Development of a physiologically-based pharmacokinetic model of the rat central nervous system

Central nervous system (CNS) drug disposition is dictated by a drug’s physicochemical properties and its ability to permeate physiological barriers. The blood–brain barrier (BBB), blood-cerebrospinal fluid barrier and centrally located drug transporter proteins influence drug disposition within the central nervous system. Attainment of adequate brain-to-plasma and cerebrospinal fluid-to-plasma partitioning is important in determining the efficacy of centrally acting therapeutics. We have developed a physiologically-based pharmacokinetic model of the rat CNS which incorporates brain interstitial fluid (ISF), choroidal epithelial and total cerebrospinal fluid (CSF) compartments and accurately predicts CNS pharmacokinetics. The model yielded reasonable predictions of unbound brain-to-plasma partition ratio (Kpuu,brain) and CSF:plasma ratio (CSF:Plasmau) using a series of in vitro permeability and unbound fraction parameters. When using in vitro permeability data obtained from L-mdr1a cells to estimate rat in vivo permeability, the model successfully predicted, to within 4-fold, Kpuu,brain and CSF:Plasmau for 81.5% of compounds simulated. The model presented allows for simultaneous simulation and analysis of both brain biophase and CSF to accurately predict CNS pharmacokinetics from preclinical drug parameters routinely available during discovery and development pathways.

A low mortality, high morbidity Reduced Intensity Status Epilepticus (RISE) model of epilepsy and epileptogenesis in the rat

Animal models of acquired epilepsies aim to provide researchers with tools for use in understanding the processes underlying the acquisition, development and establishment of the disorder. Typically, following a systemic or local insult, vulnerable brain regions undergo a process leading to the development, over time, of spontaneous recurrent seizures. Many such models make use of a period of intense seizure activity or status epilepticus, and this may be associated with high mortality and/or global damage to large areas of the brain. These undesirable elements have driven improvements in the design of chronic epilepsy models, for example the lithium-pilocarpine epileptogenesis model. Here, we present an optimised model of chronic epilepsy that reduces mortality to 1% whilst retaining features of high epileptogenicity and development of spontaneous seizures. Using local field potential recordings from hippocampus in vitro as a probe, we show that the model does not result in significant loss of neuronal network function in area CA3 and, instead, subtle alterations in network dynamics appear during a process of epileptogenesis, which eventually leads to a chronic seizure state. The model’s features of very low mortality and high morbidity in the absence of global neuronal damage offer the chance to explore the processes underlying epileptogenesis in detail, in a population of animals not defined by their resistance to seizures, whilst acknowledging and being driven by the 3Rs (Replacement, Refinement and Reduction of animal use in scientific procedures) principles.

Characterization and effects on cAMP accumulation of adrenomedullin and calcitonin gene-related peptide (CGRP) receptors in dissociated rat spinal cord cell culture

1 Adrenomedullin (AM) and calcitonin gene-related peptide (CGRP) have structural similarities, interact with each others receptors (calcitonin receptor-like receptor (CLR)/receptor-activity-modifying proteins (RAMPs)) and show overlapping biological activities. AM and CGRP receptors are chiefly coupled to cAMP production. In this study, a method of primary dissociated cell culture was used to investigate the presence of AM and CGRP receptors and their effects on cAMP production in embryonic spinal cord cells. 2 Both neuronal and non-neuronal CLR immunopositive cells were present in our model. 3 High affinity, specific [ 125I]-AM binding sites (K(d) 79±9 pM and B(max) 571±34 fmol mg -1 protein) were more abundant than specific [ 125I]-CGRP binding sites (K(d) 12±0.7 pM and B(max) 32±2 fmol mg -1 protein) in embryonic spinal cord cells. 4 Specific [ 125I]-AM binding was competed by related molecules with a ligand selectivity profile of rAM>hAM(22-52)>rCGRPα>CGRP(8-37) ≫[r-(r*,s*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl] carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1, 4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-piperidinecarboxamide (BIBN4096BS). 5 Specific [ 125I]-CGRP binding was competed by rCGRPα>rAM≥ CGRP(8-37)≥BIBN4096BS>hAM(22-52). 6 Cellular levels of cAMP were increased by AM (pEC"5"0 10.2±0.2) and less potently by rCGRPα (pEC"5"0 8.9±0.4). rCGRPα-induced cAMP accumulation was effectively inhibited by CGRP(8-37) (pA"2 7.63±0.44) and hAM(22-52) (pA"2 6.18±0.21) while AM-stimulation of cAMP levels was inhibited by CGRP(8-37) (pA"2 7.41±0.15) and AM(22-52) (pA"2 7.26±0.18). BIBN4096BS only antagonized the effects of CGRP (pA"2 8.40±0.30) on cAMP accumulation. 7 These pharmacological profiles suggest that effects of CGRP are mediated by the CGRP"1 (CLR/RAMP1) receptor in our model while those of AM are related to the activation of the AM"1 (CLR/RAMP2) receptor subtype. © 2006 Nature Publishing Group All rights reserved.

Pharmacological characterization of a receptor for calcitonin gene-related peptide on rat, L6 myocytes

1 The L6 myocyte cell line expresses high affinity receptors for calcitonin gene-related peptide (CGRP) which are coupled to activation of adenylyl cyclase. The biochemical pharmacology of these receptors has been examined by radioligand binding or adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation. 2 In intact cells at 37 degrees C, human and rat alpha- and beta-CGRP all activated adenylyl cyclase with EC50s of about 1.5 nM. A number of CGRP analogues containing up to five amino acid substitutions showed similar potencies. In membrane binding studies at 22 degrees C in 1 mM Mg2+, the above all bound to a single site with IC50s of 0.1-0.4 nM. 3 The fragment CGRP(8-37) acted as a competitive antagonist of CGRP stimulation of adenylyl cyclase with a calculated Kd of 5 nM. The Kd determined in membrane binding assays was lower (0.5 nM). 4 The N-terminal extended human alpha-CGRP analogue Tyro-CGRP activated adenylyl cyclase and inhibited [125I]-iodohistidyl-CGRP binding less potently than human alpha-CGRP (EC50 for cyclase = 12 nM, IC50 for binding = 4 nM). 5 The pharmacological profile of the L6 CGRP receptor suggests that it most closely resembles sites on skeletal muscle, cardiac myocytes and hepatocytes. The L6 cell line should be a stable homogeneous model system in which to study CGRP mechanisms and pharmacology."

The development of a novel in vitro model suitable for the prediction of bioavailability

In vitro studies of drug absorption processes are undertaken to assess drug candidate or formulation suitability, mechanism investigation, and ultimately for the development of predictive models. This study included each of these approaches, with the aim of developing novel in vitro methods for inclusion in a drug absorption model. Two model analgesic drugs, ibuprofen and paracetamol, were selected. The study focused on three main areas, the interaction of the model drugs with co-administered antacids, the elucidation of the mechanisms responsible for the increased absorption rate observed in a novel paracetamol formulation and the development of novel ibuprofen tablet formulations containing alkalising excipients as dissolution promoters.Several novel dissolution methods were developed. A method to study the interaction of drug/excipient mixtures in the powder form was successfully used to select suitable dissolution enhancing exicipents. A method to study intrinsic dissolution rate using paddle apparatus was developed and used to study dissolution mechanisms. Methods to simulate stomach and intestine environments in terms of media composition and volume and drug/antacid doses were developed. Antacid addition greatly increased the dissolution of ibuprofen in the stomach model.Novel methods to measure drug permeability through rat stomach and intestine were developed, using sac methodology. The methods allowed direct comparison of the apparent permeability values obtained. Tissue stability, reproducibility and integrity was observed, with selectivity between paracellular and transcellular markers and hydrophilic and lipophilic compounds within an homologous series of beta-blockers.

Adrenoceptors and intestinal fluid and electrolyte transport in the rat

Noradrenaline was found to significantly stimulate fluid and Na absorption across everted sacs of rat jejunum. Of a number of a1, and 2-adrenoceptor antagonists tested only prazosin significantly inhibited the stimulant effect of noradrenaline and further experiments revealed an antiabsorptive effect of prazosin alone. Theophylline reduced jejunal fluid and Na absorption and this effect was not reversed by 2-adrenoceptor stimulation in contrast to previous findings in vivo. Evidence suggests the everted sac preparation is not appropriate to the study of intestinal fluid and electrolyte transport. The investigation of Jejunal ion transport in vitro was continued using an Ussing chamber preparation. Selective 2-adrenoceptor stimulation was found to depress electrogenic anion secretion, as neurotoxin tetrodotoxin indicated that this was a direct epithelial effect. 2-adrenoceptor agonists have considerable therapeutic value as antisecretory agents and the model of rat jejunum in vitro represents a convenient experimental model for research in this area. The selective 2-adrenoceptor antagonist ICI 118551 decreased basal SCC and inhibited increases in SCC in response to isoprenaline or salbutamol indicating the presence of a 2-adrenoceptor mechanism mediating both secretory tone and increases in secretory processes. Many intestinal secretagogues elicit electrolyte secretion via the stimulation of intramural secretory nervous pathways. If these pathways involve the activation of 2-adrenoceptorsthe 2-adrenoceptor antagonists may be useful in the treatment of diarrhoeal diseases. A single pass lumen perfusion technique was used to investigate possible sympathetic tone over colonic fluid and electrolyte absorption in the rat colon in vivo. The technique employed appeared to lack the necessary resolution for this study and alternative approaches are discussed

Aminoglycoside-induced changes in the renal handling of cations in the Fischer 344 rat

Disturbances of cation homeostasis, particularly hypomagnesaemia, are a frequent consequence of treatment with aminoglycoside antibiotics. These disturbances are thought to result from renal wasting of cations and administration of gentamicin to rats has been shown to produce hypercalciuria and hypermagnesiuria. The aims of this study were to attempt to elucidate these responses in anaesthetised rats infused with gentamicin and to use this model to investigate the mechanisms of these effects. Fischer 344 rats were anaesthetised and surgically prepared for clearance experiments. Infusion of gentamicin in isotonic saline increased urinary output of calcium and magnesium while sodium and potassium output were unaffected. These elevations in calcium and magnesium excretion were explained by reduced tubular reabsorption of these cations. Both the hypercalciuric and hypermagnesiuric responses to gentamicin were extremely rapid and were sustained during drug infusion; when gentamicin infusion ceased both responses were rapidly reversible. Infusion of another aminoglycoside, tobramycin, produced very similar effects to gentamicin. The hypercalciuria and hypermagnesiuria caused by gentimicin infusion were unaffected by parathyroidectomy. The peak increases in calcium and magnesium output brought about by infusion of gentamicin with frusemide were not significantly different to the increases produced by frusemide alone. The site at which gentamicin interferes with calcium and magnesium reabsorption cannot be firmly deduced from these results. However, the known close association between calcium and sodium reabsorption in the proximal tubule implies that gentamicin is unlikely to change proximal calcium reabsorption without a similar change in proximal sodium reabsorption. The similarity between the hypercalciuric and hypermagnesiuric effects of frusemide alone and the effects of frusemide infused simultaneously with gentamicin suggests that gentamicin may act at the same site as the diuretic, the thick ascending limb of the loop of Henle.

Tetrahydrobiopterin metabolism, neurotransmitters and behaviour in the rat

Various neurotoxins were investigated to assess their suitability for developing an animal model to study partial brain BH4 deficiency, neurotransmitters and behavioural alterations. Acute dosing with lead, diethylstilboestrol (DES), amphetamine and scopolamine produced no significant changes in rat brain BH4 metabolism though total biopterins in the liver were significantly reduced by lead and DES. Acute starvation of adult rats decreased brain biopterins. This loss of biopterins may be due to enhanced oxidative catabolism of the active cofactor caused by glutathione depletion. Dietary administration of a BH4 biosynthesis inhibitor, DAHP, consistently decreased brain total biopterins in weaner rats but did not alter the levels of DA, NA, 5-HT or metabolites. However the DAHP diet also induced a marked reduction in food intake. Rats subjected to an equivalent degree of food restriction without inhibitor showed significant but less severe reductions in brain biopterins and again no effect on transmitter levels. DAHP produced a significant decrease in locomotor activity and rearing. This could not be ascribed to reduction in food intake as animals subjected to just dietary restriction showed an increase in these activities. As gross brain levels of DA, NA and 5-HT were unaltered by DAHP the behavioural changes associated with the induced deficiency in brain total biopterins might not have been mediated through the action of these compounds. Although localised changes in neurotransmitter levels may have been obscured by gross analysis it is also possible that the behaviour changes were mediated by a role of BH4 not yet elucidated. Long-term administration of a high aluminium low calcium diet to mice produced no effect on gross brain total biopterins, catecholamines, serotonin or choline acetyltransferase activity though significant behavioural changes were observed.

An in-vitro-in-vivo model for the transdermal delivery of cholecalciferol for the purposes of rodent management

The natural selection of anticoagulant resistant rats has resulted in a need for an alternative to anticoagulant rodenticides which differs in both active ingredient and in the method of dosing. Cholecalciferol toxicity to rodents using the dermal route is demonstrated using a variety of penetration enhancing formulations in two in-vitro models and finally in-vivo. A 1 ml dose of 50/50 (v/v) DMSO/ethanol containing 15% (v/v) PEG 200 and 20% (w/v) cholecalciferol was judged as 'sufficiently effective' in line with the European Union's Biocidal Products Regulation (No. 528/2012) during in-vivo studies. This dose was found to cause 100% mortality in a rat population in 64.4 h (±22 h).

Rapid tonicity induced re-localisation of endogenous aquaporin 4 in primary rat astrocytes - a therapeutic target for cytotoxic brain oedema?

It is estimated that 69-75 million people worldwide will suffer a traumatic brain injury (TBI) or stroke each year. Brain oedema caused by TBI or following a stroke, together with other disorders of the brain cost Europe €770 billion in 2014. Aquaporins (AQP) are transmembrane water channels involved in many physiologies and are responsible for the maintenance of water homeostasis. They react rapidly to changes in osmolarity by transporting water through their highly selective central pore to maintain tonicity and aid in cell volume regulation. We have previously shown that recombinant AQP1-GFP trafficking occurs in a proteinkinase C-microtubule dependant manner in HEK-293 cells in response to hypotonicity. This trafficking mechanism is also reliant on the presence of calcium and its messenger-binding protein calmodulin and results in increased cell surface expression of AQP1 in a time-scale of ~30 seconds. There is currently very little research into the trafficking mechanisms of endogenous AQPs in primary cells. AQP4 is the most abundantly expressed AQP within the brain, it is localised to the astrocytic end-feet, in contact with the blood vessels at the blood-brain-barrier. In situations where the exquisitely-tuned osmotic balance is disturbed, high water permeability can become detrimental. AQP4-mediated water influx causes rapid brain swelling, resulting in death or long term brain damage. Previous research has shown that AQP4 knock-out mice were protected from the formation of cytotoxic brain oedema in a stroke model, highlighting AQP4 as a key drug target for this pathology. As there are currently no treatments available to restrict the flow of water through AQP4 as all known inhibitors are either cytotoxic or non-specific, controlling the mechanisms involved in the regulation of AQP4 in the brain could provide a therapeutic solution to such diseases. Using cell surface biontinylation of endogenous AQP4 in primary rat astrocytes followed by neutraavidin based ELISA we have shown that AQP4 cell surface localisation increases by 2.7 fold after 5 minutes hypotonic treatment at around 85 mOsm/kg H2O. We have also shown that this rapid relocalisation of AQP4 is regulated by PKA, calmodulin, extra-cellular calcium and actin. In summary we have shown that rapid translocation of endogenous AQP4 occurs in primary rat astrocytes in response to hypotonic stimuli; this mechanism is PKA, calcium, actin and calmodulin dependant. AQP4 has the potential to provide a treatment for the development of brain oedema.