Expression and regulation of drug transporters in vertebrate neutrophils.

There remains a need to identify novel pro-resolution drugs for treatment of inflammatory disease. To date, there are no neutrophil-specific anti-inflammatory treatments in clinical use, perhaps due to our lack of understanding of how drugs access this complex cell type. Here we present the first comprehensive description and expression of both major classes of drug transporters, SLC and ABC, in resting human blood neutrophils. Moreover, we have studied the expression of these carriers in the tractable model system, the zebrafish (Danio rerio), additionally examining the evolutionary relationship between drug transporters in zebrafish and humans. We anticipate that this will be a valuable resource to the field of inflammation biology and will be an important asset in future anti-inflammatory drug design

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Early application of mechanistic PKPD modelling to guide the preclinical development of a therapeutic mAb targeting complement receptor 2 (CR2/CD21) as a novel target for the treatment of autoimmune disease

Previously held under moratorium in Chemistry department (GSK) from 10th March 2021 until 13th June 2023. The confidentiality statement on each page of this thesis DOES NOT apply.Mechanistic PKPD modelling has become the state of the art for the integration of preclinical data to improve early projection of the clinical efficacy of small molecule drug candidates. However, its application to the development of monoclonal antibody (mAb) based drug candidates has been relatively restricted to date. Typically, a key limiting factor is the necessity for a surrogate molecule to explore target pharmacology in well-established preclinical animal models. A need driven by the limited cross-species reactivity often seen with the fully human mAbs preferred for clinical development. Consequently, pharmacology is rarely established beyond target engagement during preclinical development, limiting the early projection of clinical efficacy. The present work sought to explore the application of mechanistic PKPD modelling, early in the preclinical drug discovery environment, using a surrogate mAb as a tool with which to explore the pharmacology of the novel drug target complement receptor 2 (CD21). CD21 is thought to play a key role in the regulation of humoral immunity. As such, CD21 is receiving interest as a novel mechanism by which to treat autoimmune conditions characterised by the aberrant production of destructive autoantibodies, such as systemic lupus erythematosus. Characterisation of three potential surrogate mAbs targeting murine CD21 identified nondose-linear pharmacokinetics related to on target binding, characteristic of target mediated drug disposition (TMDD). To describe this behaviour, a minimal physiologically based pharmacokinetic (mPBPK) model was developed, incorporating target abundance, kinetics and binding affinity. This model highlighted critical gaps in the understanding of CD21 kinetics, particularly regarding the origin and impact of the soluble form of the protein (sCD21). Further experiments identified that the plasma concentration of sCD21(/35) in mice remained unchanged (12.8 nM) following treatment with an anti-CD21 mAb, despite near complete loss of B cell expressed CD21. This challenged the theory that sCD21 results from ectodomain shedding of membrane receptor, implicating secretion as a predominant source of the soluble protein in mice. To explore the pharmacology of CD21 neutralisation, T-dependent immunisation was selected as a well-established acute mechanistic model of humoral immunity. To align with target biology and the clinical strategy, the protocol was optimised to comprise of a single IV immunisation with the T-dependent antigen keyhole limpet haemocyanin (KLH) at 1.2 mg/kg to C57BL/6 mice. The in vivo model was qualified using the clinically validated S1P receptor modulator Fingolimod. A relationship was established between Fingolimod blood exposure and its pharmacodynamic effect on the T-dependent antibody (IgG) response (TDAR), using an integrated PKPD model. This resulted in an estimated EC50 of 4.59 nM (SE 3.09), showing remarkable similarity to reported EC50 estimates across species (versus peripheral lymphocyte reduction). The potential to translate this model system to project clinical efficacy was also demonstrated, providing an excellent prediction of the reported efficacy of Fingolimod on KLH induced TDAR (IgG) in the clinic. Furthermore, CD21 pharmacology was explored in the mouse TDAR model using the widely published mAb 7G6 as a surrogate. A relationship was established between 7G6 plasma exposure and its pharmacodynamic effect on the KLH induced TDAR (IgM) using an integrated PKPD model, with an estimated EC50 of 0.13 nM (SE 0.02). Comparison with the measured receptor occupancy on peripheral B cells indicates this corresponds with approximately 60% target occupancy. Collectively, these data support the rationale that maintenance of mAb exposure to achieve >95% CD21 receptor occupancy at trough is an appropriate clinical benchmark. However, it also highlights that critical knowledge gaps remain with regard to CD21 and its pharmacology that would require further investigation.Mechanistic PKPD modelling has become the state of the art for the integration of preclinical data to improve early projection of the clinical efficacy of small molecule drug candidates. However, its application to the development of monoclonal antibody (mAb) based drug candidates has been relatively restricted to date. Typically, a key limiting factor is the necessity for a surrogate molecule to explore target pharmacology in well-established preclinical animal models. A need driven by the limited cross-species reactivity often seen with the fully human mAbs preferred for clinical development. Consequently, pharmacology is rarely established beyond target engagement during preclinical development, limiting the early projection of clinical efficacy. The present work sought to explore the application of mechanistic PKPD modelling, early in the preclinical drug discovery environment, using a surrogate mAb as a tool with which to explore the pharmacology of the novel drug target complement receptor 2 (CD21). CD21 is thought to play a key role in the regulation of humoral immunity. As such, CD21 is receiving interest as a novel mechanism by which to treat autoimmune conditions characterised by the aberrant production of destructive autoantibodies, such as systemic lupus erythematosus. Characterisation of three potential surrogate mAbs targeting murine CD21 identified nondose-linear pharmacokinetics related to on target binding, characteristic of target mediated drug disposition (TMDD). To describe this behaviour, a minimal physiologically based pharmacokinetic (mPBPK) model was developed, incorporating target abundance, kinetics and binding affinity. This model highlighted critical gaps in the understanding of CD21 kinetics, particularly regarding the origin and impact of the soluble form of the protein (sCD21). Further experiments identified that the plasma concentration of sCD21(/35) in mice remained unchanged (12.8 nM) following treatment with an anti-CD21 mAb, despite near complete loss of B cell expressed CD21. This challenged the theory that sCD21 results from ectodomain shedding of membrane receptor, implicating secretion as a predominant source of the soluble protein in mice. To explore the pharmacology of CD21 neutralisation, T-dependent immunisation was selected as a well-established acute mechanistic model of humoral immunity. To align with target biology and the clinical strategy, the protocol was optimised to comprise of a single IV immunisation with the T-dependent antigen keyhole limpet haemocyanin (KLH) at 1.2 mg/kg to C57BL/6 mice. The in vivo model was qualified using the clinically validated S1P receptor modulator Fingolimod. A relationship was established between Fingolimod blood exposure and its pharmacodynamic effect on the T-dependent antibody (IgG) response (TDAR), using an integrated PKPD model. This resulted in an estimated EC50 of 4.59 nM (SE 3.09), showing remarkable similarity to reported EC50 estimates across species (versus peripheral lymphocyte reduction). The potential to translate this model system to project clinical efficacy was also demonstrated, providing an excellent prediction of the reported efficacy of Fingolimod on KLH induced TDAR (IgG) in the clinic. Furthermore, CD21 pharmacology was explored in the mouse TDAR model using the widely published mAb 7G6 as a surrogate. A relationship was established between 7G6 plasma exposure and its pharmacodynamic effect on the KLH induced TDAR (IgM) using an integrated PKPD model, with an estimated EC50 of 0.13 nM (SE 0.02). Comparison with the measured receptor occupancy on peripheral B cells indicates this corresponds with approximately 60% target occupancy. Collectively, these data support the rationale that maintenance of mAb exposure to achieve >95% CD21 receptor occupancy at trough is an appropriate clinical benchmark. However, it also highlights that critical knowledge gaps remain with regard to CD21 and its pharmacology that would require further investigation

Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution

The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease