Western equine encephalitis virus (WEEV) is an arboviral pathogen naturally found in North America. The primary disease phenotype associated with WEEV infection in susceptible hosts is a relatively long prodromal period followed by viral encephalitis. By contrast, in the current work, experimental inoculation of WEEV into the peritoneum of Syrian golden hamsters produced rapid death within approximately 96 h. It was determined that direct virus killing of lymphoid cells leads to death in WEEV-infected Syrian golden hamsters, and that inflammatory cytokines have the potential to enhance virus-induced lymphoid cell destruction. It was further concluded that WEEV retains its ability to cause encephalitis in Syrian golden hamsters, if hamsters survive the early stages of virus infection or if virus is introduced directly into the CNS. Death in WEEV-infected hamsters is associated with lymphonecrotic lesions in the absence of pathological lesions in the central nervous system (CNS). Few clinical parameters were altered by WEEV infection, with the exception of circulating lymphocyte numbers. Circulating lymphocyte numbers decreased dramatically during WEEV infection, and lymphopenia was identified as a consistent indicator of eventual death. Virus infection also increased serum concentrations of the cytokines interferon and tumor necrosis factor-alpha (TNF-alpha). Hamster peritoneal macrophages exposed to WEEV expressed TNF-alpha in a dose-responsive manner. Macrophage expression of TNF-alpha could be significantly inhibited by treatment of cells with anti-inflammatory agents flunixin meglumine (FM) or dexamethasone (Dex). Anti-inflammatory treatment also protected macrophages from cytotoxicity associated with exposure to WEEV. Treatment of WEEV-infected hamsters with either FM or Dex significantly improved survival compared to placebo-treated controls. WEEV induced cytotoxicity in hamster splenocytes exposed to WEEV in a virus dose-responsive manner. Supernatant from WEEV-exposed macrophages significantly enhanced WEEV killing of splenocytes. Hamsters that survived the early stages of WEEV infection occasionally developed signs of neurological disease and died approximately 6 to 9 d after virus inoculation. These animals had histopathological lesions in the CNS consistent with alphavirus-induced encephalitis. Inoculation of WEEV directly into the CNS caused apparent encephalitic disease. Death following CNS inoculation of WEEV was rapid and concurrent with histopathological lesions in the CNS similar to lesions seen in encephalitic hamsters following peripheral inoculation.

<p>The transport of drugs across the blood-brain barrier (BBB) has been investigated in different species using morphine and morphine-6-glucuronide (M6G) as model compounds. The influence of probenecid on the BBB transport of morphine and M6G was investigated, and the consequences of meningitis and severe brain injury on the concentrations of morphine in the brain were examined. All data were obtained by microdialysis, and data analysis using mathematical models was emphasised.</p><p>Morphine is exposed to active efflux at the BBB in rats, pigs and humans. In addition, the half-life of morphine is longer in the brain than in blood in these species. These interspecies similarities show the predictive potential of the two animal models for the BBB transport of morphine in humans. In the pig the exposure of the brain to morphine was higher in the presence of meningitis than when healthy. This was interpreted as a decrease in the active efflux and an increase in the passive diffusion over the injured BBB. In contrast, there was no significant difference in the concentrations of morphine in the “better” (uninjured) or the “worse” (injured) brain tissue in brain trauma patients. The extent of the transport across the BBB is similar for morphine and M6G. However, co-administration of probenecid only increased the brain concentrations of morphine, demonstrating that morphine and M6G are substrates for different efflux transporters at the BBB. An integrated model for the analysis of data obtained by microdialysis was developed. This model makes fewer assumptions about the recovery, the protein binding and the time of the dialysate observation than a previous model and traditional non-compartmental analysis and should, therefore, yield more reliable parameter estimates.</p><p>Knowledge of the consequences of efflux transporters and disease on the brain concentrations of a drug can be useful for individualising the dosing regimen in patients. </p>

<p>The pharmacokinetics and pharmacodynamics of oxycodone and morphine was investigated and related to the transport across the blood-brain barrier (BBB) in rats. The influence of a P-glycoprotein (P-gp) inhibitor on the plasma pharmacokinetics and pharmacodynamics of oxycodone was evaluated. Microdialysis experiments were conducted to evaluate the unbound pharmacokinetics, including the rate and extent of transport across the BBB, of oxycodone and morphine. Mathematical models were used to assess the pharmacokinetics and also the relationship between pharmacokinetics and pharmacodynamics of the drugs.</p><p>Oxycodone clearance, volume of distribution at steady-state, half-life, total brain tissue concentrations and tail-flick latency were all unaffected when a P-gp inhibitor was co-administered with oxycodone as compared to a control group. The lack of differences between the groups indicates that oxycodone BBB transport is not affected by P-gp inhibition. Investigating the unbound concentrations of oxycodone in brain and blood using microdialysis revealed an exciting finding. At steady-state, the unbound concentration in brain was 3 times higher than in blood (i.e. a K_p,uu of 3), indicating that active influx is involved in the BBB transport of oxycodone. In contrast, the K_p,uu of morphine was estimated to 0.56, which is an indication that active efflux mechanisms are involved in the BBB transport of morphine. This means that based on the same unbound concentration in blood, an approximately 6-fold higher unbound concentration of oxycodone compared to morphine will be reached in the brain. Using pharmacokinetic-pharmacodynamic modelling, the unbound brain concentrations of oxycodone and morphine were correlated to the tail-flick latency in vivo. The relative potency of the drugs was found to be concentration dependent with an infliction point of 55 nM.</p><p>In summary, this thesis emphasise the importance of taking the local brain pharmacokinetics into consideration when investigating the pharmacokinetics and the pharmacokinetic-pharmacodynamic relationships of centrally acting drugs.</p>

The transport of drugs across the blood-brain barrier (BBB) has been investigated in different species using morphine and morphine-6-glucuronide (M6G) as model compounds. The influence of probenecid on the BBB transport of morphine and M6G was investigated, and the consequences of meningitis and severe brain injury on the concentrations of morphine in the brain were examined. All data were obtained by microdialysis, and data analysis using mathematical models was emphasised. Morphine is exposed to active efflux at the BBB in rats, pigs and humans. In addition, the half-life of morphine is longer in the brain than in blood in these species. These interspecies similarities show the predictive potential of the two animal models for the BBB transport of morphine in humans. In the pig the exposure of the brain to morphine was higher in the presence of meningitis than when healthy. This was interpreted as a decrease in the active efflux and an increase in the passive diffusion over the injured BBB. In contrast, there was no significant difference in the concentrations of morphine in the “better” (uninjured) or the “worse” (injured) brain tissue in brain trauma patients. The extent of the transport across the BBB is similar for morphine and M6G. However, co-administration of probenecid only increased the brain concentrations of morphine, demonstrating that morphine and M6G are substrates for different efflux transporters at the BBB. An integrated model for the analysis of data obtained by microdialysis was developed. This model makes fewer assumptions about the recovery, the protein binding and the time of the dialysate observation than a previous model and traditional non-compartmental analysis and should, therefore, yield more reliable parameter estimates. Knowledge of the consequences of efflux transporters and disease on the brain concentrations of a drug can be useful for individualising the dosing regimen in patients.

The pharmacokinetics and pharmacodynamics of oxycodone and morphine was investigated and related to the transport across the blood-brain barrier (BBB) in rats. The influence of a P-glycoprotein (P-gp) inhibitor on the plasma pharmacokinetics and pharmacodynamics of oxycodone was evaluated. Microdialysis experiments were conducted to evaluate the unbound pharmacokinetics, including the rate and extent of transport across the BBB, of oxycodone and morphine. Mathematical models were used to assess the pharmacokinetics and also the relationship between pharmacokinetics and pharmacodynamics of the drugs. Oxycodone clearance, volume of distribution at steady-state, half-life, total brain tissue concentrations and tail-flick latency were all unaffected when a P-gp inhibitor was co-administered with oxycodone as compared to a control group. The lack of differences between the groups indicates that oxycodone BBB transport is not affected by P-gp inhibition. Investigating the unbound concentrations of oxycodone in brain and blood using microdialysis revealed an exciting finding. At steady-state, the unbound concentration in brain was 3 times higher than in blood (i.e. a Kp,uu of 3), indicating that active influx is involved in the BBB transport of oxycodone. In contrast, the Kp,uu of morphine was estimated to 0.56, which is an indication that active efflux mechanisms are involved in the BBB transport of morphine. This means that based on the same unbound concentration in blood, an approximately 6-fold higher unbound concentration of oxycodone compared to morphine will be reached in the brain. Using pharmacokinetic-pharmacodynamic modelling, the unbound brain concentrations of oxycodone and morphine were correlated to the tail-flick latency in vivo. The relative potency of the drugs was found to be concentration dependent with an infliction point of 55 nM. In summary, this thesis emphasise the importance of taking the local brain pharmacokinetics into consideration when investigating the pharmacokinetics and the pharmacokinetic-pharmacodynamic relationships of centrally acting drugs.

Cronobacter species cause serious infections such as meningitis and enteritis in newborns and neonates, with the major vehicle being contaminated powdered infant formula. The main objectives of this study were i) to identify potential virulence factors, such as enterotoxin production; ii) characterize the gene(s) involved in adhesion and invasion of the human brain microvascular endothelial cells (HBMEC); and iii) determine whether strains from clinical, food, and environmental sources differ in their ability to produce surface-attached bacterial aggregates, known as biofilms. Random transposon mutagenesis was used on strains demonstrating the best adherence and invasion to blood- brain barrier cell lines (BBB). Isogenic mutants were then screened for increased or decreased adherence and invasion. Screening of the transposon library identified one isogenic mutant of a clinical strain which lost the ability to adhere to BBB cells. The transposon rescue revealed the insertion site to be within a diguanylate cyclase (DGC) gene. The major function of DGC in many Gram-negative bacteria is to synthesize cyclic diguanylate (c-di-GMP), a secondary bacterial metabolite known for regulating biofilm formation, motility, and virulence or aspects of microbial pathogenicity. Based on the findings of this study, DGC appears to play an important role in Cronobacter species’ ability to produce biofilms and may also have a role of the pathogenicity in the microorganism.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

In vivo real-time data of ultrasound and microbubble assisted blood-brain barrier disruption is centrally based on low-resolution magnetic resonance images. Additional information can be gained using online microscopic monitoring. This study presents the first ever in vivo two-photon microscopy, four-dimensional data sets of ultrasound and microbubble assisted blood-brain barrier disruption. It characterized the threshold pressures and mechanical index needed to disrupt the vasculature with 800 kHz ultrasound, and found three different leakage constants from the compromised vasculature. Furthermore, using numerical models, an ultrasound array was designed and optimized to perform specifically with our two-photon microscope. It was fabricated, fully characterized, and its performance met both the required pressure field profile and the pressure values needed for our in vivo two-photon microscopy experiments. This array is an important step in microscopically characterizing ultrasound and microbubble assisted blood-brain barrier disruption.

Cerebral ischemia occurs when blood flow to the brain is insufficient to meet metabolic demand. This can result from cerebral artery occlusion that interrupts blood flow, limits CNS supply of oxygen and glucose, and causes an infarction/ischemic stroke. Ischemia initiates a cascade of molecular events in neurons and cerebrovascular endothelial cells including energy depletion, dissipation of ion gradients, calcium overload, excitotoxicity, oxidative stress, and accumulation of ions and fluid. Blood-brain barrier (BBB) disruption is associated with cerebral ischemia and leads to vasogenic edema, a primary cause of stroke-associated mortality. To date, only a single drug has received US Food and Drug Administration (FDA) approval for treatment of acute ischemia/reperfusion injury, recombinant tissue plasminogen activator (rt-PA). While rt-PA therapy restores perfusion to ischemic brain, considerable tissue damage occurs when cerebral blood flow is re-established. Therefore, there is a critical need for novel therapeutic approaches that can "rescue" salvageable brain tissue and/or protect BBB integrity during cerebral hypoxia and subsequent reoxygenation stress (H/R). One approach that may enable neural tissue rescue following H/R is CNS delivery of drugs with brain protective effects such as HMG-CoA reductase inhibitors (i.e., statins). Our present in vivo data demonstrates that atorvastatin, a commonly prescribed statin, attenuates poly (ADP-ribose) polymerase (PARP) cleavage in the brain following H/R, suggesting neuroprotective efficacy. However, atorvastatin use as a CNS therapeutic is limited by poor blood-brain barrier (BBB) penetration. Therefore, we examined regulation and functional expression of the known statin transporter Oatp1a4 at the BBB under H/R conditions. In rat brain microvessels H/R (6% O₂, 60 min followed by 21% O₂, 10 min) increased Oatp1a4 expression. Brain uptake of taurocholate (i.e., Oap1a4 probe substrate) and atorvastatin were reduced by Oatp inhibitors (i.e., estrone-3-sulfate, fexofenadine), suggesting involvement of Oatp1a4 in brain drug delivery. Pharmacological inhibition of TGF-β/ALK5 signaling with the selective inhibitor SB431542 increased Oatp1a4 functional expression, suggesting a role for TGF-β/ALK5 signaling in Oatp1a4 regulation. Taken together, our novel data show that targeting an endogenous BBB drug uptake transporter (i.e., Oatp1a4) may be a viable approach for optimizing CNS drug delivery for treatment of diseases with an H/R component.

Šiame darbe pristatomi mechanistiniai kiekybinio struktūros ir aktyvumo ryšio modeliai, skirti vaistinių junginių savybių, charakterizuojančių jų absorbciją ir pasiskirstymą organizme prognozavimui. Nagrinėjama keletas parametrų, apibūdinančių paprastos difuzijos per biologines membranas greitį, taip pat termodinaminės konstantos, aprašančios vaistų pasiskirstymą tarp kraujo plazmos ir audinių. Ląstelinių pernašos barjerų pralaidumas buvo modeliuojamas netiesinėmis lygtimis, siejančiomis paprastos difuzijos greitį su vaistų fizikocheminėmis savybėmis, tokiomis kaip lipofiliškumas, jonizacija, vandenilinių ryšių sudarymo potencialas ir molekulių dydis. Nustatyta, kad smegenų endotelyje ir žarnyno epitelyje stebima panašaus pobūdžio difuzijos greičio priklausomybė nuo jonizacijos – katijonai ir anijonai difunduoja atitinkamai 2 ir 3 eilėmis lėčiau už neutralias molekules. Pademonstruota, kad analizuojant vaistų pasiskirstymo tarp audinių ir kraujo duomenis, būtina paversti pradines eksperimentines vertes kitais dydžiais, atspindinčiais vaistų jungimosi prie plazmos ir audinių komponentų stiprumą. Vaistų giminingumas audiniams gali būti aprašytas jų lipofiliškumu, o neigiama jonizacijos įtaka stebima tik rūgštiniams junginiams. Taip pat parodyta, kad vaistų pernašos per hematoencefalinę užtvarą kiekybinių parametrų tiesinė kombinacija leidžia 94% tikslumu klasifikuoti vaistus pagal jų prieinamumą centrinei nervų sistemai. / The objective of this work was to develop mechanistic quantitative structure activity relationship models that would facilitate the assessment of drug properties related to their absorption and distribution in the body. The analysis involved several parameters reflecting the rate of passive diffusion across brain endothelium and intestinal epithelium, and thermodynamic constants related to drug distribution between plasma and tissues. Permeation through cellular transport barriers was modeled by nonlinear equations relating the passive diffusion rate to physicochemical properties of drugs: lipophilicity, ionization, hydrogen bonding potential and molecular size. It was demonstrated that brain endothelium and intestinal epithelium exhibit a quantitatively similar pattern of permeability-ionization dependence – ionized species permeate 2-3 orders of magnitude slower than neutral molecules. Analysis of tissue to plasma partitioning data revealed the necessity to split original experimental values into separate terms reflecting plasma and tissue binding strength. Drugs’ affinity to tissues could then be described by their lipophilicity, whereas detrimental effect of ionization was only observed for acidic drugs. Finally, it was shown that a linear combination of quantitative blood-brain barrier transport parameters allows classifying drugs according to their access to central nervous system with 94% overall accuracy.