RESUMO
Analysis of pharmaceutical compounds in cerebrospinal fluid (CSF) may present challenges due to the combination of the low protein content in this matrix and relatively low drug concentrations, often corresponding to free drug concentrations in plasma, typically found in CSF. A 30% loss of AMG 579 was observed during preparation of quality control samples and further investigation determined that this loss was likely due to binding to collection tubes. This observation also highlighted the possibility of additional losses of AMG 579 that could occur during collection of clinical samples, such as binding to catheters used in the collection of CSF. Loss of AMG 579 in QC samples was reduced from 30% to 5% when the volume of CSF stored in 1.5 mL vials was increased from 0.06 mL to 1 mL. Modest but unavoidable losses of about 20% of AMG 579 were also found following perfusion through both silicone and polypropylene (Pharmed(®) BPT) collection catheters. Silicone tubing was used for CSF collection based on clinical site preference. An LC-MS/MS method was validated to quantify AMG 579 in human CSF to support clinical testing. The original range of the assay was 1-1000 ng/mL but the LLOQ was subsequently lowered to 0.1 ng/mL to better meet project requirements. Interday bias (% RE) and precision (% CV) were -4.2% and 12.3% at the LLOQ, and less than ± 0.9% and 8.3% for higher concentrations, respectively. The compound was stable in human CSF for at least 5h at room temperature, 55 days at -70 °C (-60 to -80 °C range), and through three freeze-thaw cycles. Careful selection of assay conditions and materials minimized losses of the compound during sample collection and storage. While these losses could not be entirely eliminated, practical sample collection and storage conditions were established to allow for analysis of AMG 579 in human clinical trials.
Assuntos
Benzimidazóis/líquido cefalorraquidiano , Cromatografia Líquida/métodos , Inibidores de Fosfodiesterase/líquido cefalorraquidiano , Pirazinas/líquido cefalorraquidiano , Espectrometria de Massas em Tandem/métodos , Humanos , Limite de Detecção , Manejo de Espécimes/métodos , TemperaturaRESUMO
Sensitisation to retinal S-antigen has been implicated in the pathogenesis of several clinical forms of posterior uveitis. S-antigen-like molecules have recently been demonstrated in the brain and choroid plexus of experimental animals. We used a panel of four monoclonal antibodies (MAbs), MAbF4-C1, MAbC10-C10, MAbA2-G5 and MAbA9-C6, which define specific epitopes in the amino, mid and carboxyl terminal portions of S-antigen in order to identify an S-antigen-like molecule in human choroid plexus and cerebrospinal fluid (CSF). Three MAbs, MAbF4-C1, MAbC10-C10 and MAbA9-C6, localised an S-antigen-like molecule to the cytoplasm of the epithelial cells of the human choroid plexus. Polymerase chain reaction of cDNA from choroid plexus verified the presence of S-antigen homologues in the choroid plexus. The presence of an S-antigen-like molecule in the CSF was demonstrated by western blots in seven CSF samples from patients with a variety of neuropathological disorders. It is proposed that immunological cross-reactivity and biochemical similarity between retinal S-antigen and an S-antigen-like molecule in human choroid plexus and CSF could form a basis for neurological manifestations observed in certain clinical forms of uveitides.