Palladium(0)-catalyzed intramolecular annulation of twelve 1,3-disubstituted cyclopentenes, derived from (+)-vince lactam, resulted in 5-exo cyclizations, furnishing a series of 2,5-dimethyl-1-((3R,4'S)-2H-spiro[benzofuran-3,1'-cyclopentan]-2'-en-4'-yl)-1H-pyrroles in excellent diastereoselectivities and useful isolated yields. The double bond migration process, following the arylpalladium insertion, was controlled by fine-tuning of the reaction system, providing regioselectivities of up to 98:2. The selective Mizoroki-Heck reaction was used as the key transformation for preparing two new spirocyclic monoprotected amino acids as single diastereoisomers.

The palladium(0)-catalyzed intramolecular annulation of 12 1,3-disubstituted cyclopentenes, derived from (+)-vincelactam, resulted in 5-exo cyclizations which furnished a series of 2,5-dimethyl-14(3R,4'S)-2H-spiro[benzofuran-3,1'-cyclopentan]2'-en-4'-yl)-1H-pyrroles in excellent diastereoselectivities and useful isolated yields. The double bond migration process that followed the arylpalladium insertion was controlled by a fine-tuning of the reaction system, which provided regioselectivities of up to 98:2. The selective Mizoroki-Heck reaction was used as the key transformation for preparing two new spirocyclic monoprotected amino acids as single stereoisomers.

Abstract

Background: Out of approximately 1,6 million children in Sweden, about 350 are diagnosed with cancer each year. Chemotherapeutic agents are commonly used in the treatment of various cancer types in pediatric oncology. While it is established that certain chemotherapeutic agents induce hepatotoxicity as a side effect, a comprehensive assessment of the prevalence of hepatotoxicity in pediatric oncology populations has been lacking.

Aim: This study aims to investigate the prevalence of hepatotoxicity in pediatric patients between 0-18 years with intracranial and extracranial solid tumors. 

Method: A retrospective cohort study was conducted, at Uppsala University Children’s Hospital. Data regarding administered chemotherapeutic treatment and liver values such as alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), bilirubin and lactate dehydrogenase (LD), were collected over a 4-month period from start of treatment. Tto identify the treatment cycle in which hepatotoxicity occurred the data were divided into treatment cycles. During each treatment cycle, the liver values were collected on day 1, 8, 15, 22 or adjacent days. For patients experiencing hepatotoxicity, it was graded according to Common Terminology Criteria for Adverse Events (CTCAE) version 5. 

Results: Fifty patients were included, and the study yielded four main findings: 1) Approximately 30% of pediatric oncology patients experienced hepatotoxicity, 2) Treatment adjustments were implemented, such as dose reduction and treatment pause, for those with grade 3 and 4 toxicity, 3) Hepatotoxicity tends to exhibit a higher prevalence in males, 4) Hepatotoxicity tends to exhibit a higher prevalence in younger children. 

Conclusion: The observed prevalence of hepatotoxicity exceeded initial expectations, with approximately 30% suffer from hepatotoxicity due to chemotherapeutic treatment. However, future studies with larger sample size are needed. 

Factors considered important for the interparticulate bonding structure and mechanical strength of pharmaceutical compacts were studied in this thesis.

Fractures appear to propagate mainly around rather than through grains during strength testing. Large deviations from theoretical strength values in addition to an effect of particle size were thus obtained when compaction was performed to zero porosity or obtained by extrapolation to zero porosity. When high compaction loads were used, the excess energy was to a large extent used for elastic recovery and/or alteration of the solid-state structure.

Filtering out of weak distance forces (intermolecular forces) by compaction in a liquid with a sufficiently high dielectric constant appears to provide reliable information on interparticulate bonding mechanisms. The best correlation between physiochemical properties of the liquids and the gradual decrease in tensile strength of the compacts was achieved using the dielectric constant. The weak distance forces appeared to be screened out when the liquid compaction medium had a dielectric constant of 18. The remaining tensile strength was then believed to be the result of interparticulate bonding by solid bridges for most materials. However, for most pharmaceutical materials, weak distance forces seem to dominate. Of all the materials tested, solid bridges seemed to be the most important bonding mechanism for sodium and potassium chloride. Increasing the particle size and compaction pressure of materials with the capacity to form solid bridges seemed to facilitate the bond formation process. Addition of a dry binder or milling the particles counteracted the formation of solid bridges, probably by reducing the concentration of stress at certain points in the compact, a prerequisite for the establishment of solid bridges.

Both the tablet surface area and the interparticulate distance may affect the proportion of external surface area participating in interparticulate bonding. For materials prone to develop solid bridges, the actual surface area involved in bond formation is more important than the space between the particles, i.e. compensation of the tensile strength of a tablet for the surface area and the mean interparticulate distance will probably not reflect the nature of the dominating bond type. However, for the other materials, ranking of the materials according to tensile strength adjusted for surface area and mean interparticulate distance gave a reflection of dominating interparticulate bonding type.

The molecular factors regulating interspecific interaction between the saprotrophic biocontrol fungus Phlebiopsis gigantea and the conifer pathogen Heterobasidion parviporum were investigated. We constructed cDNA libraries and used expressed sequence tag analysis for the identification and characterization of genes expressed during the self and nonself-hyphal interaction. cDNA clones from either the pathogen or biocontrol agent were arrayed on nylon membrane filters and differentially screened with cDNA probes made from mycelia forming the barrage zone during nonself-interactions, mycelia growing outside the barrage zones or monocultures. BlastX analysis of the differentially expressed clones led to the identification of genes with diverse functions, including those with potential as virulence factors, such as hydrophobins. Because of the high sequence conservation (r2 = 0.81) between P. gigantea and H. parviporum, a selected number of genes from either fungus were used to monitor the expression profile under varying interaction conditions by virtual northern blot. The results are discussed with respect to the potential role of the induced genes during the nonself-competitive interaction for space and nutrients between P. gigantea and H. parviporum.

This study explores the genetic basis of the excitability trait and its correlation with hair whorl patterns in Swedish Standardbred trotters. Understanding these connections can provide insights into equine temperament, which is crucial for breeding and managing horses in sports and recreational activities. We investigated 49 trotters, collecting data on their hair whorls and conducting behaviour tests to assess their excitability. DNA was extracted from tail hair samples, and significant SNPs were identified through a genome-wide association study (GWAS) from a previous study on horse temperaments. The novel object test was used to measure excitability levels, ranking horses on a scale from 1 (very calm) to 7 (extremely excitable). The horses were categorized based on their hair whorl types: radial, clockwise, counterclockwise, and feather. Statistical analysis using Pearson’s Chi-Squared Test revealed no significant association between whorl types and excitability scores (p-value = 0.6932). Significant SNPs were identified on chromosomes 3, 8, 15, and 23, with the top 20 SNP locations selected for each chromosome based on FST values. These SNPs were annotated using the NCBI horse genome browser (equCab3). Notably, a SNP within the DIRAS2 gene, which has been implicated in attention deficit/hyperactivity disorder (ADHD) in humans, was highlighted. This SNP was further analysed using a TaqMan assay designed for this sequence.

We present a method for interpretation of conformal prediction models. The discrete gradient of the largest p-value is calculated with respect to object space. A criterion is applied to identify the most important component of the gradient and the corresponding part of the object is visualized. The method is exemplified with data from drug discovery relating chemical compounds to mutagenicity. Furthermore, a comparison is made to already established important subgraphs with respect to mutagenicity and this initial assessment shows very useful results with respect to interpretation of a conformal predictor.

Drug transport across cells and cell membranes in the human body is crucial for the pharmacological effect of drugs. Active transport governed by transport proteins plays an important role in this process. A vast number of transport proteins with a wide tissue distribution have been identified during the last 15 years. Several important examples of their role in drug disposition and drug-drug interactions have been described to date. Investigation of drug-drug interactions at the transport protein level are therefore of increasing interest to the academic, industrial and regulatory research communities.

The gene expression of transport proteins involved in drug transport was investigated in the jejunum, liver, kidney and colon to better understand their influence on the ADMET properties of drugs. In addition, the gene and protein expression of transport proteins in cell lines, widely used for predictions of drug transport and metabolism, was examined.

The substrate and inhibitor heterogeneity of many transport proteins makes it difficult to foresee whether the transport proteins will cause drug-drug interactions. Therefore, in vitro assays for OCT1 and OATP1B1, among the highest expressed transport proteins in human liver, were developed to allow investigation of the inhibitory patterns of these proteins. These assays were used to investigate two data sets, consisting of 191 and 135 registered drugs and drug-like molecules for the inhibition of OCT1 and OATP1B1, respectively. Numerous new inhibitors of the transport proteins were identified in the data sets and the properties governing inhibition were determined. Further, antidepressant drugs and statins displayed strong inhibition of OCT1 and OATP1B1, respectively. The inhibition data was used to develop predictive in silico models for each of the two transport proteins.

The highly polymorphic nature of some transport proteins has been shown to affect drug response and may lead to an increased risk of drug-drug interactions, and therefore, the OCT1 in vitro assay was used to study the effect of common genetic variants of OCT1 on drug inhibition and drug-drug interactions. The results indicated that OCT1 variants with reduced function were more susceptible to inhibition. Further, a drug-drug interaction of potential clinical significance in the genetic OCT1 variant M420del was proposed.

In summary, gene expression of transport proteins was investigated in human tissues and cell lines. In vitro assays for two of the highest expressed liver transport proteins were used to identify previously unknown SLC transport protein inhibitors and to develop predictive in silico models, which may detect previously known drug-drug interactions and enable new ones to be identified at the transport protein level. In addition, the effect of genetic variation on inhibition of the OCT1 was investigated.

Common genetic variants of the liver-specific human organic cation transporter 1 (OCT1; SLC22A1) have reduced transport capacity for substrates such as the antidiabetic drug metformin. The effect of the reduced OCT1 function on drug interactions associated with OCT1 has not been investigated and was, therefore, the focus of the study presented here. HEK293 cells expressing human OCT1-reference or the variants R61C, V408M, M420del and G465R were first used to study the kinetics and inhibition pattern of the OCT1 substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). In the second part OCT1-mediated (14)C-metformin uptake was studied in the presence of drugs administered concomitantly with metformin. Transport studies using ASP(+) showed that the function of the variants decreased in the following order: OCT1-reference = V408M = M420del >R61C > >G465R. Variants M420del and R61C were more sensitive to drug inhibition, with IC(50) values up to 23 times lower than those of the OCT1-reference. Uptake studies using (14)C-metformin were in qualitative agreement with those using ASP(+), with the exception that a larger reduction in transport capacity was observed for M420del. Concomitantly administered drugs, such as verapamil and amitriptyline, revealed potential drug-drug interactions at clinical plasma concentrations of metformin for OCT1-M420del.

The aim of this study was to investigate the gene and protein expression profiles of important drug transporting proteins in human cell lines commonly used for studies of drug transport mechanisms. Human cell lines used to transiently or stably express single transporters (HeLa, HEK293) and leukaemia cell lines used to study drug resistance by ABC-transporters (HL-60, K562) were investigated, and compared with organotypic cell lines (HepG2, Saos-2, Caco-2 and Caco-2 TC7). For gene expression studies, real-time PCR was used, while monospecific polyclonal antibodies were generated and used to investigate protein expression by immunohistochemistry. Thirty-six transporters were studied for gene expression and nine for protein expression. The antibodies were validated using expression patterns in human tissues. Finally, the function of one ubiquitously expressed transporter, MCT1; SLC16A1 was investigated using 14C-lactic acid as a substrate. In general, the adherent cell lines (HeLa, HEK293) displayed low transporter expression and the expression patterns were barely affected by transfection. The leukaemia cell lines (K562, HL-60) and Saos-2 also had low endogenous transporter expression, while the organotypic cell lines (HepG2 and Caco-2) showed higher expression of some transporters. Comparison of gene and protein expression profiles gave poor correlations, but better agreement was obtained for antibodies with a good validation score, indicating that antibody quality was a significant variable. Importantly, the monocarboxylic acid transporting protein MCT1 was significantly expressed in all, and functional in most of the cell lines, indicating that MCT1 may be a confounding factor when the transport of small anionic drugs is investigated.

Resistance to anti-HIV protease drugs is a major problem in the design of AIDS drugs with long-term efficacy. To identify structural features associated with a certain resistance profile, the inhibitory properties of a series of symmetric and asymmetric cyclic sulfamide, cyclic urea and linear transition-state analogue inhibitors of HIV-1 protease were investigated using wild-type and mutant enzyme. To allow a detailed structure-inhibition analysis, enzyme with single, double, triple and quadruple combinations of G48V, V82A, 184V and L90M substitutions was used. Kinetic analysis of the mutants revealed that catalytic efficiency was 1-30% of that for the wild-type enzyme, a consequence of reduced kcat in all cases and an increased KM for all mutants except for the G48V enzyme. The overall structure-inhibitory profiles of the cyclic compounds were similar, and the inhibition of the V82A, 184V and G48V/L90M mutants were less efficient than of the wild-type enzyme. The greatest increase in Ki was generally observed for the 184V mutant and least for the G48V/L90M mutant, and additional combinations of mutations did not result in improved inhibition profiles for the cyclic compounds. An extended analysis of additional mutants, and including a set of linear compounds, showed that the profile was unique for each compound, and did not reveal any general structural features associated with a certain inhibition profile. The effects of structural modifications in the inhibitors, or of mutations, were not additive and they differed depending on their context. The results demonstrate the difficulties in predicting resistance, even for closely related compounds, and designing compounds with improved resistance profiles.

Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus^TM (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project.

Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters.

On average, PK parameters (Area Under the Concentration-time curve (AUC_0-tlast), Maximal concentration (C_max), half-life (t_1/2)) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC_0-tlast and around 90% of the simulations were within 10-fold error for AUC_0-tlast. Oral bioavailability (F_oral) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC_0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC_0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise.

Abstract

Suicide is a public health problem that, in addition to loss of human life, leads to extensive psychological suffering and impairment of the health of relatives. It is common that suicide occurs under the influence of mental illness such as personality disorders and depression. Depression is a serious condition that often causes severe suffering. Depression can affect all ages, i.e. children, adolescents, adults and the elderly and it is as costly as heart disease. In Sweden, depression is one of the most common psychiatric diagnoses. About 19 percent of the population (16-84 years) have been diagnosed with depression at least once in their lifetime.  Of these, almost one in three have been diagnosed more than once. The affected individual performs poorly in daily activities such as school, work and in social settings. Additionally, there is increased risk of suicide in depressed individuals. Accordingly, it is important to treat depression to reduce suffering. Depression in children was accepted as the same disease as in adults since 1980. Today, depression is treated primarily with first-line therapy SSRIs. The aim of this work was to examine the effect and safety of antidepressants in the treatment of major depresive disorder in chlidren and adolescents with special reference to suicidal activity and self-injury. To implement this study, scientific articles were obtained in Pubmed, and five articles were selected. Study 1 showed that the combination of fluoxetine and CBT, cognitive behavior therapy, had better effect than treatment with only flouxetin or with only CBT. Study 2 showed that suicidal events and ideation were least among the group treated with the combination of fluoxetine and CBT compared to the treatment with only fluoxetine or only CBT. Study 3 showed that more suicide-related events occurred among the group with previous non-suicidal self-injury, NSSI. Study 4 resulted in greater medical response and better remission in escitalopram patients compared to placebo. Study 5 showed that treatment with venlafaxine caused serious adverse events that led to many discontinuing treatment. Treatment with antidepressants, especially in combination with CBT, can reduce the severity of depression in children and adolescents and reduce suicidal ideation and suicide attempts in some patients. In cases of treatment failure a risk of suicide and self-injury remains. Previous self-injury increases the risk of future self-injury as well as the risk of future suicide attempts.

Investigation of the aerial parts of Laggera crispata and Pluchea carolonesis afforded in addition to several known compounds, three new eudesmane derivatives, 3β,4α-dihydroxy-7-epi-eudesm-11(13)-ene, 3α-(2′,3′-dihydroxy-2′-methylbutanoyl)-4,11-dihydroxy-6,7-dehydroeudesman-8-one and 3α-(3′-chloro-2′-hydroxy-2′-methylbutanoyl)cuauhtemone. The structures were elucidated by spectroscopic methods

Objective. In rheumatoid arthritis (RA), pain and joint destruction are initiated and propagated by the production of proinflammatory mediators. Synthesis of these mediators is regulated by the transcription factor NF-kappa B, which is controlled by the ubiquitin proteasome system (UPS). The present study explored the effects of the proteasome inhibitor MG132 on inflammation, pain, joint destruction, and expression of sensory neuropeptides as markers of neuronal response in a rat model of arthritis. Methods. Arthritis was induced in rats by injection of heat-killed Mycobacterium butyricum. Arthritis severity was scored, and nociception was evaluated by mechanical pressure applied to the hind paw. Joint destruction was assessed by radiologic and histologic analyses. NF-kappa B DNA-binding activity was analyzed by electromobility shift assay, and changes in the expression of the p50 NF-kappa B subunit and the proinflammatory neuropeptides substance P (SP) and calcitonin generelated peptide (CGRP) were detected by immunohistochemistry. Results. Arthritic rats treated with MG132 demonstrated a marked reduction in inflammation, pain, and joint destruction. The elevated DNA-binding activity of the NF-kappa B/p50 homodimer and p50, as well as the neuronal expression of SP and CGRP, observed in the ankle joints of arthritic rats were normalized after treatment with MG132. Conclusion. In arthritic rats, inhibition of proteasome reduced the severity of arthritis and reversed the pain behavior associated with joint inflammation. These effects may be mediated through the inhibition of NF-kappa B activation and may possibly involve the peripheral nervous system. New generations of nontoxic proteasome inhibitors may represent a novel pharmacotherapy for RA.

In the present work, the determination of omeprazole (OME) enantiomers in oral fluid and plasma samples was carried out utilizing microextraction by packed sorbent (MEPS) and liquid chromatography-tandem mass spectrometry. A chiral column with cellulose-SB phase was used for the first time for enantiomeric separation of OME with an isocratic elution system using 0.2% ammonium hydroxide in hexane-ethanol mixture (70 : 30, v/v) as the mobile phase. OME enantiomers were determined utilizing a triple quadrupole tandem mass spectrometer in positive ion mode (ESI+) monitoring mass transitions: m/z 346.3 -> 198.0 for OME and m/z 369.98 -> 252.0 for internal standard. The limits of detection and quantification of the present method for both enantiomers were 0.1 and 0.4 ng/mL, respectively. The method validation provided good accuracy and precision. The matrix effect factor was less than 5%, and no interfering peaks were observed. The interday precision values ranged from 2.2 to 7.5 (%RSD), and the accuracy of determinations varied from -9.9% to 8.3%. In addition, the pharmacokinetics (PK) of omeprazole enantiomers in healthy subjects after a single oral dose was investigated. (S)-Enantiomers showed higher levels than (R)-enantiomers throughout 24 h. It was found that the mean maximum concentrations of (R)- and (S)-omeprazole in plasma samples were about two times higher than in oral fluid.

Bakgrund: Akut koronart syndrom (AKS) är samlingsbegrepp för instabil angina, icke SThöjningsinfarkt (NSTEMI) och ST-höjningsinfarkt. AKS är en vanlig folksjukdom hos äldre och ungefär hälften av alla över 65 år har kranskärlssjukdom. Kranskärlssjukdom förorsakar cirka 30% av alla dödsfall och är den ledande dödsorsaken globalt. Huvudorsaken till AKS är ateromatös plack som rupturerar och resulterar en ocklusion av kärlen. Perkutan koronarintervention (PCI) utförs som en terapeutisk procedur för att öppna kranskärlen. Efter PCI med stent behövs dubbel trombocythämning under en begränsad tid (1 månad till 1 år) för att motverka reocklusion, därefter behandlas patienter enbart med acetylsalicylsyra (ASA). ASA är det klassiska trombocythämmande läkemedlet och utnyttjas i stort sätt inom hjärt-och kärlsjukdomar. Syfte: Att undersöka nytta-risk balansen med långtidsbehandling av ASA efter AKS jämfört med avbrytande av behandling efter ett år och om tillägg av PPI kan minska blödningsrisken som ASA medför. Metod: En systematisk litteratursökning gjordes i databaserna Pubmed och CINAHL år 2019 respektive 2020. Relevanta studietyper var metaanalyser, kohort, fallkontrollstudier och observationsstudier. Studiernas relevans och kvalitet granskades med hjälp av SBU:s granskningsmallar. Resultat: Nio studier inkluderades, där studieperioden var mellan 6-48 månader. Kvaliteten på dem inkluderade studierna bedömdes vara medelhög. Det framkom att ASA minskar successivt risken för reinfarkt och kardiovaskulär död med en RR på 0,80 i sekundärprevention. Blödningsrisken som ASA medför är högre hos äldre patienter men genom tillägg av protonpumpshämmare kan detta motverkas. Det framkom även att avbrott med ASA behandling ökar risken för reinfarkt och kardiovaskulär mortalitet med >30%. Slutsats: En livslång ASA behandling är viktigt för äldre patienter som upplevt AKS och avbrott av behandlingen ökar risken för reinfarkt och kardiovaskulär mortalitet. Detta påstående framkom 2 av dem inluderade artiklarna med hög respektiv medelhög kvalite. Ett avbrott av ASA behandlingen bör inte därför vara aktuellt för äldre. ASA:s effekt är viktigt och önskvärd efter AKS hos äldre, den sammanlagda nytta-riskbalansen är positiv och det vetenskapliga underlaget för detta påstående är måttligt starkt. Blödningsrisken kan minskas genom att kombinera PPI med ASA, dock behövs fler studier i större population för att stödja detta påstående.

Background and objective: Holistic patient care is dependent upon the accurate and reliable flow of information between care settings and healthcare professionals (HCP). Inaccurate medical records may increase the risk of patient harm as HCPs risk continuing to prescribe a medication that has been discontinued or re-prescribing a medication that has previously been discontinued. The aim of this project was to analyse the HCPs documentation of medical changes in the patients’ medical records and evaluate the extent of adherence to a predefined criteria within the regions guidelines at six general practices (GP) across Jönköping County.

Study design: A retrospective observational study, reviewing 240 medical records between June-December 2019.

Main outcome measures: The number and the type of medication changes registered, and the extent to which the documentations adhered to regional guidelines concerning the criteria of the information that ought to be included in a documentation upon a registration of a medical change.

Results: There was an average of 63 (95% CI:53.4 – 73.2) medical changes identified across the six GPs. None of the documentations completely adhered to all points in the guideline. Follow-up plans was found to be the most omitted data point having only been mentioned in 2% of the medical changes. The name of the prescribed medication was mentioned on 378 occasions however, it was only accompanied with dosage form on 44% of occasions. In 9% of the medical records that were analysed it was not found to include any medical changes at all, instead it was either filled in with a “0” or the HCPs had for instance confirmed that the patients’ prescriptions had been renewed.

Conclusions: Despite the clear guidance regarding the content of documentation for a medical change, no documentation followed the guidelines fully. Adherence to the guidelines may be enhanced by being easier to access for HCPs. As there were documents that were not classed as medical changes identified, the information regarding what is acceptable to be included in that section needs to be made clearer to the HCPs. The findings from this study can be further developed and used as a reference for future studies on this subject.

PURPOSE: To evaluate the likelihood-based methods for handling data below the quantification limit (BQL) using new features in NONMEM VI. METHODS: A two-compartment pharmacokinetic model with first-order absorption was chosen for investigation. Methods evaluated were: discarding BQL observations (M1), discarding BQL observations but adjusting the likelihood for the remaining data (M2), maximizing the likelihood for the data above the limit of quantification (LOQ) and treating BQL data as censored (M3), and like M3 but conditioning on the observation being greater than zero (M4). These four methods were compared using data simulated with a proportional error model. M2, M3, and M4 were also compared using data simulated from a positively truncated normal distribution. Successful terminations and bias and precision of parameter estimates were assessed. RESULTS: For the data simulated with a proportional error model, the overall performance was best for M3 followed by M2 and M1. M3 and M4 resulted in similar estimates in analyses without log transformation. For data simulated with the truncated normal distribution, M4 performed better than M3. CONCLUSIONS: Analyses that maximized the likelihood of the data above the LOQ and treated BQL data as censored provided the most accurate and precise parameter estimates.

Hepatocellular carcinoma (HCC) causes ~ 600,000 deaths annually, making it the second most deadly cancer form. HCC is classified into five stages and for the intermediate HCC treatment, the two most commonly used drug delivery systems (DDSs) are lipiodol-based emulsions and drug-eluting beads. The aims of this thesis were to develop in vitro methods suitable for studying these DDSs. It is important to investigate the release mechanisms and release rates with relevant in vitro methods, as this can improve the understanding of the in vivo performance. Miniaturized in vitro methods with sample reservoirs separated from the release medium by a diffusion barrier were developed and shown to be suitable for studying drug release from particle DDSs (Paper I). In Paper II these methods were further developed and used to study the release of doxorubicin (DOX) from the clinically used drug-eluting beads. DOX release rates were affected by the method set-up and the characteristics of the release medium. The choice of method and volume of release medium could improve the in vivo-likeness of the in vitro release profiles. Applied theoretical models suggested a film-controlled type of DOX release mechanism from the beads when self-aggregation, DOX-bead interaction, and DOX deprotonation were taken into account.

A micropipette-assisted microscopy method was used to further improve the understanding of the release mechanism of amphiphilic molecules from the beads (Paper III). A detailed analysis suggested an internal depletion-layer model dependent on molecular self-aggregation for the release. It was further suggested that a simple ion-exchange mechanism is unrealistic in physiological conditions.

The important pharmaceutical factors for the emulsion-based formulations were investigated in Paper IV. DOX solubility, lipid phase distribution, and emulsion stability increased when the contrast agent iohexol was added. Also, an increase in release half-life (h) was observed from emulsions with iohexol.

The in vitro methods and theoretical models presented in this thesis can be used during development and optimization of future DDSs.

Microgels, such as polymeric hydrogels, are currently used as drug delivery devices (DDSs) for chemotherapeutics and/or unstable drugs. The clinical DDS DC bead® was studied with respect to loading and release, measured as relative bead-volume, of six amphiphilic molecules in a micropipette-assisted microscopy method. Theoretical models for loading and release was used to increase the mechanistic understanding of the DDS.

It was shown that equilibrium loading was independent of amphiphile concentration. The loading model showed that the rate-determining step was diffusion of the molecule from the bulk to the bead surface (‘film control’). Calculations with the developed and applied release model on the release kinetics were consistent with the observations, as the amphiphiles distribute unevenly in the bead. The rate determining step of the release was the diffusion of the amphiphile molecule through the developed amphiphile-free depletion layer. The release rate is determined by the diffusivity and the tendency for aggregation of the amphiphile where a weak tendency for aggregation (i.e. a large cac_b) lead to faster release. Salt was necessary for the release to happen, but at physiological concentrations the entry of salt was not rate-determining. This study provides valuable insights into the loading to and release from the DDS. Also, a novel release mechanism of the clinically used DDS is suggested.

We have evaluated a miniaturized in vitro method, based on the mDISS Profiler (TM) technique that enables on-line monitoring of drug release from a 21 mu l sample with 10 ml of release medium. Four model drugs in eight clinically used formulations, including both solid and non-solid drug delivery systems, were investigated. The acquired data were compared with historical in vitro release data from the same formulations. Use of the Weibull function to describe the in vitro drug-release profiles allowed discrimination between the selected formulations with respect to the drug-release mechanisms. Comparison of the release data from the same formulation in different in vitro set-ups showed that the methodology used can affect the mechanism of in vitro release. We also evaluated the ability of the in vitro methods to predict in vivo activity by comparing simulated plasma concentration-time profiles acquired from the application of the biopharmaceutical software GI-Sim to the in vitro observations. In summary, the simulations based on the miniaturized-method release data predicted the plasma profiles as well as or more accurately than simulations based on the historical release data in 71% of the cases and this miniaturized in vitro method appears to be applicable for both solid and non-solid formulations.

The release rate of doxorubicin (DOX) from the drug-delivery system (DDS), DC Bead, was studied by 2 miniaturized in vitro methods: free-flowing and sample reservoir. The dependencies of the release mechanisms on in vitro system conditions were investigated experimentally and by theoretical modeling. An inverse relationship was found between release rates and bead size, most likely due to the greater total surface area. The release rates correlated positively with temperature, release medium volume, and buffer strength, although the release medium volume had larger effect than the buffer strength. The sample reservoir method generated slower release rates, which described the in vivo release profile more accurately than the free-flowing method. There was no difference between a pH of 6.3 or 7.4 on the release rate, implying that the slightly acidic tumor microenvironment is less importance for drug release. A positive correlation between stirring rate and release rate for all DDS sizes was observed, which suggests film controlled release. Theoretical modeling highlighted the influence of local equilibrium of protonation, self-aggregation, and bead material interactions of DOX. The theoretical release model might describe the observed larger sensitivity of the release rate to the volume of the release medium compared to buffer strength. A combination of miniaturized in vitro methods and theoretical modeling are useful to identify the important parameters and processes for DOX release from a micro gel-based DDS.

Hydrogel-based localised drug delivery minimises systemic side effects and a linear release profile ensuring a sustained drug release over time, crucial for long-term therapy. The current paper describes the use of the Copper(I)-catalyzed Azide-Alkyne Cycloaddition (CuAAc) to append azidified Dexamethasone (Dex) onto dendrons of first- and second-generation PEGs. Crosslinking with thiolated PEGs using either thiol-acrylate or nucleophilic addition reactions yielded gels containing β-thio-ether ester groups that imparted enhanced hydrolytic susceptibility. In vitro gel degradation was followed gravimetrically and expressed as swelling ratios. Thiol-acrylate crosslinked hydrogels exhibited zero-order Dex release kinetics over 11, 27, and 16 days (G1, G1-star, and G2). Crosslinking the G1-gels by nucleophilic addition also resulted in linear release and the end point was reached in 5 days. Hydrolysis was accounted as the main release mechanism for covalently bound Dex, while physically incorporated Dex showed undefined rapid burst or first-order release, with most of the drug released in the initial 1–3 days. Eluates from covalently bound Dex maintained high activity, whereas Trap-Dex gels lost activity over time, as detected by the upregulation of luciferase expression from a transformed cell line. This novel chemistry combination offers precise drug release control applicable beyond Dex to drugs with suitable nucleophilic groups.

Abstract

Background Clofazimine is a repurposed drug to treat multidrug-resistant tuberculosis (MDR-TB) and is also a cornerstone of leprosy treatment. Pharmacokinetic (PK) data of clofazimine is scarce, but a population pharmacokinetics (popPK) model of clofazimine developed on data from South African MDR-TB patients has been published. Body composition was reported to be the key covariate that impacted both drug disposition and exposure. 

Aim The aim of this master research work was to re-evaluate and optimize the published popPK model of clofazimine in Indonesian patients with MDR-TB.

Methods The pre-existing model from South Africa was used as starting point and fitted to PK data from Indonesian, 49 patients with clofazimine based treatment over 4 months. The model was evaluated and adjusted to optimize its performance. The PK parameters were estimated and accumulation of clofazimine was examined. 

Results The model estimated a large central volume of 100 L and a clearance of 11.7 L/h, the median terminal half-life was about 20 days and substantial accumulation was observed over the 4 months. Women had much longer half-life and thereby slower accumulation and lower exposures due to high body fat in comparison with men. A large variability between occasions and subjects was detected.

Conclusion The model was able to describe the central tendency of the data well, but overpredicted the random variability between patients. That could be a results of participants adherence issues or varying food intake along with the drug during the treatment. This should be investigated in future analyses to get more reliable results.

Bakgrund: Gastric bypass är en operationsmetod som leder till en fysiologisk förändring i magtarmkanalen av perorala läkemedel, i syfte till att främja viktnedgång. Tidigare studier har stärkt teorin om att ett förändrat upptag av två vanligt förekommande läkemedel i kliniken, levotyroxin och omeprazol, sker som en följd av gastric bypass operation. Absorptionen av dessa läkemedel är kliniskt relevanta då den terapeutiska effekten är avgörande för behandlingen och därmed hälsan hos en bred patientgrupp inom sjukvården. 

Syfte: Syftet med denna litteraturstudie är att reda ut hur absorptionen av levotyroxin och omeprazol påverkas efter en gastric bypass operation. 

Metod: Detta är en litterturstudie baserat på åtta orginalstudier, fyra om läkemedlet omeprazol och fyra studier om läkemedlet levotyroxin. Artiklarna identifierades efter sökningar i PubMed och Embase under april 2021. Inklusionskriterier var gastric bypass som operationsmetod samt att publikationsdatumet är inom de senaste 20 åren. Barn, äldre och patienter med samtidigt vitaminkonsumtion exkluderades. 

Resultat: Av levotyroxinartiklarna konkluderar två av dessa att en minskad absorption av läkemedlet sker efter gastric bypass. De två resterande artiklar redovisade ingen förändring gällande absorptionen. Artiklar som studerade levotyroxin upptaget i form av flytande beredning efter gastric bypass, påvisade ökat läkemedelsupptag i flytande form gentemot tablettform. I de fyra artiklar som studerades omeprazol och dess absorption, styrktes en minskad läkemedlsabsorption i två av de artiklarna, samt en observerad avsaknad av signifikant skillnad i de två resterande artiklarna. 

Slutsats: Ett fullständigt svar till frågeställningen om levotyroxin coh omeprazol leder till minskat läkemedelsupptag efter gastric bypass, kan inte återfås av denna litterturstudie på grund av motstridiga resultat. Detta resultat indikerar däremot behovet av en individuell farmakoterapi då variationen mellan olika individer gällande faktorer såsom BMI och kön, kan påverka läkemedelsupptaget. 

Introduktion: Hos äldre är smärta relativt vanligt då det med ökad ålder uppkommerproblem i muskler, leder, senor och skelett, t.ex. artros, osteoporos och sarkopeni.Socialstyrelsen utformade 2004 en mall för god läkemedelsterapi hos äldre, s.k.kvalitetsindikatorer. T.ex. får paracetamol inte överstiga en dygnsdos på 4 g/dygn ochNSAID bör endast användas vid inflammatorisk smärta. När lätta analgetika inte hjälperrekommenderas behandling med starka opioider direkt utan att först använda lättaopioider lätta opioider då dessa anses olämpliga för äldre. Vid perifer neuropatisksmärta rekommenderas i första hand TCA (amitriptylin, nortriptylin) eller gabapentin.Syfte: Det övergripande syftet är att värdera kvaliteten i äldres smärtstillandeläkemedelsbehandling genom att relatera den till rekommenderad behandling för att ökakunskapen om hur behandling med smärtstillande läkemedel ser ut inom äldrevårdenidag. Material och metoder: Läkemedelslistor från 434 patienter från olikaäldreboenden i Uppsala län inkluderas i studien. Insamlade, avidentifierade data fördesin i Excel med information som student-ID, patient-ID, ålder, kön, substansnamn,stående dos, vidbehovsdos och stående + vidbehovsdos. Resultat: Gruppen bestod av299 kvinnor och 135 män med medelåldern 85,4 år. Totalt förskrevs 485 smärtstillandeläkemedel till de 434 patienterna. 75.8 % patienter förskrevs paracetamol, 27,2 %opioider, 3,45 % NSAID, 0,92 % amitriptylin respektive 1,61 % gabapentin.Rekommenderad dygnsdos av paracetamol överskreds hos 13 patienter, av diklofenakhos en patient medan 33 patienter inte hade maximal dygnsdos angiven. Förskrivning avett analgetikum var vanligast och förekom hos 179 patienter. Två analgetika förskrevstill 119 patienter medan två, en man och en kvinna, förskrevs 5 analgetika vardera.Totalt 94 personer, 21,4 %, använde inga smärtstillande läkemedel alls. Konklusion:Studien visar att smärtstillande läkemedelsbehandling hos äldre i Uppsala län inteskiljer sig markant från studier gjorda i övriga Sverige och Europa. Dock var det någrasom, enligt Socialstyrelsen kvalitetsindikatorer, överskred dygnsdosen och stod påläkemedel som anses vara olämpliga för äldre, vilket visar att det fortfarande finnsproblem med äldres läkemedelsterapi.

Various indicators of observed-theoretical spectrum matches were compared and the resulting statistical significance was characterized using permutation resampling. Novel decoy databases built by resampling the terminal positions of peptide sequences were evaluated to identify the conditions for accurate computation of peptide match significance levels. The methodology was tested on real and manually curated tandem mass spectra from peptides across a wide range of sizes. Spectra match indicators from complementary database search programs were pro filed and optimal indicators were identified. The combination of the optimal indicator and permuted decoy databases improved the calculation of the peptide match significance compared to the approaches currently implemented in the database search programs that rely on distributional assumptions. Permutation tests using p-values obtained from software-dependent matching scores and E-values outperformed permutation tests using all other indicators. The higher overlap in matches between the database search programs when using end permutation compared to existing approaches con firmed the superiority of the end permutation method to identify peptides. The combination of effective match indicators and the end permutation method is recommended for accurate detection of peptides.

An orally administered bilayer tablet with Tamsulosin (TAM) as the sustained release (SR) and Finasteride (FIN) as immediate release (IR) was manufactured. A response surface methodology was employed to formulate bilayer tablets with individual release layers, i.e., sustained and immediate release (SR and IR). Independent variables selected in both cases comprise hydroxypropyl methylcellulose (HPMC) as SR polymer, and avicel PH102 in the inner layer while Triacetin and talc in the outer layer, respectively. Tablets were prepared by direct compression, a total of 11 formulations were prepared for inner layer TAM, and 9 formulations for outer layer FIN were designed; these formulations were evaluated for hardness, friability, thickness, %drug content, and %drug release. A central composite design was employed in response surface methodology to design and optimize the formulation. The percentage of drug released was evaluated by in-vitro USP dissolution method of optimized formulation for 0.5, 2, and 6 hrs, and results were 24.63, 52.96, and 97.68 %, respectively. Drug release data was plotted in various kinetic models using a D.D solver, where drug release was first order that is concentration dependent and was best explained by Korsmeyer–Peppa kinetics, as the highest linearity was observed (R² = 0.9693). However, a very close relationship was also noted with Higuchi kinetics (R² = 0.9358). The mechanism of drug release was determined through the Korsmeyer model, and exponent "n" was found to be 0.4, indicative of an anomalous diffusion mechanism or diffusion coupled with erosion.

Background: Opioids are common drugs for pain treatment in preterm newborn infants, in spite of several adverse effects. Constipation is a frequent problem when opioids are used in both adults and neonates. Although several studies indicate that the oral administration of naloxone hydrochloride (NH) improves intestinal motility during opioid therapy, there is still a lack of evidence in newborns. Aim: The aim of this study was to assess the efficacy of NH against reduced intestinal motility during opioid treatment. Methods: A retrospective cohort study was performed. We analysed the medical records of fifteen infants (Group 1) treated with continuous morphine (MO) infusion and fourteen infants (Group 2) treated with both oral NH (3 mu g/kg 4 times daily) and MO. Results: There was no statistically significant difference in the total MO dose. Infants treated both with NH and MO had a tendency to improve their mean stool frequency/day. A statistically significant improvement was observed in the mean total food intake (mL/kg/day) of the infants treated with NH (p = 0.014). No difference in the mean food retention between the two groups was observed. Conclusion: Orally administrated NH seems to improve intestinal motility resulting in increased food intake/day and improved stool frequency/day in premature newborn infants treated with MO. Further studies are needed to corroborate these findings.

The inoculum effect (i.e., reduction in antimicrobial activity at large starting inoculum) is a phenomenon described for various pathogens. Given that limited data exist regarding inoculum effect of Acinetobacter baumannii, we evaluated killing of A. baumannii by polymyxin B, a last-resort antibiotic, at several starting inocula and developed a pharmacokinetic-pharmacodynamic (PKPD) model to capture this phenomenon. In vitro static time-kill experiments were performed using polymyxin B at concentrations ranging from 0.125 to 128 mg/L against a clinical A. baumannii isolate at four starting inocula from 10(5) to 10(8) CFU/mL. Samples were collected up to 30 h to quantify the viable bacterial burden and were simultaneously modeled in the NONMEM software program. The expression of polymyxin B resistance genes (lpxACD, pmrCAB, and wzc), and genetic modifications were studied by RT-qPCR and DNA sequencing experiments, respectively. The PKPD model included a single homogeneous bacterial population with adaptive resistance. Polymyxin B effect was modeled as a sigmoidal E-max model and the inoculum effect as an increase of polymyxin B EC50 with increasing starting inoculum using a power function. Polymyxin B displayed a reduced activity as the starting inoculum increased: a 20-fold increase of polymyxin B EC50 was observed between the lowest and the highest inoculum. No effects of polymyxin B and inoculum size were observed on the studied genes. The proposed PKPD model successfully described and predicted the pronounced in vitro inoculum effect of A. baumannii on polymyxin B activity. These results should be further validated using other bacteria/antibiotic combinations and in vivo models.

Bakgrund: Effekten av smörjmedel på tablettens egenskaper och särskilt tabletthållfasthet är ett mycket diskuterat ämne särskilt för ämnen som har plastiska egenskaper såsom natriumklorid. Magnesiumstearat är ett välkänt smörjmedel. Natriumstearylfumarat är ett smörjmedel som har nästan samma effekt som magnesiumstearat.

Syfte: Syftet med dessa studier var dels att studera effekten av smörjmedel på tabletterbarhetsprofilen för natriumklorid, dels att få reda på om det var möjligt att förutsäga tabletterbarhetsprofilerna baserat på komprimeringsparametrar för den osmorda natriumkloriden.

Resultat: Resultaten visade att smörjmedlen orsakade en avsevärd minskning på tabletthållfasthet och därmed minskades hela profilen. Kawakita parametern (b^-1) gav en bra prediktion av det lägsta kritiska trycket för både osmord och smord natriumklorid.

α-värdet visades ha en stor inverkan på prediktionen av det högsta kritiska trycket och den maximala tabletthållfastheten. För de studerade pulvren så gav α=2 bäst överenstämmelse till experimentella data för osmord natriumklorid och α=2,7 för smord natriumklorid.

Slutsats: För det första påverkade smörjmedlet hållfastheten för natriumklorid. För det andra kunde profilen för både osmord och smord natriumklorid förutsägas med en modifiering av α-värdet.

Background: In response to the lack of consideration of patient’s kidney function when prescribing drugs, the electronic Clinical Decision Support System (CDSS), Janusmed drugs, and renal function have been developed. This system offers knowledge on prescribing drugs to patients with impaired kidney function. Studies have shown that CDSS has the potential to lower the inappropriate prescription rate for patients with impaired kidney function. However, the general problem that CDSS faces is a low implementation rate because of inadequate usability.

Aim: The aim was to evaluate the usability of the digital interface Janusmed drugs and renal function and map ways to improve the interface.Method: Think aloud sessions and semi-structured interviews were conducted with eight medical doctors and three clinical pharmacists. Data from the think-aloud sessions were structured through an affinity diagram, also called the KJ-method, to obtain a diagram of how the interface is used and possible improvements of the system. Furthermore, Nielsen’s ten usability heuristics were used to evaluate the usability of the system.

Results: A total of six violations were found in the system, but they scored low on severity (1-2 points). The affinity diagram obtained showed the following five main themes on ways to improve the system: interface/technical functions, patient data, renal function, self-assessment, and future development.

Conclusion: Janusmed drugs and renal function is a well-designed and user-friendly CDSS.However, there is still room for further improvements in the design and functionality of the interface that would increase the usability of the system

The one-humped or the dromedarian camel is a pseudo-ruminant mammal, well adapted to the hot and dry climates of the desert. Its ability to withstand torrid heat and extreme desiccation is of paramount importance to its survival. The studies presented in this thesis were designed to investigate and document the effect of dehydration in the presence or absence of angiotensin II (Ang II) AT1 receptor blocker (losartan) on blood constituents, electrolytes, hormones, neurotransmitters as well as liver and kidney enzymes in a subset of dehydrated camels and to compare them with hydrated camels. Additionally, we studied the response of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) and revealed for the first time the cardiac storage form of BNP in the camel heart. Dehydration induced significant increments in packed cell volume (PCV), white blood cells (WBC), gamma glutamyl-transferase (GGT), serum sodium, creatinine and urea levels, and a doubling in plasma cortisol and arginine vasopressin (AVP) levels. At the same time dehydration caused significant decrease in body weights, plasma insulin like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), and a 50% decrement in ANP and BNP levels. Moreover, dehydration with and without losartan resulted in significant changes in stress hormones and anti-oxidants in plasma, liver and kidney homogenates. Losartan on one hand enhanced the effect of dehydration resulting in significant increases in sodium, creatinine and urea levels. In addition losartan raised the  binding affinity of Ang II AT2 receptors in the small intestine with 8-fold and with 16-fold for liver AT1 receptors, indicating that Ang II AT1 and AT2 receptor binding sites were present in camel's small intestine while only AT1 receptor binding sites were found in the camel liver. One the other hand losartan resulted in significant decrease in body weights impaired the rise in anti-diuretic hormone and reduced aldosterone level. Finally, we showed that the proBNP is the storage form of BNP in the camel heart.

Aim: Dromedary camels are extremely well adapted to periods of water deprivation. The physiological mechanisms underlying this adaptation, however, are imperfectly understood. It is likely that the renin-angiotensin system plays an important role although few studies have addressed this possibility in the camel. Accordingly, the effects of long term dehydration alone and with angiotensin II type 1 receptor blocker, losartan, on whole blood and serum constituents were studied in camels.

Methods:Twenty eight male camels 3-4 years old were studied while under shade during summer in the Gulf-region, where the ambient temperature was above 40 degree Celsius. The camels were divided into three groups: a control group(n=6) was allowed free access to feed and water, a dehydration group (n=16) was given food ad-lib during 20days of total water deprivation, and a dehydration plus losartan (losartan) group (n=6) which received losartan 5mg/Kg daily by intravenous injection during 20 days of dehydration.  

Results: The body weight of the losartan group decreased by nearly 39.1% across dehydration whereas the reduction in body weight for the dehydration group was nearly 34.5% compared to controls. There was a significant increase in the packed cell volume (p<0.05) and leucocytes count (p<0.01) in the losartan group compared to controls. However, the mean corpuscular volume was significantly higher (p<0.05) in the dehydration group compared to controls. We observed major, statistically significant increases in serum urea (p<0.01) and creatinine (p<0.05) levels in the dehydration and losartan groups compared to controls. By the end of the period ofwater restriction, serum levels of gamma glutamyl transferase were significantly (p<0.01) lower in the losartan group compared to controls.

Conclusion: The results of our experiment show that dehydration alone or in combination with Angiotensin II receptor blocker has major effects on the biochemical and hematological parameters of the camel blood.

Background: PROTACs (proteolysis-targeting chimeras) are highly specific drugs that bind to and degrade pathogenic proteins in cells by activating the body's own degradation system. However, PROTACs have poor aqueous solubility, limiting the absorption and leading to low bioavailability when orally administrated. A potential formulation strategy for enhancing solubility is to induce supersaturation of the drug by entrapping it in an amorphous matrix. For this purpose, solubility, and supersaturation data for PROTACs are highly important, yet they are currently lacking in literature.

Aim: To characterize the solubility and supersaturation behavior of two model PROTACs in vitro.

Methods: A model drug was used to develop in vitro assays (dissolution/solubility, UV-extinction, precipitation kinetics) using µDiss Profiler™. The newly developed assays were applied to PROTACs alongside already established assays (saturation shake flask and ultracentrifugation). The solubility characterization was investigated in fasted state simulated intestinal fluid (FaSSIF) and phosphate buffer at pH 6.8.  

Results: ARV-110 showed a high degree of supersaturation in its amorphous state. FaSSIF notably increased the crystalline solubility, dissolution rates, and amorphous solubility of both PROTACs compared to phosphate buffer. Additionally, no precipitation of the supersaturated state was observed in FaSSIF, unlike phosphate buffer, where higher supersaturation led to a faster concentration decrease.

Conclusions: The high supersaturation ratio and the low precipitation tendency of amorphous PROTACs demonstrate a clear advantage in delivering PROTACs through supersaturated formulations. Additionally, the data emphasize the importance of biorelevant media in solubility and dissolution testing. The study’s findings may inform formulation strategies that favor supersaturated formulations to enhance the oral bioavailability of PROTACs.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.

Bedaquiline is a novel adenosine triphosphate synthase inhibitor anti-tuberculosis drug. Bedaquiline belongs to the class of diarylquinolines, which are antituberculosis drugs that are quite different mechanistically from quinolines and flouroquinolines. The fact that relatively similar chemical drugs produce different mechanisms of action is still not widely understood. To enhance discrimination in favor of bedaquiline, a new approach using eight-score principal component analysis (PCA), provided by a ChemGPS-NP model, is proposed. PCA scores were calculated based on 35 + 1 different physicochemical properties and demonstrated clear differences when compared with other quinolines. The ChemGPS-NP model provided an exceptional 100 compounds nearest to bedaquiline from antituberculosis screening sets (with a cumulative Euclidian distance of 196.83), compared with the different 2Dsimilarity provided by Tanimoto methods (extended connective fingerprints and the Molecular ACCess System, showing 30% and 182% increases in cumulative Euclidian distance, respectively). Potentially similar compounds from publicly available antituberculosis compounds and Maybridge sets, based on bedaquiline's eight-dimensional similarity and different filtrations, were identified too.