From Polymorph Screening to Dissolution Testing - Solid Phase Analysis during Pharmaceutical Development and Manufacturing

Solid materials can exist in different physical structures without a change in chemical composition. This phenomenon, known as polymorphism, has several implications on pharmaceutical development and manufacturing. Various solid forms of a drug can possess different physical and chemical properties, which may affect processing characteristics and stability, as well as the performance of a drug in the human body. Therefore, knowledge and control of the solid forms is fundamental to maintain safety and high quality of pharmaceuticals. During manufacture, harsh conditions can give rise to unexpected solid phase transformations and therefore change the behavior of the drug. Traditionally, pharmaceutical production has relied on time-consuming off-line analysis of production batches and finished products. This has led to poor understanding of processes and drug products. Therefore, new powerful methods that enable real time monitoring of pharmaceuticals during manufacturing processes are greatly needed. The aim of this thesis was to apply spectroscopic techniques to solid phase analysis within different stages of drug development and manufacturing, and thus, provide a molecular level insight into the behavior of active pharmaceutical ingredients (APIs) during processing. Applications to polymorph screening and different unit operations were developed and studied. A new approach to dissolution testing, which involves simultaneous measurement of drug concentration in the dissolution medium and in-situ solid phase analysis of the dissolving sample, was introduced and studied. Solid phase analysis was successfully performed during different stages, enabling a molecular level insight into the occurring phenomena. Near-infrared (NIR) spectroscopy was utilized in screening of polymorphs and processing-induced transformations (PITs). Polymorph screening was also studied with NIR and Raman spectroscopy in tandem. Quantitative solid phase analysis during fluidized bed drying was performed with in-line NIR and Raman spectroscopy and partial least squares (PLS) regression, and different dehydration mechanisms were studied using in-situ spectroscopy and partial least squares discriminant analysis (PLS-DA). In-situ solid phase analysis with Raman spectroscopy during dissolution testing enabled analysis of dissolution as a whole, and provided a scientific explanation for changes in the dissolution rate. It was concluded that the methods applied and studied provide better process understanding and knowledge of the drug products, and therefore, a way to achieve better quality.

Analysis of a finite composite plate with smooth rigid pin

A continuum method of analysis is presented in this paper for the problem of a smooth rigid pin in a finite composite plate subjected to uniaxial loading. The pin could be of interference, push or clearance fit. The plate is idealized to an orthotropic sheet. As the load on the plate is progressively increased, the contact along the pin-hole interface is partial above certain load levels in all three types of fit. In misfit pins (interference or clearance), such situations result in mixed boundary value problems with moving boundaries and in all of them the arc of contact and the stress and displacement fields vary nonlinearly with the applied load. In infinite domains similar problems were analysed earlier by ‘inverse formulation’ and, now, the same approach is selected for finite plates. Finite outer domains introduce analytical complexities in the satisfaction of boundary conditions. These problems are circumvented by adopting a method in which the successive integrals of boundary error functions are equated to zero. Numerical results are presented which bring out the effects of the rectangular geometry and the orthotropic property of the plate. The present solutions are the first step towards the development of special finite elements for fastener joints.

Effects of variance-function misspecification in analysis of longitudinal data

The approach of generalized estimating equations (GEE) is based on the framework of generalized linear models but allows for specification of a working matrix for modeling within-subject correlations. The variance is often assumed to be a known function of the mean. This article investigates the impacts of misspecifying the variance function on estimators of the mean parameters for quantitative responses. Our numerical studies indicate that (1) correct specification of the variance function can improve the estimation efficiency even if the correlation structure is misspecified; (2) misspecification of the variance function impacts much more on estimators for within-cluster covariates than for cluster-level covariates; and (3) if the variance function is misspecified, correct choice of the correlation structure may not necessarily improve estimation efficiency. We illustrate impacts of different variance functions using a real data set from cow growth.

Efficient regression analysis with ranked-set sampling

This article is motivated by a lung cancer study where a regression model is involved and the response variable is too expensive to measure but the predictor variable can be measured easily with relatively negligible cost. This situation occurs quite often in medical studies, quantitative genetics, and ecological and environmental studies. In this article, by using the idea of ranked-set sampling (RSS), we develop sampling strategies that can reduce cost and increase efficiency of the regression analysis for the above-mentioned situation. The developed method is applied retrospectively to a lung cancer study. In the lung cancer study, the interest is to investigate the association between smoking status and three biomarkers: polyphenol DNA adducts, micronuclei, and sister chromatic exchanges. Optimal sampling schemes with different optimality criteria such as A-, D-, and integrated mean square error (IMSE)-optimality are considered in the application. With set size 10 in RSS, the improvement of the optimal schemes over simple random sampling (SRS) is great. For instance, by using the optimal scheme with IMSE-optimality, the IMSEs of the estimated regression functions for the three biomarkers are reduced to about half of those incurred by using SRS.

Die Eigenschaften der Benennungen und ihr Einfluss auf deren Verwendung am Beispiel der Euro-Währung : Eine quantitative und kontrastive Analyse der Terminologie im Finnischen und im Deutschen

This dissertation deals with the terminology of the Euro currency. Its aims are to determine the characteristics of the designations in a certain LSP and to discover whether the recommendations and rules that have been given to the formation of designations and 'ideal' designations have any influence on the usage of the designations. The characteristics analysed include length of the designation, part of speech, form, formation method, constancy, monosemy, suitability to a concept system and degree of specialty. The study analyses the actual usage of the designations in texts and the implementation of the designations. The study is an adaptation of a terminometric survey and uses concept analysis and quantitative analysis as its basic methods. The frequency of each characteristic is measured in terms of statistics. It is assumed that the 'ideality' of a designation influences its usage, for example that if a designation is short, it is used more than its longer rivals (synonyms). The results are analysed in a corpus consisting of a compilation of different texts concerning the Euro. The corpus is divided according to three features: year (1998-2003), genre (judicial texts, annual reports and brochures) and language (Finnish and German). Each analysis is performed according to each of these features and compared with the others. The results indicate that some of the characteristics of the designations indeed seem to have an influence on the usage of the designations. For example, monosemy and suitability to the concept system often lead to the implementation of the designation having the ideal or certain value in these characteristics in the analysed Finnish material. In German material, an 'ideal' value in the characteristics leads to the implementation of the designations more often than in Finnish. The contrastive study indicates that, for example, suitability to a concept system leads to implementation of a designation in judicial texts more often than in other genres. The recommendations given to an 'ideal' designation are thus often acceptable, but they cannot be generalized for all languages in the same extent.

Kinetic analysis of nitroxide radical coupling reactions mediated by CuBr

In a previous paper, we described the room temperature rapid, selective, reversible, and near quantitative Cu-activated nitroxide radical coupling (NRC) technique to prepare 3-arm polystyrene stars. In this work, we evaluated the Cu-activation mechanism, either conventional atom transfer or single electron transfer (SET), through kinetic simulations. Simulation data showed that one can describe the system by either activation mechanism. We also found through simulations that bimolecular radical termination, regardless of activation mechanism, was extremely low and could be considered negligible in an NRC reaction. Experiments were carried out to form 2- and 3-arm PSTY stars using two ligands, PMDETA and Me6TREN, in a range of solvent conditions by varying the ratio of DMSO to toluene, and over a wide temperature range. The rate of 2- or 3-arm star formation was governed by the choice of solvent and ligand. The combination of Me6TREN and toluene/DMSO showed a relatively temperature independent rate, and remarkably reached near quantitative yields for 2-arm star formation after only 1 min at 25 °C.

Transcriptome analysis of Neotyphodium and Epichloë grass endophytes

Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloë festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource. They permit the interrogation of 3806 Neotyphodium genes (NchipTM microarray), and 4195 Neotyphodium and 920 Epichloë genes (EndoChipTM microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass–symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloë was performed

QTL analysis of ergot resistance in sorghum

Sorghum ergot, caused predominantly by Claviceps africana Frederickson, Mantle, de Milliano, is a significant threat to the sorghum industry worldwide. The objectives of this study were firstly, to identify molecular markers linked to ergot resistance and to two pollen traits, pollen quantity (PQ) and pollen viability (PV), and secondly, to assess the relationship between the two pollen traits and ergot resistance in sorghum. A genetic linkage map of sorghum RIL population R931945-2-2 x IS 8525 (resistance source) was constructed using 303 markers including 36 SSR, 117 AFLP™, 148 DArT™ and two morphological trait loci. Composite interval mapping identified nine, five, and four QTL linked to molecular markers for percentage ergot infection (PCERGOT), PQ and PV, respectively, at a LOD >2.0. Co-location/linkage of QTL were identified on four chromosomes while other QTL for the three traits mapped independently, indicating that both pollen and non pollen-based mechanisms of ergot resistance were operating in this sorghum population. Of the nine QTL identified for PCERGOT, five were identified using the overall data set while four were specific to the group data sets defined by temperature and humidity. QTL identified on SBI-02 and SBI-06 were further validated in additional populations. This is the first report of QTL associated with ergot resistance in sorghum. The markers reported herein could be used for marker-assisted selection for this important disease of sorghum.

Quantitative assessment of total phenol contents of European oak (Quercus petraea and Quercus robur) by diffuse reflectance NIR spectroscopy on solid wood surfaces

Near infrared spectroscopy (NIRS) combined with multivariate analysis techniques was applied to assess phenol content of European oak. NIRS data were firstly collected directly from solid heartwood surfaces: in doing so, the spectra were recorded separately from the longitudinal radial and the transverse section surfaces by diffuse reflectance. The spectral data were then pretreated by several pre-processing procedures, such as multiplicative scatter correction, first derivative, second derivative and standard normal variate. The tannin contents of sawmill collected from the longitudinal radial and transverse section surfaces were determined by quantitative extraction with water/methanol (1:4, by vol). Then, total phenol contents in tannin extracts were measured by the Folin-Ciocalteu method. The NIR data were correlated against the Folin-Ciocalteu results. Calibration models built with partial least squares regression displayed strong correlation - as expressed by high determination correlation coefficient (r2) and high ratio of performance to deviation (RPD) - between measured and predicted total phenols content, and weak calibration and prediction errors (RMSEC, RMSEP). The best calibration was provided with second derivative spectra (r2 value of 0.93 for the longitudinal radial plane and of 0.91 for the transverse section plane). This study illustrates that the NIRS technique when used in conjunction with multivariate analysis could provide reliable, quick and non-destructive assessment of European oak heartwood extractives.

Flux balance analysis of biological systems: applications and challenges

Systems level modelling and simulations of biological processes are proving to be invaluable in obtaining a quantitative and dynamic perspective of various aspects of cellular function. In particular, constraint-based analyses of metabolic networks have gained considerable popularity for simulating cellular metabolism, of which flux balance analysis (FBA), is most widely used. Unlike mechanistic simulations that depend on accurate kinetic data, which are scarcely available, FBA is based on the principle of conservation of mass in a network, which utilizes the stoichiometric matrix and a biologically relevant objective function to identify optimal reaction flux distributions. FBA has been used to analyse genome-scale reconstructions of several organisms; it has also been used to analyse the effect of perturbations, such as gene deletions or drug inhibitions in silico. This article reviews the usefulness of FBA as a tool for gaining biological insights, advances in methodology enabling integration of regulatory information and thermodynamic constraints, and finally addresses the challenges that lie ahead. Various use scenarios and biological insights obtained from FBA, and applications in fields such metabolic engineering and drug target identification, are also discussed. Genome-scale constraint-based models have an immense potential for building and testing hypotheses, as well as to guide experimentation.

Detecting quantitative trait loci affecting beef tenderness on bovine chromosome 7 near calpastatin and lysyl oxidase

From a study of 3 large half-sib families of cattle, we describe linkage between DNA polymorphisms on bovine chromosome 7 and meat tenderness. Quantitative trait loci (QTL) for Longissimus lumborum peak force (LLPF) and Semitendonosis adhesion (STADH) were located to this map of DNA markers, which includes the calpastatin ( CAST) and lysyl oxidase (LOX) genes. The LLPF QTL has a maximum lodscore of 4.9 and allele substitution of approximately 0.80 of a phenotypic standard deviation, and the peak is located over the CAST gene. The STADH QTL has a maximum lodscore of 3.5 and an allele substitution of approximately 0.37 of a phenotypic standard deviation, and the peak is located over the LOX gene. This suggests 2 separate likelihood peaks on the chromosome. Further analyses of meat tenderness measures in the Longissimus lumborum, LLPF and LL compression (LLC), in which outlier individuals or kill groups are removed, demonstrate large shifts in the location of LLPF QTL, as well as confirming that there are indeed 2 QTL on bovine chromosome 7. We found that both QTL are reflected in both LLPF and LLC measurements, suggesting that both these components of tenderness, myofibrillar and connective tissue, are detected by both measurements in this muscle.

Mapping quantitative trait loci for resistance to Pratylenchus thornei from synthetic hexaploid wheat in the International Triticeae Mapping Initiative (ITMI) population

Root-lesion nematode (Pratylenchus thornei) is a serious pathogen of wheat in many countries. The International Triticeae Mapping Initiative (ITMI) population of recombinant inbred lines (RILs) was assessed for resistance to P. thornei to determine the chromosome locations of the resistance genes. The ITMI population is derived from a cross between the resistant synthetic hexaploid wheat W-7984 and a susceptible bread wheat cultivar Opata 85. Two years of phenotypic data for resistance to P. thornei were obtained in replicated glasshouse trials. Quantitative trait locus (QTL) analysis was performed using available segregation and map data for 114 RILs. A QTL on chromosome 6DS showed consistent effects for reduced nematode numbers (partial resistance) across years and accounted for 11% and 23% of the phenotypic variation. A second QTL for P. thornei resistance on chromosome 2BS accounted for an additional 19% and 5%. Restriction fragment length polymorphism (RFLP) and simple sequence repeat (SSR) markers associated with the QTLs are physically located in regions rich in major genes at the distal ends of the short chromosome arms of 6D and 2B. SSR markers with potential for marker-assisted selection of P. thornei resistance effective in different genetic backgrounds have been identified.

Identification of quantitative trait loci for resistance to two species of root-lesion nematode (Pratylenchus thornei and P. neglectus) in wheat

Pratylenchus thornei and P. neglectus are two species of root-lesion nematode that cause substantial yield losses in wheat. No commercially available wheat variety has resistance to both species. A doubled-haploid population developed from a cross between the synthetic hexaploid wheat line CPI133872 and the bread wheat Janz was used to locate and tag quantitative trait loci (QTLs) associated with resistance to both P. thornei and P. neglectus. Wheat plants were inoculated with both species of nematode in independent replicated glasshouse trials repeated over 2 years. Known locations of wheat microsatellite markers were used to construct a framework map. After an initial single-marker analysis to detect marker-trait linkages, chromosome regions associated with putative QTLs were targetted with microsatellite markers to increase map density in the chromosome regions of interest. In total, 148 wheat microsatellite markers and 21 amplified fragment length polymorphism markers were mapped. The codominant microsatellite marker Xbarc183 on the distal end of chromosome 6DS was allelic for resistance to both P. thornei and P. neglectus. The QTL were designated QRlnt.lrc-6D.1 and QRlnn.lrc-6D.1, for the 2 traits, respectively. The allele inherited from CPI133872 explained 22.0-24.2% of the phenotypic variation for P. thornei resistance, and the allele inherited from Janz accounted for 11.3-14.0% of the phenotypic variation for P. neglectus resistance. Composite interval mapping identified markers that flank a second major QTL on chromosome 6DL (QRlnt.lrc-6D.2) that explained 8.3-13.4% of the phenotypic variation for P. thornei resistance. An additional major QTL associated with P. neglectus resistance was detected on chromosome 4DS (QRlnn.lrc-4D.1) and explained a further 10.3-15.4% of the phenotypic variation. The identification and tagging of nematode resistance genes with molecular markers will allow appropriate allele combinations to be selected, which will aid the successful breeding of wheat with dual nematode resistance.

Spectral analysis of pair-correlation bandwidth: Application to cell biology images

Images from cell biology experiments often indicate the presence of cell clustering, which can provide insight into the mechanisms driving the collective cell behaviour. Pair-correlation functions provide quantitative information about the presence, or absence, of clustering in a spatial distribution of cells. This is because the pair-correlation function describes the ratio of the abundance of pairs of cells, separated by a particular distance, relative to a randomly distributed reference population. Pair-correlation functions are often presented as a kernel density estimate where the frequency of pairs of objects are grouped using a particular bandwidth (or bin width), Δ>0. The choice of bandwidth has a dramatic impact: choosing Δ too large produces a pair-correlation function that contains insufficient information, whereas choosing Δ too small produces a pair-correlation signal dominated by fluctuations. Presently, there is little guidance available regarding how to make an objective choice of Δ. We present a new technique to choose Δ by analysing the power spectrum of the discrete Fourier transform of the pair-correlation function. Using synthetic simulation data, we confirm that our approach allows us to objectively choose Δ such that the appropriately binned pair-correlation function captures known features in uniform and clustered synthetic images. We also apply our technique to images from two different cell biology assays. The first assay corresponds to an approximately uniform distribution of cells, while the second assay involves a time series of images of a cell population which forms aggregates over time. The appropriately binned pair-correlation function allows us to make quantitative inferences about the average aggregate size, as well as quantifying how the average aggregate size changes with time.

Hip power analysis in individuals with transfemoral amputation: A different strategy different from stabilisation during gait stance

Introduction and Objectives Joint moments and joint powers during gait are widely used to determine the effects of rehabilitation programs as well as prosthetic fitting. Following the definition of power (dot product of joint moment and joint angular velocity) it has been previously proposed to analyse the 3D angle between both vectors, αMw. Basically, joint power is maximised when both vectors are parallel and cancelled when both vectors are orthogonal. In other words, αMw < 60° reveals a propulsion configuration (more than 50% of the moment contribute to positive power) while αMw > 120° reveals a resistance configuration (more than 50% of the moment contribute to negative power). A stabilisation configuration (less than 50% of the moment contribute to power) corresponds to 60° < αMw < 120°. Previous studies demonstrated that hip joints of able-bodied adults (AB) are mainly in a stabilisation configuration (αMw about 90°) during the stance phase of gait. [1, 2] Individuals with transfemoral amputation (TFA) need to maximise joint power at the hip while controlling the prosthetic knee during stance. Therefore, we tested the hypothesis that TFAs should adopt a strategy that is different from a continuous stabilisation. The objective of this study was to compute joint power and αMw for TFA and to compare them with AB. Methods Three trials of walking at self-selected speed were analysed for 8 TFAs (7 males and 1 female, 46±10 years old, 1.78±0.08 m 82±13 kg) and 8 ABs (males, 25±3 years old, 1.75±0.04, m 67±6 kg). The joint moments are computed from a motion analysis system (Qualisys, Goteborg, Sweden) and a multi-axial transducer (JR3, Woodland, USA) mounted above the prosthetic knee for TFAs and from a motion analysis system (Motion Analysis, Santa Rosa, USA) and force plates (Bertec, Columbus, USA) for ABs. The TFAs were fitted with an OPRA (Integrum, AB, Gothengurg, Sweden) osseointegrated implant system and their prosthetic designs include pneumatic, hydraulic and microprocessor knees. Previous studies showed that the inverse dynamics computed from the multi-axial transducer is the proper method considering the absorption at the foot and resistance at the knee. Results The peak of positive power at loading response (H1) was earlier and lower for TFA compared to AB. Although the joint power is lower, the 3D angle between joint moment and joint angular velocity, αMw, reveals an obvious propulsion configuration (mean αMw about 20°) for TFA compared to a stabilisation configuration (mean αMw about 70°) for AB. The peaks of negative power at midstance (H2) and of positive power at preswing / initial swing (H3) occurred later, lower and longer for TFA compared to AB. Again, the joint powers are lower for TFA but, in this case, αMw is almost comparable (with a time lag), demonstrating a stabilisation (almost a resistance for TFA, mean αMw about 120°) and a propulsion configuration, respectively. The swing phase is not analysed in the present study. Conclusion The analysis of hip joint power may indicate that TFAs demonstrated less propulsion and resistance than ABs during the stance phase of gait. This is true from a quantitative point of view. On the contrary, the 3D angle between joint moment and joint angular velocity, αMw, reveals that TFAs have a remarkable propulsion strategy at loading response and almost a resistance strategy at midstance while ABs adopted a stabilisation strategy. The propulsion configuration, with αMw close to 0°, seems to aim at maximising the positive joint power. The configuration close to resistance, with αMw far from 180°, might aim at unlocking the prosthetic knee before swing while minimising the negative power. This analysis of both joint power and 3D angle between the joint moment and the joint angular velocity provides complementary insights into the gait strategies of TFA that can be used to support evidence-based rehabilitation and fitting of prosthetic components.