Objective: Determine if varying ultrasound frequency affects the delivery of 10% hydrocortisone concentrations during phonophoresis. Utilize intramuscular microdialysis probe for drug collection, thus improving the experimental model. Methods: Thirty one (10 in groups 1 and 2, 11 in group 3) healthy subjects participated in this study. Interventions: Subjects were randomly assigned to one of three treatment groups receiving 10 minute ultrasound treatments applied to a standardized area of the gastrocnemius muscle of the right leg. The ultrasound was performed over the treated area using a 10% hydrocortisone compound mixed with standard ultrasound gel. The contralateral limb served as the control (no mixed compound or treatment) for all groups. Group one received sham ultrasound. Medicated gel was placed on the treatment site, the sound head moved, but no ultrasound was applied. Group two received 45 KHz at .056 w/cm2. Group three received 1 MHz at 1.0 w/cm2 at a 50 % duty cycle. Results: There was no difference in cortisol concentration change during treatment between the three treatment groups on the treated limbs (sham = 1.1 ±7.5 ng/ml, 45 KHz = 1.1 ± 1.5 ng/ml, 1 MHz = 4.1 ± 7.8 ng/ml; F2,22 = .34, P = .72) or control limbs (sham = 1.65 ± 6.6 ng/ml, 45 KHz = -1.3 ± 2.7 ng/ml, 1 MHz = 0.37 ± 8.1 ng/ml; F2,22 = .67, P = .546). No difference was found in cortisol concentration change during treatment between the treatment limbs and the control limbs (treatment = 2.1 ± 6.2 ng/ml, control = 0.20 ± 5.9 ng/ml; F1,22 = .9, P = .35). The following factors were found to influence cortisol concentrations levels in dialysate collected during treatment: depth of muscle in the treatment limbs (F1,22 = 6.4, P = .02), microdialysis probe depth in the control limbs (F1,22 = 4.1, P = .05), and pre treatment cortisol level in the control limbs (F1,22 = 10.1, P = .004. Conclusions: There was no evidence altering ultrasound frequency from 45 KHz to 1 MHZ enhanced the delivery of 10% hydrocortisone to treatment tissues under these experimental conditions.

Context: Transdermal delivery of hydrocortisone by phonophoresis is used for the treatment of musculoskeletal conditions. Research shows hydrocortisone and other white or opaque topical preparations transmit ultrasound energy poorly. Effective transmission of ultrasound is important in phonophoresis. Main Outcome measured: Samples of subcutaneous interstitial fluid were collected during and for 20 minutes following phonophoresis treatment. Cortisol concentrations were analyzed by an enzyme linked immune-assay (ELISA) test. Objective: Determine the subcutaneous cortisol concentration after two different phonophoresis treatments using a 2.5% hydrocortisone preparation. Design: Randomized design in which 22 healthy participants were assigned to receive a phonophoresis treatment where: 1) hydrocortisone cream was rubbed in completely prior to phonophoresis or 2) hydrocortisone powder was compounded with an ultrasound coupling gel. Test Subjects: 22 healthy individuals were recruited: 13 females with a mean age of 21 years and 9 males with a mean age of 21.8 years. Intervention: Phonophoresis consisted of pulsed ultrasound at 1 MHz, 1.0 w/cm2, and a 50% duty cycle. The treatment duration was 10 minutes and was localized over the distal gastrocnemius muscle. Results: We observed no significant difference in subcutaneous cortisol concentration between the two phonophoresis treatments (p=0.05). Also no significant difference was detected between pre and post-treatment cortisol levels within each individual treatment group. Conclusions: Our data indicates that completely rubbing a topical hydrocortisone application into the skin prior to placement of ultrasound gel does not result in increased transdermal delivery of cortisol when compared with the use of a compound of ultrasound gel and hydrocortisone powder applied topically to the skin.

Le peptide YSNSG est un cyclopeptide antagoniste de l’intégrine alpha v beta 3 actif par inhibition de la migration tumorale et de l'angiogénèse. Ces propriétés ont été démontrées in vitro sur diverses lignées cellulaires et in vivo chez des souris C57BL/6 avec xénogreffe de mélanome. Nos travaux se sont intéressés à la pharmacologie et à la pharmacocinétique du YSNSG. Dans un premier temps, nous avons étudié sa pharmacocinétique plasmatique et tissulaire (cerveau et sous-cutané) chez des rats Wistar en utilisant la microdialyse tissulaire. Elle a permis la détermination des principaux paramètres plasmatiques et tissulaire. Le taux de pénétration du peptide au niveau tissulaire est apparu élevé au niveau sous-cutané et faible au niveau cérébral. L’analyse compartimentale de ces données a permis de valider un modèle de type PB-PK pour simuler différents débits de perfusion permettant d’atteindre au niveau tissulaire des concentrations actives. Dans un second temps, nous avons étudiés la distribution intra—tumorale du peptide YSNSG sur le modèle de souris C57BL/6 xénogreffé. A l’aide de la microdialyse tissulaire, nous avons étudié la distribution du peptide YSNSG au sein de deux régions intra-tumorales distinctes, respectivement au centre et en périphérie de la tumeur pour discuter du rôle de l’hétérogénéité intra-tumorale dans la distribution intra-tissulaire des anticancéreux. Notre approche n’a pas permis de mettre en évidence de différence significative dans la distribution du peptide entre les deux régions mais a permis de montrer une faible distribution. Dans une troisième et dernière approche, nous présentons les résultats de la nanoencapsulation du peptide YSNSG à l’aide de particules de type PLGA +/- F-68 à l’aide de méthodes spectroscopiques (Raman), microscopiques (confocale) et in vivo (comparaison de l’efficacité du peptide encapsulé ou non sur l’inhibition de la croissance tumorale sur le modèle animal précédemment utilisé). / Peptide YSNSG is a cyclopeptide addressed against integrins growth tumor inhibition and neoangiogenesis. These properties were highlighted in vitro on several cell lines and in vivo in syngeneic mice C57BL/6 with B16F1 melanoma xenograft. In order to describe the pharmacology and pharmacokinetic of peptide YSNSG we first studied plasma and tissue pharmacokinetic (cerebral and subcutaneous) in healthy Wistar rats using microdialysis. We finded main plasma and tissue parameters and a high penetration rate in subcutaneous and a low penetration rate of peptide YSNSG in cerebral tissue, respectively. Compartment analysis of our data allowed assessing a physiologically-based pharmacokinetic model in order to simulate different administration flow rate required to reach pharmacological active concentrations. Secondly we investigated the intra-tumoral distribution of peptide YSNSG the same syngeneic mice C57BL/6 model previously described, using microdialysis. Distribution was assessed in two distinct regions of melanoma tumor (central and peripheral) for the study of the impact of intra-tumoral heterogeneity on YSNSG peptide distribution. This approach did not found any significant difference in term of penetration rate and, moreover, found a low penetration rate in the melanoma tumor in discordance with previous subcutaneous distribution in healthy rat. In a third chapter we describe results of nanoencapsulation tests of YSNSG peptide using PLGA +/- F-68 with spectroscopic method (Raman), microscopic (confocal) and in vivo experimentation (growth tumor inhibition level in the same mice model with or without nanoencapsulation of YSNSG peptide).

Necrotizing enterocolitis (NEC), a severe inflammatory disorder of the gastrointestinal tract with high morbidity and mortality, affects primarily preterm infants. The diagnosis represents a challenging task, and no biomarker has been found to aid early diagnosis with high accuracy. Microdialysis has been widely used to detect metabolites of anaerobic metabolism, enabling a local and early detection of ischemia. This thesis aims to evaluate the possibility of detecting intestinal ischemic stress in experimental and clinical  NEC, by use of rectal intraluminal microdialysis. Intraluminal rectal microdialysis was performed on rats subjected to total intestinal ischemia. Metabolites of ischemia were detectable in both ileum and rectum, with raised glycerol concentrations and lactate/pyruvate ratios. Elevated concentrations of glycerol correlated to increasing intestinal histopathological injury. Experimental early NEC was induced in newborn rat pups, by hypoxia/re-oxygenation treatment. Development of NEC was confirmed by histopathology. Elevated glycerol concentrations were detected by rectal microdialysis. The genetic alterations following experimental NEC in rat pups were studied with microarray. Immunohistochemistry staining was performed for tight junction proteins claudin-1 and claudin-8. Several genes were altered in experimental NEC, mainly genes regulating tight junctions and cell adhesion. Immunohistochemistry revealed reduced expression of claudin-1. A prospective study was conducted on preterm infants with a gestational age of less than 28 weeks. The infants were admitted to a neonatal intensive care unit, and observed during a 4-week period. Rectal microdialysis was performed twice a week, and blood was drawn for analysis of I-FABP. A total of 15 infants were included in the study, whereof four infants developed NEC, and 11 served as controls. Rectal glycerol and I-FABP displayed high concentrations, which varied considerably during the observation periods, both in NEC and controls. No differences in either glycerol or I-FABP concentrations were seen in the NEC-group vs. the controls. In conclusion, rectal microdialysis can detect metabolites of intestinal ischemia, both in experimental and clinical NEC. Rectal microdialysis is safe and could provide a valuable non-invasive aid to detect hypoxia-induced intestinal damage or ischemic stress in extremely preterm infants. In this study however, it was not possible to predict the development of clinical NEC using microdialysis or I-FABP.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.

Although microdialysis (MD) and solid-phase microextraction (SPME) are widely used sampling techniques, a comparison study has not been performed to date. The goal of the research presented was not only to address this issue but also to develop new analytical methods that were more suitable for in vivo study using MD and SPME. A new calibration method called kinetic microdialysis was developed for in vivo sampling. Two MD probes with different flow rates were simultaneously inserted into the symmetric parts of sampling system. A simple empirical equation was proposed to calculate the analyte concentrations in the sample matrix using two different dialysate concentrations. Several factors that influenced the correction factor in this equation were discussed. An excellent correlation was observed between the calculated and theoretical value. This method was subsequently applied for in vivo sampling, for the measurement of pesticide allocation in the different leaves of a jade plant (Crassula ovata). Compared to the other reported MD calibration methods, this novel approach offers several advantages including simplicity, speed, robustness, and increased accuracy. The on-fiber standardization technique for solid-coated SPME was studied and a theoretical model is proposed for the isotropic behavior of adsorption and desorption, based on Fick’s law of diffusion and the Langmuir model. The isotropy of the adsorption and desorption of analytes onto and from the surface of porous solid SPME fiber was validated with the use of a commercially available fiber, a 50 um carbowax/templated resin (CW/TPR) for carbamate pesticide analysis in various in vitro sample matrices. Time constants were comparable for the adsorption and desorption processes. Equilibrium constants and fiber capacities were calculated with the Langmuir Isotherm Model. A kinetic method was developed to calibrate adsorption using desorption. This calibration corrected for the sample matrix effects and minimized displacement effects as a pre-equilibrium extraction. The technique was successfully applied to the analysis of pesticides in river water and white wine. This developed method could be potentially applied for in vivo study. A new kinetic calibration was developed using dominant pre-equilibrium desorption by SPME. The calibration was based on isotropism between absorption and desorption, which was proved theoretically and experimentally in an aqueous solution and semi-solid matrix. This approach allows for the calibration of absorption using desorption to compensate for matrix effects. Moreover, concentration profiles are initially proposed to verify isotropism between the absorption and desorption, while providing a linear approach to obtain time constants for the purpose of quantitative analysis. This linear approach is more convenient, robust and accurate than the non-linear version with the previously used time profiles. Furthermore, the target analytes are used as the internal standards, thus radioactive or deuterated internal standards are not necessary. In addition, dominant pre-equilibrium desorption utilizes the pre-equilibrium approach and offers a shorter sample preparation time, which is typically suitable for in vivo sampling. This kinetic calibration method was successfully applied to prepare samples of polycyclic aromatic hydrocarbons (PAHs) in a flow-through system and in vivo pesticide sampling in a jade plant (Crassula ovata). Previous field studies utilizing SPME predominantly focused on volatile compounds in air or water. Earlier in vivo sampling studies utilizing SPME were limited to liquid matrices, namely blood. In this study, SPME was developed for in vivo laboratory and field sampling of pharmaceuticals in fish muscle. Pre-equilibrium extraction was used to shorten in vivo sampling time. The use of pre-equilibrium desorption rates are proposed as a means to calibrate pre-equilibrium extractions. Excellent linearity was found between the free concentrations determined by SPME from the muscle of living fish and the waterborne concentrations of several pharmaceuticals. It is also firstly proposed a simple SPME method to determine free and total concentrations simultaneously in a living tissue using the known protein binding value. The utility of in vivo SPME sampling under field conditions was evaluated in wild fish collected from a number of different river locations under varying degrees of influence from municipal wastewater effluents. Diphenhydramine and diltiazem were detected in the muscle of fish downstream of a local wastewater treatment plant. Based on this study, SPME technique has demonstrated several important advantages for laboratory and field in vivo sampling. The development of a rapid, robust, easy to deploy technique which combines sampling, extraction and concentration into one step is a potentially important tool for use in vivo field-based sampling. MD and SPME methods have been developed and compared through in vitro and in vivo study. For in vitro study (juice, milk and orange jelly), both methods offered accurate and precise results (recovery: 88-105% with RSD < 15%) for complex sample matrices by standard addition method. The limits of quantification (LOQs) of the two methods developed were below the tolerance levels in milk set by the United Nations Food and Agriculture Organization (FAO). Compared to MD, the fully automated SPME procedure offered several advantages including high-throughput and more efficient sampling, less labor intensity, and capability for batch analysis. For in vivo study, kinetic calibrations were performed using retrodialysis and in-fiber standardization techniques for MD and SPME, respectively. Quantitative analysis was performed to measure pesticide concentrations in living tissue, i.e., the leaves of a living jade plant (Crassula ovata). Although both techniques provided sampling with minimal perturbation to the system under study, SPME was more sensitive, precise and accurate, suitable for field sampling and had a wider application than MD. It demonstrated that SPME has the potential to replace MD for in vivo study.

In order to investigate the early ultrastructural lesions at the first sign of lameness in the oligofructose (OF) model of laminitis, the disease was induced in four horses, while another four horses were sham-treated controls. Minor lesions were detected in lamellar samples examined by light microscopy. Examination by transmission electron microscopy (TEM) showed excessive waviness, breaks and separation of portions of lamellar basement membrane (BM) in the treated horses. There was also disintegration and disappearance of hemidesmosomes (HD) and epidermal basal cell (EBC) cytoskeleton, and an increase in the distance between the EBC plasmalemma and the centre of the BM. A link was thus established between the first clinical signs of lameness and ultrastructural changes in the lamellar dermo-epidermal interface. This implied that pathogenesis was underway well before clinical signs (24 h) and that successful therapy would need to be instituted earlier than previously considered. Earlier therapy may be facilitated if delivery of efficacious drugs to the foot was achievable. A treatment modality that delivered effective concentrations of anti laminitic drugs to the target organ (the epidermal lamellae) was thus an objective of this study. Hoof lamellar tissue from five ponies treated with prolonged euglycaemic hyperinsulinaemia and four control (sham-treated) ponies were harvested and processed for TEM. Lamellae from treated ponies showed attenuation and elongation of secondary epidermal lamellae (SEL), HD number reduction and infiltration of leukocytes. Unlike carbohydrate induced laminitis in horses, there was no global separation at the lamellar dermal/epidermal interface in ponies. Two unique lamellar lesions found in this induction model was mitosis among EBCs and thickening of the BM, not normally characteristic of acute laminitis. The pathophysiology of hyperinsulinaemic laminitis remains unresolved but if insulin, delivered directly to the foot, induced laminitis several pathophysiological questions would be answered. In particular, it would emphasise the laminitogenic potential of insulin alone in the pathogenesis of laminitis. It also allows the treatment foot to be compared with the remaining three that act as internal controls. A modality that delivered drugs like insulin to the target organ (the epidermal lamellae) was needed and was an objective of this study. A microdialysis (MD) method, based on continuous sampling of the lamellar extracellular fluid (ECF), was developed to monitor lamellar drug concentrations. MD probes were implanted in the hoof lamellar tissue of six normal Standardbred horses under local anaesthesia. A bolus intravenous (IV) dose (5 mg/kg BWT) of gentamicin sulfate was injected into the jugular vein. MD and blood samples were collected at different time points during 24 h, and calibrated and analyzed using an ELISA method for gentamicin. During the first 8 h, the concentration of gentamicin was significantly higher in blood than lamellar ECF, a result that is reversed when lamellar MD is repeated during IO infusion of gentamicin. The results showed that this modestly invasive method was a useful tool to monitor changes in the lamellar ECF during drug delivery or during laminitis development. Knowledge of the anatomy and dynamics of blood circulation in the equine foot is fundamental to understand laminitis pathophysiology. Using histology, decalcification, diaphanization, computed tomography (CT), micro CT and gelatin-India Ink vascular perfusion, the normal anatomy of the dorsal part of distal phalanx (DP) and its vascular relationship to hoof lamellae was characterised. The results showed a close relationship between the distal phalangeal and lamellar circulations and raised the possibility of accessing the lamellar circulation via the DP and the possibility that IO perfusion (IOP) of the DP could deliver drugs to the lamellae. IOP of the DP with methyl methacrylate (MMA) corrosion casting material resulted in filling of the lamellar and sublamellar vascular network and incomplete filling of lamellar capillaries. Perfusion of common digital artery with a suspension of barium sulfate resulted in filling of lamellar arteries but not capillaries. Perfusion of the common digital vein resulted in filling of lamellar veins but not capillaries. Perfusion with barium sulfate partitioned veins from arteries because particle size prevented entry into capillaries. IOP with barium sulfate filled only veins revealing that vascular egress from the DP was venous. This study showed that a retrograde venous connection exists between the DP and lamellar circulations with the potential for lamellar drug delivery. Intra-arterial (IA) and IO infusion results using gelatin-India Ink were markedly improved when cadaver limbs were subjected to cyclic loading within the physiological range. Without loading lamellar capillaries failed to fill no matter what the injection pressure. Cyclic loading cadaver limbs 6 times resulted in complete lamellar capillary filling and suggested that cyclic limb loading contributed to perfusion of lamellar capillaries normally in horses. To evaluate IO delivery of drugs to hoof lamellae in the standing, conscious horse, gentamicin solution (25 mg/mL) was slowly infused (20 µL/min) through an IO bone screw. Lamellar ECF was collected via a lamellar MD probe and blood was collected from the jugular vein. Gentamicin was 50-100 times more concentrated in lamellar ECF than in blood. This study introduces a potential method for delivery of drugs into the lamellar tissue in the standing, conscious horse. Laminitis pathology occurs before clinical signs and can be induced by insulin as well as enteric OF overload. Thus therapy delivered to the target of laminitis, the hoof lamellae, has an improved chance of success if delivered promptly, safely and at high concentrations. A validated drug delivery and lamellar analysis system that achieves these criteria, was the discovery of this project and is now available to combat laminitis.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 5 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.