry. Manganese concentrations in MWB were significantly lower (p < 0.01) in IUGR cases than in appropriate for gestational age (AGA) cases (mean ± S.D.; 16.7 ± 4.8 and 19.1 ± 5.9 μg/l, respectively). Conversely, UCB concentrations of Mn were significantly higher (p < 0.05) in IUGR newborns than AGA newborns (44.7 ± 19.1 and 38.2 ± 13.1 μg/l, respectively). Logistic regression analysis demonstrated significant relationships of the mother whole blood and the umbilical cord blood concentrations of Mn in IUGR cases (OR = 0.868, 1.044, respectively). The study suggests that manganese concentrations in MWB and UCB might induce different effects on birth weight in healthy mothers. Because intrauterine growth restriction is a multi-factorial problem, further epidemiological and clinical studies on larger numbers of subjects are needed to confirm the findings in the present study.     

Isolation and characterisation of acanmyotoxin-2 and acanmyotoxin-3, myotoxins from the venom of the death adder Acanthophis sp. Seram

Death adder (genus Acanthophis) venoms display neurotoxic activity but were thought to be devoid of myotoxic components. Studies from our laboratory have shown that some species (i.e. Acanthophis rugosus and Acanthophis sp. Seram) posses venom with myotoxic activity [Wickramaratna JC, Fry BG, Aguilar M, Kini RM, Hodgson WC. Isolation and pharmacological characterisation of a phospholipase A₂ myotoxin from the venom of the Irian Jayan death adder (A. rugosus). Br J Pharmacol 2003;138:333–342; Wickramaratna JC, Fry BG, Hodgson WC. Species-dependent variations in the in vitro myotoxicity of death adder (Acanthophis) venoms. Toxicol Sci 2003;74:352–360]. The present study describes the isolation and characterisation of two myotoxins (acanmyotoxin-2 and acanmyotoxin-3) from A. sp. Seram venom. Venom was fractionated into approximately 12 major peaks using reverse phase high performance liquid chromatography. Two components caused concentration (0.1–1 μM) dependent inhibition of direct (2 ms, 0.1 Hz, supramaximal V) twitches and an increase in baseline tension in the chick biventer cervicis nerve-muscle. Histological examination of the muscle confirmed damage. PLA₂ activity was detected in both acanmyotoxin-2 (390.2 ± 19.7 μmol/(min mg); n = 4) and acanmyotoxin-3 (14.2 ± 7.7 μmol/(min mg); n = 4). In comparison, A. sp. Seram whole venom had a specific activity of 461.3 ± 90.4 μmol/(min mg) (n = 3). Mass spectrometry analysis indicated acanmyotoxin-2 had a mass of 13,082 Da and acanmyotoxin-2 13,896 Da. Acanmyotoxin-2 and acanmyotoxin-3 accounted for approximately 7 and 4% of total venom composition, respectively. N-terminal sequencing of the first 30 amino acids of each toxin indicated they shared some sequence homology with known myotoxins. In conclusion, clinicians should be aware that symptoms of envenoming by some species of death adder may include signs of myotoxicity as well as neurotoxicity. Future studies will investigate the efficacy of the current antivenom treatment against the myotoxic components of A. sp. Seram venom.     

High-performance liquid chromatographic assay for morphine in small plasma samples: application to pharmacokinetic studies in rats

In order to perform a reliable pharmacokinetic study of morphine during subchronic treatment in rats, an easy, rapid, sensitive and selective analytical method was proposed and validated. The analyte and internal standard (naloxone) were extracted from plasma samples (100 μL) by a single solid-phase extraction method prior to reverse-phase high performance liquid chromatography (HPLC) along with electrochemical detection (ED). Standard calibration graphs were linear within a range of 3.5–1000 ng/mL (r = 0.999). The intra-day coefficients of variation (CV) were in the range of 5.8–8.5% at eight concentration levels (7–1000 ng/mL) and the inter-day coefficient of variation ranged from 7.4 to 8.8%. The intra-day assay accuracy was in the range of −5–10% and the inter-day assay accuracy ranged from 3.0 to 9.3% of relative error (RE). The limit of quantification was 3.5 ng/mL using a plasma sample of 100 μL (15.8% of CV and 12.8% of RE). Plasma samples were stable for 2 months at −20 °C. This method was found to be suitable for pharmacokinetic studies in rats, after subcutaneous administration of morphine (5.6 mg/kg per day) in subchronic treatment for 6 and 12 days.     

Effect of injection routes on pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin in rats

Pharmacokinetics and lactone/carboxylate equilibrium of 9-Nitrocamptothecin (9-NC) were compared after intravenous (i.v.) and intramuscular (i.m.) injection at a dose of 1.5 mg/kg 9-NC solution. The concentrations of three different forms of 9-NC, namely lactone, carboxylate and total 9-NC, were measured by HPLC analysis. Injection routes were demonstrated to have significant effect on pharmacokinetics of 9-NC. Compared with i.v. injection route, mean residence time (MRT) of 9-NC three forms was significantly prolonged following i.m. route (p < 0.05). The AUC_0–∞ ratios of i.m. to i.v. route were calculated to be 102 ± 43%, 273 ± 221% and 150 ± 62% for lactone, carboxylate and total 9-NC, respectively. Compared with i.v. injection route, although AUC_0–∞ was barely changed, MRT of lactone 9-NC was dramatically prolonged 4.5-fold after i.m. injection, which may account for the reported improved antitumor efficacy. However, the results of the present study also demonstrated that i.m. injection route increased both AUC_0–∞ and MRT of carboxylate 9-NC more significantly. Since the carboxylate form of CPT analogs including 9-NC is associated with their unwanted toxicity, i.m. injection route might lead to severe toxicity compared with i.v. route. Lactone/carboxylate equilibrium was also significantly influenced by injection routes. Based on the AUC_0–∞ measurements, the lactone 9-NC constituted 50 ± 8% and 32 ± 7% of circulating total 9-NC after i.v. or i.m. administration, respectively (p < 0.01).     

Deramciclane in the treatment of generalized anxiety disorder: A placebo-controlled, double-blind, dose-finding study

Deramciclane, a camphor derivative, is a novel anxiolytic agent with a unique mechanism of action. It acts as a potent and specific antagonist at serotonin 5-HT_2A/2C receptors, and exhibits anxiolytic efficacy in animal models. The aim of this double-blind, placebo-controlled, parallel-group study was to evaluate the efficacy, safety, and tolerability of a range of doses of deramciclane in patients with generalized anxiety disorder (GAD). Adult patients with a diagnosis of GAD (DSM-IV) and a Hamilton Anxiety Rating Scale (HAM-A) total score ≥ 18; a score ≥ 2 for the HAM-A items ‘Anxious Mood’ and ‘Tension’; a score ≥ 4 on the Clinical Global Impression of Severity of Illness (CGI-S) Scale; and a score ≤ 20 on the Montgomery–Åsberg Depression Rating Scale (MADRS) were enrolled in the study. Following a 1–2 week placebo run-in period, patients were randomized to receive deramciclane (10, 30, or 60 mg/day in two divided doses) or placebo for 8 weeks, followed by a 2-week placebo wash-out period. The primary efficacy measure was change in HAM-A score from baseline to week 8. Adverse events were monitored throughout the study. Withdrawal reactions were assessed at the end of the study (week 8) and during the placebo wash-out period using the Physician's Withdrawal Checklist (34 items). In the intent-to-treat population (n = 208), both the deramciclane 30 mg/day and 60 mg/day doses provided clinically relevant improvements in HAM-A total score after 8 weeks of treatment, reaching statistical significance compared with placebo in the 60 mg/day dose group (p = 0.024) and a clear trend in the 30 mg/day group (p = 0.059). On the HAM-A psychic anxiety factor, significant improvements were seen in patients in the deramciclane 30 mg/day and 60 mg/day treatment groups compared with those in the placebo group (p < 0.05). Adverse events were reported at a similar frequency across all four treatment groups; the most commonly reported adverse event was headache. No withdrawal reactions were observed on abrupt discontinuation of deramciclane. In conclusion, deramciclane 60 mg/day showed significant evidence of efficacy for the treatment of GAD in adult patients. The efficacy for the 30 mg/day dose was close to the larger dose although not significant in the primary analysis, and there was no significant evidence of efficacy for the 10 mg/day dose. Deramciclane was safe and well-tolerated up to the 60 mg/day dose over an 8-week period.     

Development and validation of a novel LC non-derivatization method for the determination of amikacin in pharmaceuticals based on evaporative light scattering detection

A novel method for the direct determination of the aminoglycoside antibiotic amikacin and its precursor component kanamycin was developed and validated, based on reversed phase LC with evaporative light scattering detector (ELSD). ELSD response to amikacin was found to be enhanced by: (a) use of ion-pairing acidic reagents of increased molecular mass, (b) increase of mobile phase volatility and (c) decrease of peak width and asymmetry (obtained by controlling the mobile phase acidity and/or ratio of organic solvent to water). Utilizing a Thermo Hypersil BetaBasic C₁₈ column, the selected optimized mobile phase was water–methanol (60:40, v/v), containing 3.0 ml l⁻¹ nonafluoropentanoic acid (18.2 mM) (isocratic elution with flow rate of 1.0 ml min⁻¹). ELSD experimental parameters were: nitrogen pressure 3.5 bar, evaporation temperature 50 °C, and gain 11. Amikacin was eluted at 8.6 min and kanamycin at 10.4 min with a resolution of 1.5. Logarithmic calibration curves were obtained from 7 to 77 μg ml⁻¹ (r > 0.9995) for amikacin and 8 to 105 μg ml⁻¹ (r > 0.998) for kanamycin, with a LOD equal to 2.2 and 2.5 μg ml⁻¹, respectively.

In amikacin sulfate pharmaceutical raw materials, the simultaneous determination of sulfate (t_R = 2.3 min, LOD = 1.8 μg ml⁻¹, range 5–40 μg ml⁻¹, %R.S.D. = 1.1, r > 0.9997), kanamycin and amikacin was feasible. No significant difference was found between the results of the developed LC–ELSD method and those of reference methods, while the mean recovery of kanamycin from spiked samples (0.5%, w/w) was 97.3% (%R.S.D. ≤ 2.0, n = 6). Further, the developed method was applied for the determination of amikacin in pharmaceutical formulations (injection solutions) without any interference from the matrix (recovery from spiked samples ranged from 95.6 to 103.8%).     

Validation of a reverse-phase high performance liquid chromatographic method for concurrent assay of a weak base (salmeterol xinafoate) and a pharmacologically active steroid (fluticasone propionate)

The analysis of weakly basic drugs such as salmeterol xinafoate (SX) by reverse-phase liquid chromatography remains a problem, particularly when present in combination with other drugs such as steroids and weak acids. This study describes the validation of an assay for a weakly basic drug, salmeterol (SB), its weakly acidic counter-ion, 1-hydroxy-2-naphthoic acid (XA), and the neutral glucocorticoid, fluticasone propionate (FP) using a second-generation silica stationary phase (Inertsil ODS-2). The assay utilized an Inertsil ODS-2 base-deactivated 250 mm × 4.6 mm, 5 μm HPLC column, with 75:25 methanol:0.6% aqueous ammonium acetate as the mobile phase. Under these near neutral conditions, SB demonstrated a good peak shape (tailing factor = 1.21 ± 0.02, n = 85). The method provided a short analysis time: XA, t_R = 2.96 min; SB, t_R = 5.23 min and FP, t_R = 7.01 min. The assay displayed good sensitivity for both XA (LOD for SX = 0.22 μg mL⁻¹) and SB (LOD for SX = 0.26 μg mL⁻¹). The limit of detection for FP was 0.19 μg mL⁻¹. Neither of the drugs was found to interfere in the determination of the other and the assay accuracy (% recovery) was high (the recoveries were: 99.58 ± 1.85% for XA, 99.49 ± 1.88% for SB and 100.24 ± 1.28% for FP). The assay reproducibility was determined with a mean coefficient of variance for the five calibration concentrations of XA = 0.71 ± 0.18%; SB = 1.11 ± 0.64% and FP = 0.92 ± 0.14%. Analysis of a pressurized metered dose inhaler formulation demonstrated recovery of the analytes that are within pharmacopoeial limits. It was shown that RP-HPLC was suitable for the high throughput analysis of the combination of SX and FP.