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.

Understanding solid-state transformations during dehydration: new insights using vibrational spectroscopy and multivariate modelling

Many active pharmaceutical ingredients (APIs) have both anhydrate and hydrate forms. Due to the different physicochemical properties of solid forms, the changes in solid-state may result in therapeutic, pharmaceutical, legal and commercial problems. In order to obtain good solid dosage form quality and performance, there is a constant need to understand and control these phase transitions during manufacturing and storage. Thus it is important to detect and also quantify the possible transitions between the different forms. In recent years, vibrational spectroscopy has become an increasingly popular tool to characterise the solid-state forms and their phase transitions. It offers several advantages over other characterisation techniques including an ability to obtain molecular level information, minimal sample preparation, and the possibility of monitoring changes non-destructively in-line. Dehydration is the phase transition of hydrates which is frequently encountered during the dosage form production and storage. The aim of the present thesis was to investigate the dehydration behaviour of diverse pharmaceutical hydrates by near infrared (NIR), Raman and terahertz pulsed spectroscopic (TPS) monitoring together with multivariate data analysis. The goal was to reveal new perspectives for investigation of the dehydration at the molecular level. Solid-state transformations were monitored during dehydration of diverse hydrates on hot-stage. The results obtained from qualitative experiments were used to develop a method and perform the quantification of the solid-state forms during process induced dehydration in a fluidised bed dryer. Both in situ and in-line process monitoring and quantification was performed. This thesis demonstrated the utility of vibrational spectroscopy techniques and multivariate modelling to monitor and investigate dehydration behaviour in situ and during fluidised bed drying. All three spectroscopic methods proved complementary in the study of dehydration. NIR spectroscopy models could quantify the solid-state forms in the binary system, but were unable to quantify all the forms in the quaternary system. Raman spectroscopy models on the other hand could quantify all four solid-state forms that appeared upon isothermal dehydration. The speed of spectroscopic methods makes them applicable for monitoring dehydration and the quantification of multiple forms was performed during phase transition. Thus the solid-state structure information at the molecular level was directly obtained. TPS detected the intermolecular phonon modes and Raman spectroscopy detected mostly the changes in intramolecular vibrations. Both techniques revealed information about the crystal structure changes. NIR spectroscopy, on the other hand was more sensitive to water content and hydrogen bonding environment of water molecules. This study provides a basis for real time process monitoring using vibrational spectroscopy during pharmaceutical manufacturing.

Investigating physical properties of solid dosage forms during pharmaceutical processing : Process analytical applications of vibrational spectroscopy

In order to improve and continuously develop the quality of pharmaceutical products, the process analytical technology (PAT) framework has been adopted by the US Food and Drug Administration. One of the aims of PAT is to identify critical process parameters and their effect on the quality of the final product. Real time analysis of the process data enables better control of the processes to obtain a high quality product. The main purpose of this work was to monitor crucial pharmaceutical unit operations (from blending to coating) and to examine the effect of processing on solid-state transformations and physical properties. The tools used were near-infrared (NIR) and Raman spectroscopy combined with multivariate data analysis, as well as X-ray powder diffraction (XRPD) and terahertz pulsed imaging (TPI). To detect process-induced transformations in active pharmaceutical ingredients (APIs), samples were taken after blending, granulation, extrusion, spheronisation, and drying. These samples were monitored by XRPD, Raman, and NIR spectroscopy showing hydrate formation in the case of theophylline and nitrofurantoin. For erythromycin dihydrate formation of the isomorphic dehydrate was critical. Thus, the main focus was on the drying process. NIR spectroscopy was applied in-line during a fluid-bed drying process. Multivariate data analysis (principal component analysis) enabled detection of the dehydrate formation at temperatures above 45°C. Furthermore, a small-scale rotating plate device was tested to provide an insight into film coating. The process was monitored using NIR spectroscopy. A calibration model, using partial least squares regression, was set up and applied to data obtained by in-line NIR measurements of a coating drum process. The predicted coating thickness agreed with the measured coating thickness. For investigating the quality of film coatings TPI was used to create a 3-D image of a coated tablet. With this technique it was possible to determine coating layer thickness, distribution, reproducibility, and uniformity. In addition, it was possible to localise defects of either the coating or the tablet. It can be concluded from this work that the applied techniques increased the understanding of physico-chemical properties of drugs and drug products during and after processing. They additionally provided useful information to improve and verify the quality of pharmaceutical dosage forms

Molecular Genetic Background of Tumours in Hereditary Leiomyomatosis and Renal Cell Cancer Syndrome

Hereditary Leiomyomatosis and Renal Cell Cancer (HLRCC) is a hereditary tumour predisposition syndrome. Its phenotype includes benign cutaneous and uterine leiomyomas (CLM, ULM) with high penetrance and rarer renal cell cancer (RCC), most commonly of papillary type 2 subtype. Over 130 HLRCC families have been identified world-wide but the RCC phenotype seems to concentrate in families from Finland and North America for unknown reasons. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. FH encodes the enzyme fumarase from mitochondrial citric acid cycle. Fumarase enzyme activity or type or site of the FH mutation are unassociated with disease phenotype. The strongest evidence for tumourigenesis mechanism in HLRCC supports a hypoxia inducible factor driven process called pseudohypoxia resulting from accumulation of the fumarase substrate fumarate. In this study, to assess the importance of gene- or exon-level deletions or amplifications of FH in patients with HLRCC-associated phenotypes, multiplex ligation-dependent probe amplification (MLPA) method was used. One novel FH mutation, deletion of exon 1, was found in a Swedish male patient with an evident HLRCC phenotype with CLM, RCC, and a family history of ULM and RCC. Six other patients with CLM and 12 patients with only RCC or uterine leiomyosarcoma (ULMS) remained FH mutation-negative. These results suggest that copy number aberrations of FH or its exons are an infrequent cause of HLRCC and that only co-occurrence of benign tumour types justifies FH-mutation screening in RCC or ULMS patients. Determination of the genomic profile of 11 HLRCC-associated RCCs from Finnish patients was performed by array comparative genomic hybridization. The most common copy number aberrations were gains of 2, 7, and 17 and losses of 13q12.3-q21.1, 14, 18, and X. When compared to aberrations of sporadic papillary RCCs, HLRCC-associated RCCs harboured a distinct DNA copy number profile and lacked many of the changes characterizing the sporadic RCCs. The findings suggest a divergent molecular pathway for tumourigenesis of papillary RCCs in HLRCC. In order to find a genetic modifier of RCC risk in HLRCC, genome-wide linkage and identical by descent (IBD) analysis studies were performed in Finnish HLRCC families with microsatellite marker mapping and SNP-array platforms. The linkage analysis identified only one locus of interest, the FH gene locus in 1q43, but no mutations were found in the genes of the region. IBD analysis yielded no convincing haplotypes shared by RCC patients. Although these results do not exclude the existence of a genetic modifier for RCC risk in HLRCC, they emphasize the role of FH mutations in the malignant tumourigenesis of HLRCC. To study the benign tumours in HLRCC, genome-wide DNA copy number and gene expression profiles of sporadic and HLRCC ULMs were defined with modern SNP- and gene-expression array platforms. The gene expression array suggests novel genes involved in FH-deficient ULM tumourigenesis and novel genes with putative roles in propagation of sporadic ULM. Both the gene expression and copy number profiles of HLRCC ULMs differed from those of sporadic ULMs indicating distinct molecular basis of the FH-deficient HLRCC tumours.

Genetic Mechanisms of Tumourigenesis in von Hippel-Lindau-Associated Tumours with the Emphasis on Capillary Hemangioblastoma

The von Hippel-lindau (VHL) disease is a dominantly inherited neoplastic disorder which predisposes patients to multiple tumours including capillary haemangioblastomas (CHBs), pheochromocytomas (PCCs), renal cell carcinomas (RCCs). CHBs are the most common manifestations of VHL disease, occurring sporadically or as a manifestation of VHL disease. Inactivation of the VHL gene at 3p25-26 is believed to cause both familial and sporadic VHL-associated tumours and germ-line mutation of the VHL gene have been detected in 100% of the CHBs studied. However, a limited number of sporadic CHBs, PCCs display VHL inactivation. Other molecular alterations involved in tumourigenesis of sporadic CHBs, PCCs remain largely unknown. The purpose of the present work was to search for genetic alterations, or other mechanisms of inactivation, in addition to the VHL gene, that may be important in the development of VHL-associated tumours. Though less satisfactory than cure, prevention and early detection are the most promising and feasible means reducing cancer morbidity and mortality. This work is based on the view that increasing knowledge about the molecular events underlying tumour development will eventually aid in early detection and lead to improved treatment. We evaluated a large set of VHL-associated patients, searched for a clinical and radiologic signs of the disease. We succesfully performed a germ-line mutation analysis and characterised three patient groups, VHL, suspect VHL and sporadic, a germ-line mutation analysis revealed a 50% mutation rate only in the VHL groups, no sporadic or suspect cases displayed any mutation. We also utilized comparative genomic hybridization (CGH) to screen for DNA copy number changes in both sporadic and VHL-associated CHB. Our analysis revealed (27%) DNA copy number losses. The most common finding was loss of chromosomal arm 6q, seen in (23%) cases, No differences were noted between VHL-associated and sporadic tumours. Furthermore a loss of heterozygosity (LOH) study on chromosome 3p and 6q was done with the purpose to determine allele losses not observable by CGH, and to uncover the location of putative tumour suppressor genes important in CHB and PCC tumourigenesis. We identified loss of chromosome 6q and a minimal deleted area at 6q23-24 in CHBs. We also showed LOH at 6q23-24 in PCCs and identified the ZAC1 (6q24-25) as a candidate gene, ZAC1 is a maternally imprinted tumour suppressor gene with anti proliferative properties. To study further the role of ZAC inactivation in CHBs, we investigated LOH, promoter hypermethylation and expression status of the ZAC1 gene in mainly sporadic CHBs. Our LOH analysis revealed that the majority of the tumours with allele loss. The gene promoter methylation analysis similarly detected predominance of the methylated ZAC sequence in almost all tumours. Immunohistochemistry exhibited a strongly reduced expression of ZAC in stromal cells of all CHBs studied. Our current results indicate that the absence of the unmethylated, ZAC1 promoter sequence was highly concurrent with LOH for the ZAC1 region or 6q loss. This observation together with lack of ZAC expression, points to preferential loss of the non imprinted, expressed ZAC allele in CHB, in summary, our series of studies reveal a new chromosomal region 6q, emphasizes the importance of ZAC1 gene in the development of CHB and PCC, particularly in non-VHL associated cases.

Computational studies on new chemical species in the gas-phase and the solid-state.

There is intense activity in the area of theoretical chemistry of gold. It is now possible to predict new molecular species, and more recently, solids by combining relativistic methodology with isoelectronic thinking. In this thesis we predict a series of solid sheet-type crystals for Group-11 cyanides, MCN (M=Cu, Ag, Au), and Group-2 and 12 carbides MC2 (M=Be-Ba, Zn-Hg). The idea of sheets is then extended to nanostrips which can be bent to nanorings. The bending energies and deformation frequencies can be systematized by treating these molecules as an elastic bodies. In these species Au atoms act as an 'intermolecular glue'. Further suggested molecular species are the new uncongested aurocarbons, and the neutral Au_nHg_m clusters. Many of the suggested species are expected to be stabilized by aurophilic interactions. We also estimate the MP2 basis-set limit of the aurophilicity for the model compounds [ClAuPH_3]_2 and [P(AuPH_3)_4]^+. Beside investigating the size of the basis-set applied, our research confirms that the 19-VE TZVP+2f level, used a decade ago, already produced 74 % of the present aurophilic attraction energy for the [ClAuPH_3]_2 dimer. Likewise we verify the preferred C4v structure for the [P(AuPH_3)_4]^+ cation at the MP2 level. We also perform the first calculation on model aurophilic systems using the SCS-MP2 method and compare the results to high-accuracy CCSD(T) ones. The recently obtained high-resolution microwave spectra on MCN molecules (M=Cu, Ag, Au) provide an excellent testing ground for quantum chemistry. MP2 or CCSD(T) calculations, correlating all 19 valence electrons of Au and including BSSE and SO corrections, are able to give bond lengths to 0.6 pm, or better. Our calculated vibrational frequencies are expected to be better than the currently available experimental estimates. Qualitative evidence for multiple Au-C bonding in triatomic AuCN is also found.

Spectroscopy and photochemistry of hydrogen peroxide and its complexes in solid rare gases

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Agreeable Agreement : An Examination into the Quest for a Solid Consensus in Light of Bilateral Doctrinal Dialogues among Anglicans, Lutherans and Roman Catholics

The purpose of this study is to analyse the development and understanding of the idea of consensus in bilateral dialogues among Anglicans, Lutherans and Roman Catholics. The source material consists of representative dialogue documents from the international, regional and national dialogues from the 1960s until 2006. In general, the dialogue documents argue for agreement/consensus based on commonality or compatibility. Each of the three dialogue processes has specific characteristics and formulates its argument in a unique way. The Lutheran-Roman Catholic dialogue has a particular interest in hermeneutical questions. In the early phases, the documents endeavoured to describe the interpretative principles that would allow the churches to together proclaim the Gospel and to identify the foundation on which the agreement in the church is based. This investigation ended up proposing a notion of basic consensus , which later developed into a form of consensus that seeks to embrace, not to dismiss differences (so-called differentiated consensus ). The Lutheran-Roman Catholic agreement is based on a perspectival understanding of doctrine. The Anglican-Roman Catholic dialogue emphasises the correctness of interpretations. The documents consciously look towards a common future , not the separated past. The dialogue s primary interpretative concept is koinonia. The texts develop a hermeneutics of authoritative teaching that has been described as the rule of communion . The Anglican-Lutheran dialogue is characterised by an instrumental understanding of doctrine. Doctrinal agreement is facilitated by the ideas of coherence, continuity and substantial emphasis in doctrine. The Anglican-Lutheran dialogue proposes a form of sufficient consensus that considers a wide set of doctrinal statements and liturgical practices to determine whether an agreement has been reached to the degree that, although not complete , is sufficient for concrete steps towards unity. Chapter V discusses the current challenges of consensus as an ecumenically viable concept. In this part, I argue that the acceptability of consensus as an ecumenical goal is based not only the understanding of the church but more importantly on the understanding of the nature and function of the doctrine. The understanding of doctrine has undergone significant changes during the time of the ecumenical dialogues. The major shift has been from a modern paradigm towards a postmodern paradigm. I conclude with proposals towards a way to construct a form of consensus that would survive philosophical criticism, would be theologically valid and ecumenically acceptable.

Bone Health in Children and Adolescents with Juvenile Idiopathic Arthritis and Solid Organ Transplant

Osteoporosis is a skeletal disorder characterized by compromised bone strength that predisposes to increased fracture risk. Childhood and adolescence are critical periods for bone mass gain. Peak bone mass is mostly acquired by the age of 18 years and is an important determinant of adult bone health and lifetime risk for fractures. Medications, especially glucocorticoids (GCs), chronic inflammation, decreased physical activity, hormonal deficiencies, delayed puberty, and poor nutrition may predispose children and adolescents with a chronic disease to impaired bone health. In this work, we studied overall bone health, the incidence and prevalence of fractures in children and adolescents who were treated for juvenile idiopathic arthritis (JIA) or had undergone solid organ transplantation. The first study cohort included 62 patients diagnosed with JIA and treated with GCs. The epidemiology of fractures after transplantation was investigated in 196 patients and a more detailed analysis of bone health determinants was performed on 40 liver (LTx) and 106 renal (RTx) transplantation patients. Bone mineral density (BMD) and vertebral morphology were assessed by dual-energy x-ray absorptiometry. Standard radiographs were obtained to detect vertebral fractures and to determine bone age; BMD values were adjusted for skeletal maturity. Our study showed that median BMD values were subnormal in all patient cohorts. The values were highest in patients with JIA and lowest in patients with LTx. Age at transplantation influenced BMD values in LTx but not RTx patients; BMD values were higher in patients who had LTx before the age of two years. BMD was lowest during the immediate posttransplantation years and increased subnormally during puberty. Delayed skeletal maturation was common in all patient groups. The prevalence of vertebral fractures ranged from 10% to 19% in the cohorts. Most of the fractures were asymptomatic and diagnosed only at screening. Vertebral fractures were most common in LTx patients. Vitamin D deficiency was common in all patient groups, and only 3% of patients with JIA and 25% of transplantation patients were considered to have adequate serum vitamin D levels. The total cumulative weight-adjusted dose of GC was not associated with BMD values in JIA or LTx patients. The combination of female gender and age over 15 years, parathyroid hormone concentration over 100 ng/L, and cumulative weight-adjusted methylprednisolone dose over 150 mg/kg during the three preceding years were found to be important predictors for low lumbar spine BMD in RTx patients. Based on the high prevalence of osteoporosis in the study cohorts more efforts should be put to prevention and early diagnosis of osteoporosis in these pediatric patients.