Development of a novel in vitro system for nasal drug delivery development

There is currently, no ideal system for studying nasal drug delivery in vitro. The existing techniques such as the Ussing chamber and cell culture all have major disadvantages. Most importantly, none of the existing techniques accurately represent the interior of the nasal cavity, with its airflow and humidity; neither do they allow the investigation of solid dosage forms.The work in this thesis represents the development of an in vitro model system in which the interior characteristics of the nasal cavity are closely represented, and solid or minimal volume dosage forms can be investigated. The complete nasal chamber consists of two sections: a lower tissue, viability chamber and an upper nasal chamber. The lower tissue viability chamber has been shown, using existing tissue viability monitoring techniques, to maintain the viability of a number of epithelial tissues, including porcine and rabbit nasal tissue, and rat ileal and Payers' patch tissue. The complete chamber including the upper nasal chamber has been shown to provide tissue viability for porcine and rabbit nasal tissue above that available using the existing Ussing chamber techniques. Adaptation of the complete system, and the development of the necessary experimental protocols that allow aerosol particle-sizing, together with videography, has shown that the new factors investigated, humidity and airflow, have a measurable effect on the delivered dose from a typical nasal pump. Similarly, adaptation of the chamber to fit under a confocal microscope, and the development of the necessary protocols has shown the effect of surface and size on the penetration of microparticulate materials into nasal epithelial tissues. The system developed in this thesis has been shown to be flexible, in allowing the development of the confocal and particle-sizing systems. For future nasal drug delivery studies, the ability to measure such factors as the size of the delivered system in the nasal cavity, the depth of penetration of the formulation into the tissue are essential. Additionally, to have access to other data such as that obtained from drug transport in the same system, and to have the tissue available for histological examination represents a significant advance in the usefulness of such an in vitro technique for nasal delivery.

Biological barriers to the nasal delivery of peptide drugs

The nasal absorption of larger peptide and protein drugs is generally low. The importance of the mucus layer and enzymic degradation in reducing absorption were investigated. Reversed-phase high-performance liquid chromatographic (HPLC) methods were developed to assay a variety of compounds. Pig gastric mucus (PGM) was selected to investigate the importance of the mucus layer. A method of treating and storing PGM was developed and evaluated which was representative of the gel in vivo. The nature of the mucus barrier was evaluated in vitro with three-compartment diffusion cells and a series of compounds with differing physicochemical properties. Mucus retarded the diffusion of all the compounds with molecular weight and charge exerting a marked effect. Binding to mucus was investigated by a centrifugation method. All of the compounds tested were found to bind to mucus with the exception of the negatively charged molecule benzoic acid. The small peptides did not demonstrate greater binding to mucus than any of the other compounds evaluated. The effect of some absorption enhancers upon the rate of diffusion of tryptophan through mucus was determined in vi tro. At the concentrations employed the enhancers EDTA, N-acetylcysteine and taurodeoxycholic acid exerted no effect, whilst taurocholic acid and cholic acid, were found to slightly reduce the rate of diffusion. The intracellular and luminal proteolytic activity of the nose was investigated in the sheep animal model with a nasal mucosal homogenate and a nasal wash preparation respectively and a series of chemically similar peptides. Hydrolysis was also investigated with the proteolytic enzymes carboxypeptidase A, cytosolic leucine aminopeptidase and microsomal leucine aminopeptidase. Sheep nasal mucosa possesses significant peptide hydrolase activity capable of degrading all the substrates tested. Considerable variation in susceptibility was observed. Degradation occurred excl us i ve ly at the pept ide bond between the aromatic amino ac id and glycine, indicating some specificity for aromatic amino acids. Hydrolysis profiles indicated the presence of both aminopeptidase and carboxypeptidase enzymes. The specific activity of the microsomal fraction was found to be greater than the cytosolic fraction. Hydrolysis in the nasal wash indicated the presence of either luminal or loosely-bound proteases, which can degrade peptide substrates. The same specificity for aromatic amino acids was observed and aminopeptidase activity demonstrated. The specific activity of the nasal wash was smaller than that of the homogenate.

Variation and change in the use of hesitation markers in Germanic languages

In this study, we investigate crosslinguistic patterns in the alternation between UM, a hesitation marker consisting of a neutral vowel followed by a final labial nasal, and UH, a hesitation marker consisting of a neutral vowel in an open syllable. Based on a quantitative analysis of a range of spoken and written corpora, we identify clear and consistent patterns of change in the use of these forms in various Germanic languages (English, Dutch, German, Norwegian, Danish, Faroese) and dialects (American English, British English), with the use of UM increasing over time relative to the use of UH. We also find that this pattern of change is generally led by women and more educated speakers. Finally, we propose a series of possible explanations for this surprising change in hesitation marker usage that is currently taking place across Germanic languages.

The effect of ageing on in vivo human ciliary muscle morphology and contractility

Purpose. To assess the effect of ageing on in vivo human ciliary muscle morphology and contractility during accommodation. Methods. Seventy-nine subjects, aged 19–70 years were recruited. High-resolution images were acquired of nasal and temporal ciliary muscle in the relaxed state, and at stimulus vergence levels of -4 and -8 D, using anterior segment optical coherence tomography (AS-OCT). Objective refractions and axial lengths were also recorded. Linear regression analysis was performed to determine the effect of age on nasal and temporal ciliary muscle morphologic characteristics. Results. Ciliary muscle anterior length decreased significantly with age both nasally (R = 0.461, P = 0.001) and temporally (R = 0.619, P < 0.001) in emmetropic eyes. In a subset of 37 participants, ciliary muscle maximum width increased significantly with age, by 2.8 µm/year nasally (R = 0.54, P < 0.001) and 3.0 µm/year temporally (R = 0.44, P = 0.007), while the distance from the inner apex of the ciliary muscle to the scleral spur decreased significantly with age on both the nasal and temporal aspects (R = 0.47; P = 0.004 and R = 0.43; P = 0.009, respectively). During accommodation, changes to ciliary muscle thickness and length remained constant throughout life. Conclusions. The human ciliary muscle undergoes age-dependent changes in morphology that suggest an antero-inwards displacement of muscle mass, particularly in emmetropic eyes. However, the morphologic changes observed appear not to affect the ability of the muscle to contract during accommodation, even in established presbyopes, thus supporting a lenticular model of presbyopia development.

In vivo analysis of ciliary muscle morphologic changes with accommodation and axial ametropia

Purpose. To use anterior segment optical coherence tomography (AS-OCT) to analyze ciliary muscle morphology and changes with accommodation and axial ametropia. Methods. Fifty prepresbyopic volunteers, aged 19 to 34 years were recruited. High-resolution images were acquired of nasal and temporal ciliary muscles in the relaxed state and at stimulus vergence levels of -4 and -8 D. Objective accommodative responses and axial lengths were also recorded. Two-way, mixed-factor analyses of variance (ANOVAs) were used to assess the changes in ciliary muscle parameters with accommodation and determine whether these changes are dependent on the nasal–temporal aspect or axial length, whereas linear regression analysis was used to analyze the relationship between axial length and ciliary muscle length. Results. The ciliary muscle was longer (r = 0.34, P = 0.02), but not significantly thicker (F = 2.84, P = 0.06), in eyes with greater axial length. With accommodation, the ciliary muscle showed a contractile shortening (F = 42.9. P < 0.001), particularly anteriorly (F = 177.2, P < 0.001), and a thickening of the anterior portion (F= 46.2, P < 0.001). The ciliary muscle was thicker (F = 17.8, P < 0.001) and showed a greater contractile response on the temporal side. Conclusions. The accommodative changes observed support an anterior, as well as centripetal, contractile shift of ciliary muscle mass.

Influence of corneal asymmetry on the phakometric measurement of ocular surface alignment

Phakometric measurements of corneal and crystalline lens surface alignment are influenced by corneal asymmetry in which the corneal apex does not coincide with the limbal centre. The purpose of this study was to determine the horizontal separation (e) between these corneal landmarks. Measurements were made in 60 normal eyes (30 subjects) using the Orbscan Ilz corneal analysis workstation. Our results show that both corneal landmarks typically coincide, so that e = 0, but that inter-subject variations of about ±1 mm can be expected (so that the corneal apex may fall nasal or temporal to the visual axis). This suggests that no correction for corneal asymmetry is required when estimating average amounts of ocular alignment from samples of eyes but that the measurement of e is strongly recommended for measurements in individual eyes. © 2004 The College of Optometrists.

Development and evaluation of a novel intranasal spray for the delivery of amantadine

The aim of this study was to develop and characterize an intranasal delivery system for amantadine hydrochloride (AMT). Optimal formulations consisted of a thermosensitive polymer Pluronic® 127 and either carboxymethyl cellulose or chitosan which demonstrated gel transition at nasal cavity temperatures (34 ± 1°C). Rheologically, the loss tangent (Tan δ) confirmed a 3-stage gelation phenomena at 34 ± 1°C and non-Newtonian behavior. Storage of optimized formulation carboxymethyl cellulose and optimal formulation chitosan at 4°C for 8 weeks resulted in repeatable release profiles at 34°C when sampled, with a Fickian mechanism earlier on but moving toward anomalous transport by week 8. Polymers (Pluronic® 127, carboxymethyl cellulose, and chitosan) demonstrated no significant cellular toxicity to human nasal epithelial cells up to 4 mg/mL and up to 1 mM for AMT (IC50: 4.5 ± 0.05 mM). Optimized formulation carboxymethyl cellulose and optimal formulation chitosan demonstrated slower release across an in vitro human nasal airway model (43%-44% vs 79 ± 4.58% for AMT). Using a human nasal cast model, deposition into the olfactory regions (potential nose-to-brain) was demonstrated on nozzle insertion (5 mm), whereas tilting of the head forward (15°) resulted in greater deposition in the bulk of the nasal cavity.

Influence of accommodation and refractive status on the peripheral refractive profile

AIM: The aim of the study was to determine, objectively and non-invasively, whether changes in accommodative demand modify differentially the peripheral refraction in emmetropic and myopic human eyes. METHODS: Forty subjects (19 male, 21 female) aged 20-30 years (mean 22.7 (SD 2.8) years), 21 emmetropes (mean spherical equivalent refractive error (MSE) -0.13 (SD 0.29) D) and 19 myopes (MSE -2.95 (SD 1.76) D) participated in the study. Ametropia was corrected with soft contact lenses (etafilcon A, 58% water content). Subjects viewed monocularly a stationary, high contrast (85%) Maltese cross at 0.0, 1.0, 2.0 and 3.0 D of accommodative demand and at 0, 10, 20 and 30 degrees field angle (nasal and temporal) through a +3.0 D Badal optical system. Static recordings of the accommodation response were obtained for each accommodative level, at each field angle, with an objective, open-view, infrared optometer. RESULTS: Peripheral mean spherical equivalent (M) data showed that the emmetropic cohort exhibited relative myopic shifts into the periphery, while the myopic group showed hypermetropic shifts. Increasing accommodative demand did not alter the peripheral refractive profile in either the temporal (p = 0.25) or nasal (p = 0.07) periphery with no differential accommodative effect between refractive groups in either the temporal (p = 0.77) or nasal (p = 0.73) field. Significant shifts in the J(0) astigmatic component were seen in the temporal (p<0.0005) and nasal (p<0.0005) fields with increasing eccentricity. Interaction effects between eccentricity and accommodative demand illustrated that increasing accommodative demand significantly altered the peripheral refractive profile in the temporal J(0) astigmatic component (p<0.0005). The nasal periphery, however, failed to show such an effect (p = 0.65). CONCLUSIONS: Alterations in peripheral refraction augmented by changes in ocular accommodation are relatively unaffected by refractive error for young, healthy human eyes.

Three-dimensional modeling of the human eye based on magnetic resonance imaging

PURPOSE. A methodology for noninvasively characterizing the three-dimensional (3-D) shape of the complete human eye is not currently available for research into ocular diseases that have a structural substrate, such as myopia. A novel application of a magnetic resonance imaging (MRI) acquisition and analysis technique is presented that, for the first time, allows the 3-D shape of the eye to be investigated fully. METHODS. The technique involves the acquisition of a T2-weighted MRI, which is optimized to reveal the fluid-filled chambers of the eye. Automatic segmentation and meshing algorithms generate a 3-D surface model, which can be shaded with morphologic parameters such as distance from the posterior corneal pole and deviation from sphericity. Full details of the method are illustrated with data from 14 eyes of seven individuals. The spatial accuracy of the calculated models is demonstrated by comparing the MRI-derived axial lengths with values measured in the same eyes using interferometry. RESULTS. The color-coded eye models showed substantial variation in the absolute size of the 14 eyes. Variations in the sphericity of the eyes were also evident, with some appearing approximately spherical whereas others were clearly oblate and one was slightly prolate. Nasal-temporal asymmetries were noted in some subjects. CONCLUSIONS. The MRI acquisition and analysis technique allows a novel way of examining 3-D ocular shape. The ability to stratify and analyze eye shape, ocular volume, and sphericity will further extend the understanding of which specific biometric parameters predispose emmetropic children subsequently to develop myopia. Copyright © Association for Research in Vision and Ophthalmology.

Posterior retinal contour in adult human anisomyopia

PURPOSE. It is well documented that myopia is associated with an increase in axial length or, more specifically, in vitreous chamber depth. Whether the transverse dimensions of the eye also increase in myopia is relevant to further understanding of its development. METHODS. The posterior retinal surface was localized in two-dimensional space in both eyes of young adult white and Taiwanese-Chinese iso- and anisomyopes (N = 56), from measured keratometry, A-scan ultrasonography, and central and peripheral refraction (±35°) data, with the aid of a computer modeling program designed for this purpose. Anisomyopes had 2 D or more interocular difference in their refractive errors, with mean values in their more myopic eyes of -5.57 D and in their less myopic eyes of -3.25 D, similar to the means of the two isomyopic groups. The derived retinal contours for the more and less myopic eyes were compared by way of investigating ocular shape changes that accompany myopia, in the posterior region of the vitreous chamber. The presence and size of optic disc crescents were also investigated as an index of retinal stretching in myopia. RESULTS. Relative to the less myopic eyes of anisometropic subjects, the more myopic eyes were more elongated and also distorted into a more prolate shape in both the white and Chinese groups. However, the Chinese eyes showed a greater and more uniform relative expansion of the posterior retinal surface in their more myopic eyes, and this was associated with larger optic disc crescents. The changes in the eyes of whites displayed a nasal-temporal axial asymmetry, reflecting greater enlargement of the nasal retinal sector. CONCLUSIONS. Myopia is associated with increased axial length and a prolate shape. This prolate shape is consistent with the proposed idea that axial and transverse dimensions of the eye are regulated differently. The observations that ocular shape changes are larger but more symmetrical in Chinese eyes than in eyes of whites warrant further investigation.

The percutaneous absorption of ionisable compounds

The effects of ionisation on transdermal drug delivery using excised human epidermis (HS) and silastic rubber (SR) as model permeation barriers were investigated in vitro using Franz-type absorption cells. Suspensions and solutions of salicylic acid (SA), the model ionogenic permeant, were used as donors and the variables studied were vehicle pH and trans-membrane pH-gradients. For solutions, the pH effect was related to the level of ionisation of the drug and the degree of saturation of the solution. With suspensions, the observed permeation rate was unaffected by pH. The penetration profiles through HS and SR were similar, although the overall flux through HS was about 70% of that observed through SR. Pretreatment of the membranes with various enhancer regimens, including oleic acid, Azone and N, N-dimethylamides in propylene glycol (PG) and isopropyl myristate (IPM) promoted the penetration of SA. SR was not a suitable model for enhancer pretreatment using IPM as a vehicle as the membrane was significantly disrupted by this vehicle. The results from comparable experiments with and without a trans-membrane pH-gradient did not have a significant effect upon flux or flux enhancement after pretreatment with the above enhancers. A theoretical model for the extraction coefficients of weak acids was derived using the partition coefficients of the ionised and unionised species, pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. The distribution of this solute between aqueous and oily phases, with and without added enhancer, was measured as a function of pH. The extraction coefficients determined were consistent with the model and showed that the behaviour of the system can be explained without referral to ion-pair mechanisms. Phosphonoacetate is an effective antiviral agent. However, as it is charged at physiological pH, its permeation across cell membranes is limited. To assess the improvement of the transport properties of this molecule, mono-, di- and tri-ester prodrugs were examined. These were assessed for stability and subsequent breakdown with respect to pH by HPLC. In vitro percutaneous absorption was observed using the triester, but not the ionic mono- or di-esters. The triester absorption could be potentiated using a range of enhancers with oleic acid being the most effective. Cyclodextrins (CD) have a role as absorption enhancers for peptide compounds across nasal epithelium. One potential mode of action is that CDs include these compounds, protect them from enzymic attack and thereby increase their residence time in the nasal epithelium. This study investigated the potential of CDs to protect ester prodrugs from enzymatic breakdown and prevent production of poorly transportable ionic species. Using a range of CD to ester molar ratios (10:1 to 2500:1) a small, but measurable, protection for the model esters (parabens) against esterase attack was observed. Possible mechanisms for this phenomenon are that CDs include the ester, making it unavailable for hydrolysis, the CDs may also affect the esterase in some way preventing access for the ester into the active site.