RESUMO
Thiamin pyrophosphate (TPP) is an essential co-factor in amino acid and carbohydrate metabolic pathways. The TPP-related vitamin B1 biosynthetic pathway is found in most bacterial, plant and lower eukaryotic processes; however, it is not present in humans. In bacterial thiamin synthesis and salvage pathways, the 5-(hydroxyethyl)-methylthiazole kinase (ThiM) is essential in the pathway forming TPP. Thus, ThiM is considered to be an attractive antibacterial drug target. Here, we determined the crystal structures of ThiM from pathogenic Klebsiella pneumoniae (KpThiM) and KpThiM in complex with its substrate 5-(hydroxyethyl)-4-methylthiazole (TZE). KpThiM, consisting of an α-ß-α domain, shows a pseudosymmetric trimeric formation. TZE molecules are located in the interface between the KpThiM subunits in the trimer and interact with Met49 and Cys200. Superimposition of the apo and TZE-complexed structures of KpThiM show that the side chains of the amino acids interacting with TZE and Mg2+ have a rigid configuration. Comparison of the ThiM structures shows that KpThiM could, in terms of sequence and configuration, be different from other ThiM proteins, which possess different amino acids that recognize TZE and Mg2+. The structures will provide new insight into the ThiM subfamily proteins for antibacterial drug development.
Assuntos
Proteínas de Bactérias/metabolismo , Clormetiazol/análogos & derivados , Klebsiella pneumoniae/metabolismo , Proteínas Quinases/metabolismo , Tiamina/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Vias Biossintéticas , Clormetiazol/química , Clormetiazol/metabolismo , Cristalografia por Raios X , Humanos , Infecções por Klebsiella/microbiologia , Klebsiella pneumoniae/química , Modelos Moleculares , Conformação Proteica , Proteínas Quinases/química , Multimerização Proteica , Especificidade por SubstratoRESUMO
BACKGROUND: Synaptic dysfunction is a key event in pathogenesis of neurodegenerative diseases such as Alzheimer's disease (AD) where synapse loss pathologically correlates with cognitive decline and dementia. Although evidence suggests that aberrant protein production and aggregation are the causative factors in familial subsets of such diseases, drugs singularly targeting these hallmark proteins, such as amyloid-ß, have failed in late stage clinical trials. Therefore, to provide a successful disease-modifying compound and address synaptic dysfunction and memory loss in AD and mixed pathology dementia, we repurposed a clinically proven drug, CMZ, with neuroprotective and anti-inflammatory properties via addition of nitric oxide (NO) and cGMP signaling property. RESULTS: The novel compound, NMZ, was shown to retain the GABAA potentiating actions of CMZ in vitro and sedative activity in vivo. Importantly, NMZ restored LTP in hippocampal slices from AD transgenic mice, whereas CMZ was without effect. NMZ reversed amnestic blockade of acetylcholine receptors by scopolamine as well as NMDA receptor blockade by a benzodiazepine and a NO synthase inhibitor in the step-through passive avoidance (STPA) test of learning and working memory. A PK/PD relationship was developed based on STPA analysis coupled with pharmacokinetic measures of drug levels in the brain: at 1 nM concentration in brain and plasma, NMZ was able to restore memory consolidation in mice. CONCLUSION: Our findings show that NMZ embodies a promising pharmacological approach targeting synaptic dysfunction and opens new avenues for neuroprotective intervention strategies in mixed pathology AD, neurodegeneration, and dementia.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Clormetiazol/análogos & derivados , Reposicionamento de Medicamentos/métodos , Agonistas de Receptores de GABA-A/farmacologia , Hipocampo/efeitos dos fármacos , Potenciação de Longa Duração/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Nootrópicos/farmacologia , Animais , Proteína de Ligação a CREB/metabolismo , GMP Cíclico/metabolismo , Modelos Animais de Doenças , Agonistas de Receptores de GABA-A/farmacocinética , Masculino , Camundongos , Camundongos Transgênicos , Fármacos Neuroprotetores/farmacocinética , Óxido Nítrico/metabolismo , Nootrópicos/farmacocinética , Transdução de Sinais/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Sinapses/patologia , Xenopus laevisRESUMO
BACKGROUND AND PURPOSE: Chlormethiazole (CMZ), a clinical sedative/anxiolytic agent, did not reach clinical efficacy in stroke trials despite neuroprotection demonstrated in numerous animal models. Using CMZ as a lead compound, neuroprotective methiazole (MZ) analogues were developed, and neuroprotection and GABA(A) receptor dependence were studied. EXPERIMENTAL APPROACH: Eight MZs were selected from a novel library, of which two were studied in detail. Neuroprotection, glutamate release, intracellular calcium and response to GABA blockade by picrotoxin were measured in rat primary cortical cultures using four cellular models of neurodegeneration. GABA potentiation was assayed in oocytes expressing the α1ß2γ2 GABA(A) receptor. KEY RESULTS: Neuroprotection against a range of insults was retained even with substantial chemical modification. Dependence on GABAA receptor activity was variable: at the extremes, neuroprotection by GN-28 was universally sensitive to picrotoxin, while GN-38 was largely insensitive. In parallel, effects on extracellular glutamate and intracellular calcium were associated with GABA(A) dependence. Consistent with these findings, GN-28 potentiated α1ß2γ2 GABA(A) function, whereas GN-38 had a weak inhibitory effect. Neuroprotection against moderate dose oligomeric Aß1â42 was also tolerant to structural changes. CONCLUSIONS AND IMPLICATIONS: The results support the concept that CMZ does not contain a single pharmacophore, rather that broad-spectrum neuroprotection results from a GABA(A)-dependent mechanism represented by GN-28, combined with a mechanism represented in GN-38 that shows the least dependence on GABA(A) receptors. These findings allow further refinement of the neuroprotective pharmacophore and investigation into secondary mechanisms that will assist in identifying MZ-based compounds of use in treating neurodegeneration.
Assuntos
Clormetiazol/análogos & derivados , Clormetiazol/farmacologia , Agonistas GABAérgicos/farmacologia , Degeneração Neural/prevenção & controle , Fármacos Neuroprotetores/farmacologia , Receptores de GABA-A/efeitos dos fármacos , Animais , Disponibilidade Biológica , Encéfalo/metabolismo , Cálcio/metabolismo , Hipóxia Celular , Cromatografia Líquida de Alta Pressão , Agonistas de Aminoácidos Excitatórios/toxicidade , Feminino , Glucose/deficiência , Ácido Glutâmico/metabolismo , Ácido Glutâmico/toxicidade , Camundongos , Camundongos Endogâmicos C57BL , N-Metilaspartato/toxicidade , Neurônios/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Gravidez , Cultura Primária de Células , Ratos , Ratos Sprague-Dawley , Xenopus laevis , Ácido gama-Aminobutírico/farmacologiaRESUMO
OBJECTIVE: Clomethiazole is virtually completely eliminated by hepatic metabolism. This study was designed to assess the impact of liver impairment on its elimination and sedative effects. METHODS: Eight patients with mild liver impairment (Child-Pugh grade A), eight patients with moderate/severe liver impairment (Child-Pugh grade B/C) and eight healthy subjects of similar age were given 68 mg/kg clomethiazole edisilate according to a 24-h infusion scheme aimed at producing minimum sedation as it was intended for clinical use in patients with stroke. Concentrations of clomethiazole and its active alpha-carbon hydroxylated metabolite NLA-715 were followed in plasma and urine for 96 h and 24 h, respectively. Sedation was monitored using a scale from 1 to 6. RESULTS: The fraction excreted unchanged in urine was less than 0.2% for clomethiazole and less than 0.4% for NLA-715. Urine concentrations of clomethiazole were strongly correlated (r(2)=0.60) to plasma concentrations and approximately equal to unbound plasma concentrations. Plasma levels of NLA-715 increased steadily during the infusion, eventually reaching mean levels exceeding those of clomethiazole in all groups. Plasma clearance of clomethiazole in subjects with mildly impaired liver function was not statistically different from that of healthy controls (40 l/h vs 44 l/h). In subjects with moderate/severe liver impairment, there was a 50% reduction in clearance. Sedation was not observed except in two subjects in the Child-Pugh A group showing mild sedation. CONCLUSION: The reduced clomethiazole clearance in patients with moderate/severe liver impairment seems to call for a reduction of clomethiazole dosage. However, sedation was not observed in this group at the investigated dose level.
Assuntos
Clormetiazol/análogos & derivados , Clormetiazol/farmacologia , Clormetiazol/farmacocinética , Hipnóticos e Sedativos/farmacologia , Hipnóticos e Sedativos/farmacocinética , Hepatopatias/metabolismo , Área Sob a Curva , Cafeína/farmacocinética , Clormetiazol/efeitos adversos , Relação Dose-Resposta a Droga , Feminino , Meia-Vida , Humanos , Hipnóticos e Sedativos/efeitos adversos , Verde de Indocianina/farmacocinética , Infusões Intravenosas , Rim/metabolismo , Rim/fisiopatologia , Hepatopatias/fisiopatologia , Masculino , Pessoa de Meia-Idade , Fármacos Neuroprotetores/efeitos adversos , Fármacos Neuroprotetores/farmacocinética , Fármacos Neuroprotetores/farmacologia , Tiazóis/sangue , Tiazóis/urina , Fatores de TempoRESUMO
We have investigated the neuroprotective properties of AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl-1-phenyl-methylamine], a novel compound structurally related to clomethiazole. Administration (i.p.) of (+/-)-AR-A008055 60 min after 5 min of global cerebral ischaemia in gerbils produced a dose-dependent protection of the hippocampus from damage. Both enantiomers [(R)-(+)-AR-A008055 and (S)-(-)- AR-A008055] at 600 micromol/kg produced similar protection to that following clomethiazole (600& micromol/kg) and both produced similar and sustained neuroprotection, at 4, 7 and 21 days post-insult. When infused intravenously over a 2-h period, both enantiomers produced concentration-dependent neuroprotection, with the enantiomers providing similar protection at every plasma concentration (50-200 nmol/ml). The efficacy of (S)-(-)-AR-A008055 was similar to clomethiazole, but it was slightly less potent. Ischaemia-induced glutamate efflux from rat brain cortical prisms in vitro was inhibited by both isomers (100 microM). The inhibitory effect of (R)-(+)-AR-A008055 was blocked by bicuculline (10 microM) and picrotoxin (100 microM), while the effect of (S)-(-)-AR-A008055 was only antagonised by picrotoxin. This indicated that (S)-(-)-AR-A008055, like clomethiazole, is able to open the GABA(A)-chloride channel in the absence of endogenous GABA. (R)-(+)-AR-A008055 was more potent than (S)-(-)-AR-A008055 in enhancing the concentration of GABA in the medium following 30 min exposure of tissue to the ischaemic conditions, suggesting that it is an effective GABA uptake inhibitor. This action may explain both its effect on glutamate efflux in vitro and its neuroprotective effect in vivo.
Assuntos
Isquemia Encefálica/metabolismo , Clormetiazol/análogos & derivados , Clormetiazol/farmacologia , Ácido Glutâmico/metabolismo , Hipocampo/efeitos dos fármacos , Metilaminas/farmacologia , Fármacos Neuroprotetores/farmacologia , Tiazóis/farmacologia , Ácido gama-Aminobutírico/metabolismo , Animais , Feminino , Gerbillinae , Hipocampo/metabolismo , Masculino , Fármacos Neuroprotetores/química , Ratos , Ratos Wistar , Acidente Vascular Cerebral/metabolismoRESUMO
AR-A008055 [(+/-)-1-(4-methyl-5-thiazolyl)-1-phenylmethylamine] is structurally related to clomethiazole and has been used to probe the mechanism of the neuroprotective effect of clomethiazole. Clomethiazole, (+/-)-AR-A008055 and (S)-(-)-AR-A008055 all displaced [35S]-t-butyl-bicyclophosphorothionate ([35S]TBPS) from rat cerebral cortex tissue (IC50 values: GABA, 8.1+/-0.04 microM; clomethiazole, 130+/-30 microM; (+/-)-AR-A008055, 494+/-7 microM; (S)-(-)-AR-A008055, 221+/-14 microM. (R)-(+)-AR-A008055 was without significant effect (IC50>1000 microM). None of the compounds interacted with NMDA or AMPA receptors or with sodium or calcium (N, P/Q) channels. Brain penetration of both enantiomers following their i.p. administration was excellent, with brain and plasma concentrations being similar. Clomethiazole dose-dependently inhibited spontaneous locomotor activity in rats and was approximately 10 times more sedative than either enantiomer of AR-A008055. Clomethiazole was more potent than (S)-(-)-AR-A008055 in the "pull-up" test (muscle relaxation) and in producing loss of righting reflex, while (R)-(+)-AR-A008055 had little effect. The time animals remained on a Rota-rod was of the order: clomethiazole<(S)-(-)-AR-A008055<(R)-(+)-AR-A008055. (S)-(-)-AR-A008055 (210 micromol/kg) raised seizure threshold to pentylenetetrazole (i.v.) by 119+/-21%. The (R)-(+)- enantiomer was not anticonvulsant. Overall, (S)-(-)-AR-A008055 exhibited a similar pharmacology to clomethiazole. However, its sedative and muscle relaxant effects were substantially less than clomethiazole, emphasising that these properties are not directly related to neuroprotective efficacy. The current data suggest that the proposed GABA uptake inhibitory property of (R)-(+)-AR-A008055 fails to produce significant sedative, myorelaxant or anticonvulsant activity.
Assuntos
Encéfalo/efeitos dos fármacos , Clormetiazol/farmacologia , Moduladores GABAérgicos/farmacologia , Metilaminas/farmacologia , Atividade Motora/efeitos dos fármacos , Relaxamento Muscular/efeitos dos fármacos , Receptores de GABA-A/metabolismo , Tiazóis/farmacologia , Animais , Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Encéfalo/metabolismo , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Clormetiazol/análogos & derivados , Clormetiazol/sangue , Convulsivantes/metabolismo , Relação Dose-Resposta a Droga , Moduladores GABAérgicos/sangue , Moduladores GABAérgicos/química , Hipnóticos e Sedativos/química , Hipnóticos e Sedativos/farmacologia , Masculino , Atividade Motora/fisiologia , Relaxantes Musculares Centrais/química , Relaxantes Musculares Centrais/farmacologia , Relaxamento Muscular/fisiologia , RatosRESUMO
A single dose of clomethiazole (600 micromol kg(-1) i.p.) has previously been shown to be neuroprotective in the gerbil model of global ischaemia. In gerbils, clomethiazole (600 micromol kg(-1)) injection produced a rapid appearance (peak within 5 min) of drug in plasma and brain and similar clearance (plasma t(1/2): 40 min) from both tissues. The peak brain concentration (226+/-56 nmol g(-1)) was 40% higher than plasma. One major metabolite, 5-(1-hydroxyethyl-2-chloro)-4-methylthiazole (NLA-715) and two minor metabolites 5-(1-hydroxyethyl)-4-methylthiazole (NLA-272) and 5-acetyl-4-methylthiazole (NLA-511) were detected in plasma and brain. Evidence suggested that clomethiazole is metabolized directly to both NLA-715 and NLA-272. Injection of NLA-715, NLA-272 or NLA-511 (each at 600 micromol kg(-1)) produced brain concentrations respectively 2.2, 38 and 92 times greater than seen after clomethiazole (600 micromol kg(-1)). Clomethiazole (600 micromol kg(-1)) injected 60 min after a 5 min bilateral carotid artery occlusion in gerbils attenuated the ischaemia-induced degeneration of the hippocampus by approximately 70%. The metabolites were not neuroprotective at this dose. In mice, clomethiazole (600 micromol kg(-1)) produced peak plasma and brain concentrations approximately 100% higher than in gerbils, drug concentrations in several brain regions were similar but 35% higher than plasma. Clomethiazole (ED(50): 180 micromol kg(-1)) and NLA-715 (ED(50): 240 micromol kg(-1)) inhibited spontaneous locomotor activity. The other metabolites were not sedative (ED(50) >600 micromol kg(-1)). These data suggest that the neuroprotective action of clomethiazole results from an action of the parent compound and that NLA-715 contributes to the sedative activity of the drug. British Journal of Pharmacology (2000) 129, 95 - 100
Assuntos
Clormetiazol/farmacologia , Clormetiazol/farmacocinética , Hipnóticos e Sedativos/farmacologia , Fármacos Neuroprotetores/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/patologia , Clormetiazol/análogos & derivados , Clormetiazol/sangue , Clormetiazol/metabolismo , Gerbillinae , Masculino , Atividade Motora/efeitos dos fármacos , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Tiazóis/sangue , Tiazóis/metabolismoAssuntos
Anticonvulsivantes/toxicidade , Clormetiazol/análogos & derivados , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/congênito , Hipnóticos e Sedativos/toxicidade , Animais , Animais Recém-Nascidos , Clormetiazol/toxicidade , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/fisiopatologia , Feminino , Masculino , Gravidez , Ratos , Fatores SexuaisAssuntos
Clormetiazol/análogos & derivados , Delírio/induzido quimicamente , Síndrome de Abstinência a Substâncias , Transtornos de Ansiedade/tratamento farmacológico , Clormetiazol/administração & dosagem , Clormetiazol/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , AutoadministraçãoRESUMO
An improved high-performance liquid chromatographic method for the determination of chlormethiazole levels in plasma is described. The drug is extracted from plasma using commercially available reversed-phase extraction columns; recovery values obtained using Sep-Pak C18 and Bond Elut C1, C2, C4, C6, C8, C18 columns are compared. Separation was achieved by reversed-phase chromatography, using a mobile phase consisting of 0.025 M sodium acetate buffer, pH 4.5-acetonitrile (67:33) at a flow-rate of 1.6 ml/min in conjunction with a 15-cm Jones Chromatography Apex ODS column. The analytical column was protected by a Waters Assoc. Guard-Pak module containing a Guard-Pak CN insert. Using ultraviolet detection at 254 nm chlormethiazole levels in the region of 50 ng/ml can be measured with only 500 microliter of plasma.
Assuntos
Clormetiazol/sangue , Clormetiazol/análogos & derivados , Cromatografia Líquida de Alta Pressão , Feminino , Sangue Fetal/análise , Humanos , Indicadores e Reagentes , Recém-Nascido , Nitrazepam/sangueRESUMO
A clinical analysis of 476 cases of delirium tremens is presented. The sample covers virtually all cases of delirium tremens that occurred in the Helsingborg Health Service District during the period 1975-1980. A retrospective analysis of the incidence of delirium tremens over the observation period shows that there was an increase in the number and severity of cases developing delirium tremens when non-cross-tolerant drugs, such as carbamazepine and neuroleptics, were used as first-line treatment for acute alcohol withdrawal. The results also show that when chlormethiazole (a drug exhibiting a certain degree of cross-tolerance with ethanol) was used either as the first-line treatment for all cases or as the first-line treatment for predetermined "high risk" patients, the incidence of delirium tremens was greatly reduced. The favourable findings when chlormethiazole treatment was initiated at an early stage in the "high risk" patients suggest that chlormethiazole has a protective action against delirium tremens. Moreover, in the occasional case in which delirium tremens occurred during chlormethiazole treatment, the disorder was milder and relatively easy to treat.
Assuntos
Delirium por Abstinência Alcoólica/prevenção & controle , Clormetiazol/uso terapêutico , Psicoses Alcoólicas/prevenção & controle , Alcoolismo/reabilitação , Clormetiazol/análogos & derivados , Esquema de Medicação , Quimioterapia Combinada , Tolerância a Medicamentos , Humanos , Estudos Retrospectivos , SuéciaRESUMO
The effect of hemineurin on the clinical picture and the course of severe forms of delirium tremens was studied over time in 28 male patients who were simultaneously examined for the enzymic activity of the liver and parameters of the cholinergic system. The activity of alanine (AlAT) and aspartate aminotransferases (AsAT), sorbite dehydrogenase (SorDH), fructose-1-phosphate aldolase, blood acetylcholine (AC) levels, the activity of acetylcholine esterase in the whole blood (ACWB) and acetylcholine esterase of red blood cells (ACRC) determined in the course of treatment showed that hemineurin, in addition to a marked sedative effect, contributed to rapid normalization of liver function and carbohydrate metabolism. The effect of hemineurin helped to restore the synthesis and acetylation of CoA, to stimulate AC metabolism, and to set a relative balance of the mediator systems, ensuring the prevention of dangerous complications (acute cardiovascular insufficiency, cerebral edema) in severe cases of delirium tremens.
Assuntos
Delirium por Abstinência Alcoólica/tratamento farmacológico , Delirium por Abstinência Alcoólica/fisiopatologia , Clormetiazol/análogos & derivados , Fígado/fisiopatologia , Sistema Nervoso Parassimpático/fisiopatologia , Psicoses Alcoólicas/tratamento farmacológico , Psicoses Alcoólicas/fisiopatologia , Acetilcolina/sangue , Acetilcolinesterase/sangue , Adulto , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Clormetiazol/uso terapêutico , Frutose-Bifosfato Aldolase/sangue , Humanos , L-Iditol 2-Desidrogenase/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
Dopamine (DA) sensitivity, assessed through maximal growth hormone (GH) response to stimulation by apomorphine (APO) (0.18-0.24 mg iv) was studied in 16 chronic alcoholics newly admitted after a period of heavy alcohol intake. Repeated hormonal tests were thereafter performed during a 2-month period under strictly controlled conditions to avoid relapse into alcohol consumption. Eight healthy volunteers with alcohol consumption slightly less than that of the general population were used as controls. It was found that DA sensitivity in the early abstinence phase was higher than later in the 2-month recovery period but not significantly different from control values. The relatively higher DA sensitivity in the early abstinence phase might be responsible for a lower threshold for psychotic symptoms and neuroleptic-induced extrapyramidal side effects. The results of this study give further evidence of a prolonged recovery phase after heavy alcohol intake.
Assuntos
Alcoolismo/fisiopatologia , Hormônio do Crescimento/metabolismo , Adeno-Hipófise/fisiopatologia , Receptores Dopaminérgicos/fisiologia , Adulto , Alcoolismo/tratamento farmacológico , Ansiolíticos/uso terapêutico , Anticonvulsivantes/uso terapêutico , Apomorfina , Benzodiazepinas , Clormetiazol/análogos & derivados , Clormetiazol/uso terapêutico , Etanol/efeitos adversos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Síndrome de Abstinência a Substâncias/fisiopatologiaRESUMO
The dynamics of the interaction of clomethiazole edisylate (1) with polyvinyl chloride and cellulose propionate, the main plastics used in the manufacture of infusion bags and sets, was examined. An experimental system in which the plastic was either open or closed to the environment was used to determine the relative contribution of the sorption and permeation processes to loss from solutions of clomethiazole edisylate (I) in contact with the plastic infusion systems. Sorption by the plastic infusion materials accounted for most of the drug loss, while permeation into the external environment accounted for the remainder. The sorption and permeation into and through polyvinyl chloride was temperature dependent. The diffusion coefficient and permeation rate constant both increased with temperature, while the polyvinyl chloride-water partition coefficients were independent of temperature. The activation energy for the diffusion in polyvinyl chloride was 13.5 kcal/mol. The permeability of the infusion bag plastic and the evaporation across an unstirred air boundary layer adjacent to the external surface of the plastic both appeared to contribute to the overall diffusional resistance encountered in the permeation process. The plastic-water partition coefficients are independent of initial concentration, suggesting that the concentration-dependent loss of the drug from solutions stored in plastic infusion bags and burets is a result of the greater diffusivity of the drug in the plastic at the higher initial concentrations. Plasticization of the polymers by the drug is indicated by the increase in the diffusivity of the drug in polyvinyl chloride and cellulose propionate, the increase in the rate and extent of sorption of a radiolabeled marker (diazepam) by the plastic, and the decreased stiffness of polyvinyl chloride exposed to higher concentrations of the drug.