Drug Discovery 2023: ELRIG

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Abstracts

Topics: Organoids, GI Toxicity, Next-Generation Sequencing, Biomarker Platforms, IBD.

The Use of Intestinal Organoids as a Preclinical Screen to Assess Gastrointestinal (GI) Toxicity and Barrier Integrity.

V. Ubertini, S. Hall, F. Ponthan, and J. Wilson.

Gastrointestinal (GI) toxicity is a common and often severe limiting factor in the development of therapeutic drugs. Symptoms include diarrhoea, dehydration and ulceration which increase susceptibility to infection, partly due to damage of crypt and/or villus structures in the small intestine, impairing the barrier function. As novel targeted therapies are emerging, assessment of their potential GI toxicity remains crucial.

Small intestinal (SI) organoids are 3D in vitro models of the epithelium which recapitulate the structure and function of the small intestine.

We have further developed and validated the organoid model as a screening tool to predict GI toxicity and subsequent mucosal regeneration in four species: mouse, rat, canine, and human. The culture conditions have been designed to mimic the stem cell niche and allow cell differentiation and proliferation to occur. All intestinal lineages were present in the organoids derived from each species and the epithelial hierarchy closely resembled that observed in vivo.

The toxic effects of therapeutic drugs on the organoids were assessed by quantification of the total cell viability upon treatment, coupled to morphometric analysis of the organoids (size, area, perimeter, and branching efficiency). Immunofluorescence and RNAseq can be used to identify the targeted cells and the toxicity mechanisms of the drugs.

To further assess the effect of drugs on barrier integrity, we have developed transwell-grown cell monolayers derived from normal human small intestinal organoids. This model allows direct access to both apical and basal surfaces, which is not possible when using organoids where their geometry prevents access to the apical surface.

The monolayer formation was followed by live imaging and Trans-Epithelial Electrical Resistance (TEER) measurements and treatments with toxic drugs were applied after TEER reached a plateau. The direct effect on barrier integrity was evaluated by TEER reduction, FITC-Dextran permeability increase and analysis of TJ proteins by immunofluorescence. Furthermore, effect on cell death was assessed by measuring the levels of Lactate Dehydrogenase (LDH) released in the medium by dying cells.

In conclusion, our models provide an innovative in vitro solution to study the toxic effects of drugs on the small intestine and to analyse direct effects on barrier integrity. The monolayer assay may also be useful for developing models of transient TJ impairment to improve the bioavailability of orally administered drugs.

Monitoring Microbial Diversity in Healthy Volunteers Using 16s Metagenomic Sequencing.

J. McConnell, G. Beale and C. Booth

Metagenomic sequencing can be used to sequence DNA from bacteria and fungi present in a sample, enabling the composition of the community to be assessed. In this study, 16S ribosomal RNA, a highly conserved gene containing nine hypervariable regions, was targeted using PCR and analysed using next generation sequencing. This allowed the bacterial genii present within faecal, buccal and saliva samples to be discriminated. In the faecal arm, participants collected samples before and after consuming probiotic drinks. In the buccal and saliva arm, participants collected samples before and after using mouthwash.

Bacterial DNA was extracted, quality checked, and run through an NGS library preparation specific to the V3-V4 region of 16S. Reads were mapped to a consensus database, and species compared by principal component analysis. The buccal and saliva samples clustered, while the faecal samples formed a separate distribution, suggesting the samples contained significantly different microbial distributions. The buccal and saliva samples showed an increased portion of Streptococcus and Hemophilus, while the faecal samples showed a greater abundance of Prevotella and Bacteroides. To compare species distribution, the Shannon-Weiner (SW) Index was calculated. While there was some variation, several participants showed significantly increased SW diversity post treatment, suggesting the treatment was effective.

Microbial diversity is a well characterised marker of health at sites including the gut and oral cavity. This study indicates that it is possible to assess the microbiome in these regions, and that the range of genii present can be modified. The high proportion of Streptococcus and Hemophilus present in the buccal samples is particularly interesting as these genii have been shown to mechanistically interact, contributing to their in vivo survival. These data show utility in future studies using microbiome diversity as a read out for drug efficacy and toxicity, enabling less invasive monitoring of volunteer health and a personalised approach to drug design.

Evaluation of Drug Target Engagement on Pharmacodynamic Biomarkers in Human Plucked Scalp Hair.

G. Tudor, F. Ponthan, A. Boanas, M. Brown, and C. Booth

Plucked hair is a valuable surrogate tissue to monitor pharmacodynamic (PD) responses in a clinical trial. Hair collection is minimally invasive, simple and is amenable to frequent sampling. We have developed a method to assess target expression changes following exposure of plucked hairs to chemotherapeutic agents and other potential therapeutics. Successful ex vivo responses provide proof of concept data prior to clinical studies.

Since hairs are epithelial appendages, many signalling pathways active in other epithelial tissues, including cancers, are also present in hairs. Highly proliferative scalp hair can be utilised as a surrogate tissue to measure proliferation, phosphorylation and DNA damage responses after treatment. Furthermore, hairs are highly vascularised, suggesting that Test Articles may be delivered efficiently to the hair in a similar manner to other highly vascularised tissues, including many tumour types.

Human plucked hair (either from the scalp or beard) was placed in maintenance media in the presence of conventional or targeted chemotherapeutic agents, known for their different mechanisms of action (Gemcitabine, Epirubicin, Carboplatin, Tarceva), for up to 24 hours. Hairs were then fixed post-exposure and longitudinal sections immunohistochemically labelled for various markers including p-ERK1/2, p-AKT, p-Chk1, g-H2AX, Ki67 and androgen receptor. Quantitative image analysis to measure the level and intensity of labelling was performed using an Aperio ScanScope®. The relative number of positively labelled cell nuclei, or relative tissue area (depending on the labelling pattern of the target), of the hair outer root sheath labelled was analysed.

The expected changes in labelling were observed.  For example, Gemcitabine, which is linked to DNA polymerase inhibition, increased p-Chk1 labelling after 4-8 hours and strongly induced both p53 and g-H2AX after 24 hours. The topoisomerase II inhibitor Epirubicin also strongly induced g-H2AX expression after 24 hours, whereas the alkylating agent carboplatin had a more moderate effect at the concentrations used. Tarceva, an inhibitor of the EGF pathway, decreased levels of both p-ERK1/2 and p-AKT after only 10 minutes.

We have demonstrated that plucked hair is a valuable PD biomarker tissue for various chemotherapy agents. Proof of concept studies performed ex vivo can inform the design of clinical validation studies by indicating optimum markers and timepoints. Further, each hair is an independent unit thereby allowing independent replicate tissues (hairs) to be sampled.

Long-Term Experience of Using Anti-il-12/23 P40 Igg as a Reference Therapeutic in Adoptive T-Cell Transfer Models of Colitis.

A. Horwell, W. Howard, M. Brown, and J. Wilson.

Epistem has run more than 50 studies to evaluate therapeutic candidates for treatment of IBD using adoptive T cell transfer models.  Biologics, tyrosine kinase inhibitors and syphingosine-1-phosphate receptor modulators are the three classes of agent that have shown the greatest efficacy in these models.  Anti-IL-12/23 p40 IgG was the most used reference therapeutic and has been employed in both prophylactic and intervention studies.  The therapeutic use of anti-IL-12/23 p40 IgG and the importance of IL-12/IL-23 signalling in mouse models of IBD have been reported previously by others [DOI: 10.1084/jem.187.8.1225; DOI: 10.1172/jci21404].

Most commonly, we have run studies for 28 days-post CD4⁺CD62L⁺ T cell transfer with prophylactic administration of anti-p40 IgG once per week (from day of T cell transfer).  In this setting, treatment resulted in a (51.3 ± 3.56) % reduction in disease severity (relative to IgG control-treated mice; n = 22 studies), based on large bowel weight:length ratio.   In an intervention setting (n = 7 studies), where administration was delayed to 14 days-post T cell transfer, a (41.6 ± 2.2) % reduction in disease severity was observed in response to anti-p40 IgG; extension of intervention studies up to 45 days-post T cell transfer did not impact on the efficacy of the anti-p40 IgG.

Blinded histopathology analysis of the large bowel demonstrated regional differences in both colitis severity and the efficacy of anti-IL-12/23 p40 IgG.  In prophylactic studies, baseline disease was scored at 5.24 ± 0.53 (on a scale of 0 to 12; n = 22) across the whole of the large bowel, in mice treated with rat IgG2aκ.  In contrast, administration of anti-IL-12/23 p40 IgG was associated with a reduction in colitis severity score to 1.54 ± 0.35, a decrease of (70.7 ± 7.4) %.  On a regional basis, anti-IL-12/23 p40 IgG reduced the colitis severity by more than 80 % in both the proximal and distal thirds of the large bowel, whereas in the middle third, the reduction in disease severity was (52.7 ± 6.9) %.  For intervention studies of 28 days duration, the reduction in disease severity in response to anti-IL-12/23 p40 was lower than in prophylactic studies, with an overall decrease of (56.8 ± 6.1) % and a reduction of (38.5 ± 7.3) % in the mid large bowel.

The efficacy of anti-IL-12/23 p40 was associated with improvements in a range of disease endpoints including reductions in spleen weight, inflammatory cytokines, the number of circulating leukocytes, and infiltrating leukocytes in the intestinal mucosa.  Anti-IL-12/IL-23 p40 therapy was effective across a range of donor/recipient strain pairings, including BALB/c & CB17/Icr-Prkdc^scid/IcrIcoCrl; C57BL/6J & B6.Cg-Prkdc^scid/SzJ and C57BL6/NTac & B6.129S6-Rag2^{tm1Fwa N12}.