An ion-pairing, reversed-phase liquid chromatography method to assess the cross-contamination of cancer chemotherapy infusions prepared in a dual-operator aseptic isolator.

UNLABELLED: Cytotoxics are usually prepared in a centralized pharmacy unit in a controlled hospital environment. Despite the rigorous operating procedures used for such preparations, contamination is theoretically possible - for example due to vial switches. Therefore products ought to be checked in order to determine whether quality control measures are adequate. Numerous strategies have been applied locally to ensure the safety of both patients and operators but the efficacy of these methodologies has not previously been examined. The aim of this study was to develop an analytical method sensitive enough to detect traces of anti-cancer drugs, in order to evaluate cross-contamination between infusions prepared in a dual-operator isolator in the dedicated pharmacy unit. We developed a high performance liquid chromatography (HPLC) method with ultraviolet (UV) detection to identify and quantify the following seven drugs: 5-Fluorouracil, Cytarabine, Gemcitabine, Irinotecan, Doxorubicin, Epirubicin, and Daunorubicin. We assessed the levels of cross-contamination in 20 random preparations. We achieved separation of the seven drugs in less than 28 min, with a lower limit of quantification capable of detecting cross-contamination. An assessment of 20 preparations revealed no cross-contamination. We developed a reproducible and sensitive HPLC method which could be a potentially useful tool for use in practice. We checked the level of cross-contamination in anti-cancer drug infusions and confirmed that the process in current use was safe. This study is the first to assess cross-contamination in anti-cancer preparations. This work is the first step in an extensive programme of quality control, whose aim is to ensure the safety of both patients and operators. Copyright © 2015 John Wiley & Sons, Ltd. KEY FINDINGS: Development of a reproducible and sensitive HPLC method capable of detecting seven anticancer drugs. This method could be used alongside MS detection, to check for biological contamination of nursing and pharmacy staff with anticancer drugs. No cross-contamination was detected in cancer chemotherapy infusions prepared in a dual-operator aseptic isolator.

CRF1 receptor antagonists do not reverse pharmacological disruption of prepulse inhibition in rodents.

RATIONALE: As enhanced corticotropin-releasing factor (CRF) transmission is associated with induction of sensorimotor gating deficits, CRF1 receptor antagonists may reverse disrupted prepulse inhibition (PPI), an operational measure of sensorimotor gating. OBJECTIVES: To determine the effects of CRF1 receptor antagonists in pharmacological models of disrupted PPI and to determine if long-term elevated central CRF levels alter sensitivity towards PPI disrupting drugs. METHODS: CP154,526 (10-40 mg/kg), SSR125543 (3-30 mg/kg) and DMP695 (40 mg/kg) were tested on PPI disruption provoked by D-amphetamine (2.5, 3 mg/kg), ketamine (5, 30 mg/kg) and MK801 (0.2, 0.5 mg/kg) in Wistar rats, C57Bl/6J and CD1 mice, and on spontaneously low PPI in Iffa Credo rats and DBA/2J mice. PPI-disrupting effects of D-amphetamine (2.5-5 mg/kg) and MK801 (0.3-1 mg/kg) were examined in CRF-overexpressing (CRFtg) mice, which display PPI deficits. Finally, we determined the influence of CP154,526 on D-amphetamine-induced dopamine outflow in nucleus accumbens and prefrontal cortex of CRFtg mice using in vivo microdialysis. RESULTS: No CRF1-antagonists improved PPI deficits in any test. CRFtg mice showed blunted PPI disruption in response to MK801, but not D-amphetamine. Further, D-amphetamine-induced dopamine release was less pronounced in CRFtg versus wild-type mice, a response normalized by pretreatment with CP154,526. CONCLUSION: The inability of CRF1 receptor antagonists to block pharmacological disruption of sensorimotor gating suggests that the involvement of CRF1 receptors in the modulation of dopaminergic and glutamatergic neurotransmission relevant for sensory gating is limited. Furthermore, the alterations observed in CRFtg mice support the notion that long-term elevated central CRF levels induce changes in these neurotransmitter systems.

Synchrotron X-ray and ab initio studies of beta-Si3N4.

Almost absorption- and extinction-free single-crystal synchrotron X-ray diffraction data were measured at 150, 200 and 295 K for beta-Si3N4, silicon nitride, at a wavelength of 0.7 A. The true symmetry of this material has been the subject of minor controversy for several decades. No compelling evidence favouring the low-symmetry P6(3) model was identified in this study.

SSR181507, a putative atypical antipsychotic with dopamine D2 antagonist and 5-HT1A agonist activities: improvement of social interaction deficits induced by phencyclidine in rats.

Social behaviour is frequently impaired in schizophrenic patients, and current antipsychotics appear poorly effective in alleviating this deficit. SSR181507 is a selective dopamine D2 receptor antagonist and 5-HT1A receptor agonist [Neuropsychopharmacology 28 (2003) 2064] with an atypical antipsychotic profile and additional antidepressant/anxiolytic activities [Neuropsychopharmacology 28 (2003) 1889]. Here, we sought to assess the efficacy of SSR181507, and of reference antipsychotics and antidepressant/anxiolytics, to counteract phencyclidine (PCP)-induced social interaction deficit in rats. Pairs of unfamiliar rats were placed for 10 min each day into a dimly lit arena, during four consecutive days. On the test day (5th day), each pair was placed into the arena 30 min after i.p. treatment with PCP (or vehicle) and a challenge compound or vehicle (same for both rats, i.p. or s.c.). The time spent in social interaction was scored during 10 min. PCP (1 mg/kg) decreased social interaction time by about 35%. This effect was fully antagonized by pre-treatment with SSR181507 (1 mg/kg). In contrast, neither haloperidol (0.05 and 0.1 mg/kg) nor clozapine (0.3 and 1 mg/kg) antagonized this PCP-induced deficit. The selective 5-HT1A receptor agonist 8-OH-DPAT (0.025 and 0.05 mg/kg s.c.), but not the anxiolytic diazepam (0.75 and 1.5 mg/kg), also improved social interaction impairment in PCP-treated rats: this would indicate that the 5-HT1A receptor agonist properties of SSR181507 are responsible for the reversal of PCP-induced social deficit. These data suggest that, in addition to its atypical antipsychotic profile and antidepressant/anxiolytic activities, SSR181507 has a potential therapeutic activity in another key feature of schizophrenia poorly controlled by current antipsychotics, namely deterioration in social functioning.

Decreased densities of dopamine and serotonin transporters and of vesicular monoamine transporter 2 in severely kainic acid lesioned subregions of the striatum.

Fifteen days after a striatal kainic acid (KA) injection, we have examined presynaptic modifications of dopamine and serotonin terminals in the striatum through (i) autoradiographic labeling of dopamine, serotonin and vesicular monoamine transporters respectively with 3H-mazindol, 3H-citalopram and 3H-dihydrotetrabenazine, and (ii) determination of the contents in dopamine, serotonin and their metabolites. Acetylcholinesterase histochemical labeling enabled the definition of severely and moderately KA-lesioned subregions within the striatum. A significant decrease of the three transporters labeling density was observed only in the severely lesioned subregions. The strong decrease in serotonin transporter labeling revealed here has not been described until now. Besides, the striatal contents of homovanillic acid (dopamine metabolite) and 5-hydroxyindolacetic acid (serotonin metabolite) were significantly increased in the lesioned striatum. The whole data evidence an incomplete sparing of dopamine and serotonin terminals in the striatum 15 days after a KA injection, especially in the areas where the degeneration of postsynaptic neurons was the most extensive.

Haloperidol-induced catalepsy is absent in dopamine D(2), but maintained in dopamine D(3) receptor knock-out mice.

We have previously found that mice homozygous for the deletion of the dopamine D(2) receptor gene (D(2)(-/-) mice) do not present spontaneous catalepsy when tested in a "bar test". In the present study, we sought to analyse the reactivity of D(2) receptor mutant mice to the cataleptogenic effects of dopamine D(2)-like or D(1)-like receptor antagonists. In parallel, we assessed the cataleptogenic effects of these antagonists in dopamine D(3) receptor mutant mice. D(2)(-/-) mice were totally unresponsive to the cataleptogenic effects of the dopamine D(2)-like receptor antagonist haloperidol (0.125-2 mg/kg i.p.), while D(2)(+/-) mice, at the highest haloperidol doses tested, showed a level of catalepsy about half that of wild-type controls. The degree of haloperidol-induced catalepsy was thus proportional to the level of striatal dopamine D(2) receptor expression (0.50, 0.30 and 0.08 pmol/mg protein as measured at 0.25 nM [3H]spiperone for D(2)(+/+), D(2)(+/-) and D(2)(-/-) mice, respectively). However, D(2)(-/-) and D(2)(+/-) mice were as sensitive as their wild-type counterparts to the cataleptogenic effects of the dopamine D(1)-like receptor antagonist R-(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4, 5-tetrahydro-1H-3-benzazepine hydrochloride (SCH 23390: 0.03-0.6 mg/kg s.c.). Striatal dopamine D(1) receptor expression (as measured using [3H]SCH 23390 binding) was not significantly affected by the genotype. The ability of SCH 23390 to induce catalepsy in D(2)(-/-) mice suggests that their resistance to haloperidol-induced catalepsy is due to the absence of dopamine D(2) receptors, and not to the abnormal striatal synaptic plasticity that has been shown by others to occur in these mice. In agreement with the observation that dopamine D(2) and dopamine D(1) receptor expression was essentially identical in D(3)(+/+), D(3)(+/-) and D(3)(-/-) mice, dopamine D(3) receptor homozygous and heterozygous mutant mice, on the whole, did not differ from their controls in the time spent in a cataleptic position following administration of either haloperidol (0.5-2 mg/kg i.p.) or SCH 23390 (0.03-0.6 mg/kg s.c.). Also, dopamine D(3) receptor mutant mice were no more responsive than wild-type controls when co-administered subthreshold doses of haloperidol (0.125 mg/kg) and SCH 23390 (0.03 mg/kg), suggesting that dopamine D(3) receptor knock-out mice are not more sensitive than wild-types to the synergistic effects of concurrent blockade of dopamine D(2) and dopamine D(1) receptors in this model. These results suggest that the dopamine D(2) receptor subtype is necessary for haloperidol to produce catalepsy, and that the dopamine D(3) receptor subtype appears to exert no observable control over the catalepsy produced by dopamine D(2)-like, D(1)-like and the combination of D(1)-like and D(2)-like receptor antagonists.

Decreased locomotor activity after microinjection of dopamine D2/D3 receptor agonists and antagonists into lobule 9/10 of the cerebellum: a D3 receptor mediated effect?

The restricted localization of dopamine (DA) D3 receptors in the rat cerebellum lobule 9/10 appears to provide a method for investigating the in vivo selectivity of dopaminergic compounds for the D3 receptor subtype. Sprague-Dawley rats implanted with a cannula aimed at lobule 9/10 were microinjected with DA receptor ligands and immediately placed into activity chambers to record their spontaneous locomotor activity for short term (0 to 20 min) and delayed (20 to 40 min) effects. The DA D2/D3 receptor agonists quinelorane (0.1 to 2.5 microg) and 7-OH-DPAT (0.1 to 10 microg) decreased locomotor activity in the first 20 min post-microinjection. In contrast, the DAD1, receptor agonist 6-Br-APB (0.1 to 10 microg) did not affect locomotor activity during this time period, but markedly increased locomotion between 20 and 40 min at the highest dose tested. The DA receptor antagonists haloperidol and raclopride (1 to 10 microg) were also found to reduce locomotor activity. Furthermore, quinelorane and 7-OH-DPAT, but not haloperidol, when microinjected into lobules 1/2 or 6/7 (where no DA D3 receptors have been detected) decreased locomotor scores. These results show that both DA receptor agonists and antagonists decrease locomotor activity when microinjected into lobule 9/10 of the cerebellum. Additionally, DA receptor agonists can reduce spontaneous locomotion when microinjected outside of lobule 9/10. This would suggest that, at least for quinelorane and 7-OH-DPAT, the locomotor decreasing effects following microinjection into cerebellar lobule 9/10 may not be mediated by activity at DA D3 receptors, and that this behavioural assay is unlikely to provide a means for studying the in vivo pharmacology of the DA D3 receptor.

Dopamine D2 receptor knock-out mice are insensitive to the hypolocomotor and hypothermic effects of dopamine D2/D3 receptor agonists.

The dopamine (DA) D2-like family of receptors is comprised of three subtypes, the D2, D3, and D4 receptors. It has been suggested that the potency of DA receptor agonists to produce hypothermia and hypolocomotion in rodents correlates more strongly with the in vitro affinity for, or potency (mitogenesis test) at the D3 than at the D2 subtype. However, it has recently been reported that when tested in DA D3 receptor knock-out mice, several DA D2/D3 receptor agonists (7-OH-DPAT, PD 128907 and quinelorane) induced levels of hypothermia and decreases of locomotor activity similar to those obtained in control (wild-type) mice. These results do not argue in favour of an implication of DA D3 receptors in these in vivo effects. In order to investigate whether the DA D2 receptor is the subtype that mediates hypothermia and hypolocomotion produced by DA D2/D3 receptor agonists, we tested the effects of ip administration of the DA D2/D3 receptor agonists 7-OH-DPAT and PD 128907, on core temperature and locomotor activity in DA D2 receptor knock-out mice (homozygotes: D2(-/-) and heterozygotes: D2(+/-)), and in wild-type (D2(+/+)) mice. 7-OH-DPAT (0.1-3 mg/kg) and PD 128907 (1-10 mg/kg) induced hypothermia and decreased locomotion in D2(+/+) mice, but had no effects in D2(-/-) mice; the magnitude of the hypothermic and locomotor-reducing effects of these two agonists in D2(+/+) mutants was approximately half that of D2(+/+) mice. During the first 10 min in the activity chambers, the level of spontaneous locomotor activity of D2(-/-) individuals was almost 50% below that of D2(+/+) mice; basal locomotor activity of D2(+/-) mice was between that of D2(-/-) and D2(+/+) individuals. Neither type of mutant showed spontaneous catalepsy or deficits in forelimb muscle strength (grip-strength test). These results show that the presence of DA D2 receptors is necessary for the expression of the locomotor- and core temperature-decreasing effects of DA D2/D3 receptor agonists such as 7-OH-DPAT and PD 128907.

Dopamine D3 receptor agonists produce similar decreases in body temperature and locomotor activity in D3 knock-out and wild-type mice.

The function of the dopamine (DA) D3 receptor, a member of the D2-like family, has not been firmly established. It has been reported that the potency of DA receptor agonists in producing hypothermia and hypolocomotion in rodents correlates more strongly with the in vitro affinity for, or potency (mitogenesis test) at the D3 than the D2 subtype. In order to investigate further the role of D3 receptors in hypothermia and hypolocomotion, we tested the effects of ip administration of three DA receptor agonists reported to be selective for the D3 receptor subtype (7-OH-DPAT, quinelorane and PD 128907) on core temperature and spontaneous locomotor activity in homozygous (D3-/-), heterozygous (D3+/-) mutant and wild-type (D3+/+) mice. Quinelorane (0.003-0.3 mg/kg), PD 128907 (1-10 mg/kg) and 7-OH-DPAT (0.1-3 mg/kg) induced hypothermia and decreased locomotion to a similar extent in the three genotypes. Additionally, the putatively selective DA D3 receptor antagonist PNU 99194A (3-20 mg/kg i.p.) increased locomotor activity in habituated mice and reversed the hypothermia induced by 30 microg/kg of quinelorane, with no apparent difference between D3-/-, D3+/- and D3+/+ genotypes. The spontaneous level of locomotor activity of mutants (D3-/- or D3+/-) was found to be either at, below, or above that of controls, with no consistent trend between different batches of mice. These results show that the presence of DA D3 receptors is not necessary for the expression of these effects induced by the three agonists or the antagonist supposedly selective for the D3 receptor subtype. This raises the question of the involvement of the D3 receptor in these behavioural effects and the issue of the in vivo selectivity of these four compounds for the D3 receptor subtype. Alternatively, possible adaptive mechanisms taking place in D3-/- mice might have compensated for the absence of DA D3 receptors.

Effect of the delta-aminolevulinate dehydratase polymorphism on the accumulation of lead in bone and blood in lead smelter workers.

Lead inhibition of the zinc metalloenzyme delta-aminolevulinate dehydratase (ALAD) is one of the most sensitive indicators of blood lead levels. ALAD is polymorphic, with about 20% of Caucasians expressing the rarer ALAD2 allele. Previous studies indicated that this polymorphism may be a genetic factor in lead transport, metabolism, and/or distribution. Whole blood lead, serum lead, and ALAD genotype were determined for 381 lead smelter workers, including 70 workers expressing the ALAD2 allele, whose blood lead elevations were observed for more than 20 years of employment. The same employees demonstrated higher serum lead levels. Using a cumulative blood lead index (CBLI) for each worker, based on individual blood lead histories, and in vivo X-ray fluorescence measurements of bone lead to estimate total lead body burden, the slopes of linear relations of bone lead to CBLI were greater for workers homoallelic for ALAD1, indicating more efficient uptake of lead from blood into bone. This effect was most significant in calcaneus bone and for workers hired since 1977 [ALAD1-1: 0.0528 +/- 0.0028 and ALAD1-2 or 2-2: 0.0355 +/- 0.0031 (P < 0.001)]. Decreased transfer of blood lead into bone in individuals expressing the ALAD2 allele contrasted with increased blood lead. Thus the ALAD genotype affected lead metabolism and potentially modified lead delivery to target organs including the brain; however, the ALAD genotype did not significantly affect the net accumulation of lead in bone.

Accumulated body burden and endogenous release of lead in employees of a lead smelter.

Bone lead levels for 367 active and 14 retired lead smelter workers were measured in vivo by X-ray fluorescence in May-June 1994. The bone sites of study were the tibia and calcaneus; magnitudes of concentration were used to gauge lead body burden. Whole blood lead readings from the workers generated a cumulative blood lead index (CBLI) that approximated the level of lead exposure over time. Blood lead values for 204 of the 381 workers were gathered from workers returning from a 10-month work interruption that ended in 1991; their blood level values were compared to their tibia and calcaneus lead levels. The resulting relations allowed constraints to be placed on the endogenous release of lead from bone in smelter works. Calcaneus lead levels were found to correlate strongly with those for tibia lead, and in a manner consistent with observations from other lead industry workers. Relations between bone lead concentration and CBLI demonstrated a distinctly nonlinear appearance. When the active population was divided by date of hire, a significant difference in the bone lead-CBLI slope emerged. After a correction to include the component of CBLI existing before the workers' employment at the smelter was made, this difference persisted. This implies that the transfer of lead from blood to bone in the workers has changed over time, possibly as a consequence of varying exposure conditions.

Locomotor sensitization and decrease in [3H]mazindol binding to the dopamine transporter in the nucleus accumbens are delayed after chronic treatments by GBR12783 or cocaine.

Rats were treated once daily for 15 consecutive days with either cocaine or the specific dopamine uptake inhibitor 1-[2- (diphenylmethoxy)ethyl]-4-(3-phenyl-2-(propenyl)-piperazine (GBR12783) at a dose (10 mg/kg) that given acutely increases locomotor activity. Two or 14 days after the last administration, the motor stimulant responses of rats to a challenge dose (5 mg/kg) of the drug administered previously were compared with the motor stimulant responses of rats daily injected with solvent. A sensitization to the acute stimulant locomotor effect of these drugs was only observed 14 days after cessation of chronic treatments. After this withdrawal period, autoradiographic analysis revealed a significant decrease in the desipramine-insensitive [3H]mazindol binding to the dopamine transporter in the shell of the nucleus accumbens. No change was noticed in other regions with high dopamine content: core of nucleus accumbens, striatum, olfactory tubercle, substantia nigra and ventral tegmental area. Absence of concomitant decrease in [3H]dihydrotetrabenazine labeling, which indicates lack of effect on vesicular monoamine transporters, suggests that the decrease in accumbal [3H]mazindol binding did not result from a cytotoxic effect on corresponding dopamine neurons. In addition, 14 days after the last administration of GBR12783, the levels of dopamine and metabolites (dihydroxy-phenylacetic acid, homovanillic acid) and the ability of acute GBR12783 to synergize with haloperidol-induced increase in these metabolites were not modified either in the whole nucleus accumbens or in the striatum.

Locomotor sensitization to [D-Trp(11)]neurotensin after repeated injections of the dopamine uptake inhibitor GBR12783 in rats.

Rats received one daily i.p. injection of the dopamine uptake inhibitor GBR12783 (1-[2-(diphenylmethoxy)ethyl]4-(3-phenyl-2-(propenyl)-piperazine) (10mg/kg) or vehicle for 9 days. Fourteen days after discontinuing treatment, their locomotor activity was assessed after injection of GBR12783 (5 mg/kg) or vehicle, then 6 days later, after i.c.v. injection of [D-Trp(11)]neurotensin (750 ng) or saline. A sensitization to the stimulant locomotor effects of both GBR12783 and [D-Trp(11)]neurotensin occurred in rats exposed to the actimeter following the 1st, 5th and 9th injections of GBR12783. Rats without prior experience of the activity cages before the challenge tests showed no sensitization to either GBR12783 or [D-Trp(11)] neurotensin. Our data suggest that a similar mechanism may underlie the locomotor sensitization to GBR12783 and the heterosensitization to [D-Trp(11)]neurotensin.

Evaluation of a new non-isotopic sandwich hybridization for the detection of HIV1-specific PCR products.

A new non-isotopic sandwich hybridization assay was developed to detect human immunodeficiency virus type 1 (HIV1) provirus amplified by the polymerase chain reaction. The sensitivity and specificity of this new technique using 96-well microplates as the support for the sandwich hybridization procedure and stable enzyme-linked oligomer as the detection probe were compared with those of Southern hybridization using a 32P-labelled oligomer probe. Three laboratories studied 437 peripheral blood mononuclear cell samples from 294 different subjects including both HIV1-seropositive and -seronegative individuals. The non-isotopic assay exhibited a sensitivity of 99.5% and a specificity of 99.1% when compared with the Southern procedure. In addition, the non-isotopic assay gives clear numeric data, is safe when handling, and is especially adapted to large-scale analyses.

Invariance of the density of dopamine uptake sites and dopamine metabolism in the rat brain after a chronic treatment with the dopamine uptake inhibitor GBR 12783.

A chronic treatment (10 mg/kg, twice daily during 9 days) with the dopamine uptake inhibitor GBR 12783 was performed in rats at a dose increasing their locomotor activity. Forty-eight hours after the last administration, animals were sacrificed and 3H mazindol binding was performed on brain slices. Autoradiographic analysis revealed no change in this binding relatively to control animals in regions with high dopamine contents: striatum, nucleus accumbens, olfactory tubercle, substantia nigra and ventral tegmentum area. The treatment did not either modify the levels of dopamine (DA) and metabolites (HVA, DOPAC) both in the striatum and the nucleus accumbens. Thus, early after the end of the treatment, the chronic blockade of the dopamine uptake complex regulates neither the dopamine uptake complex nor the dopamine metabolism.