Van Daele, Marieke
(2024)
The safety pharmacology of Syk inhibitors: new preclinical screens to characterise cardiovascular adverse drug effects.
PhD thesis, University of Nottingham.
Abstract
Since the implementation of ICH S7A, the field of safety pharmacology has advanced radically. Current approaches for detection of cardiovascular (CV) adverse drug reactions (ADR), including evaluating mean arterial pressure (MAP) and heart rate (HR) often identify those safety risks. Nonetheless, high attrition rates due to safety issues remain an important challenge in drug development. Conventional analysis (MAP or HR) uses a minority of the recorded high-fidelity waveform data, overlooking a plethora of information. Potentially in-depth analysis of the CV waveform could provide early and mechanistically relevant markers to detect subtle CV ADR and aid in closing the current gaps in safety pharmacology. The Symmetric Projection Attractor Reconstruction (SPAR) is a novel in-depth wave analysis method that uses the entire waveform to generate an attractor which reflects and enlarges subtle changes in the wave. In this thesis, the application of SPAR on blood pressure and blood flow waves in CV safety pharmacology was explored. To perform this investigation, two tyrosine kinase inhibitors were selected and presented as an example of compounds with varying CV safety liabilities: (1) fostamatinib that targets Syk but is associated with the onset of hypertension due to off-target inhibition of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) and (2) entospletinib, a second-generation compound designed to not bind VEGFR2 and to achieve Syk inhibition without the onset of cardiovascular safety liabilities.
First, the cardiovascular effects of these two Syk inhibitors were assessed using radiotelemetry in rats, as is currently the golden standard for MAP and HR assessments in in vivo preclinical safety studies. This study showed that fostamatinib indeed caused a significant increase in MAP, while not affecting HR or pulse pressure (PP). Entospletinib did not affect MAP, HR or PP significantly.
Second, the blood pressure waveform data obtained in the radiotelemetry studies were analysed in-depth using SPAR. This analysis yielded insights that were not obtained from the conventional wave analysis. Firstly, SPAR metrics detected fostamatinib-induced CV effects within 8 h after fostamatinib administration, while MAP only changed significantly from the second day of the study. The changes in SPAR metrics indicated a fostamatinib-induced vasoconstriction, consistent with the anticipated mechanism of BP elevation. In addition, entospletinib changed the SPAR metrics in a similar way as fostamatinib, suggesting that this second-generation compound might display subtle effects on the cardiovascular system that were not apparent from MAP, HR or PP.
Third, the two Syk inhibitors were assessed in the Doppler flowmetry model to interrogate their effect on the regional vascular conductances (VC) of the renal, mesenteric or hindquarters bed in rats. Subsequently, obtained blood flow waves were analysed using SPAR. Fostamatinib induced a decrease in renal and hindquarters VC, partially reflecting VC changes that are typically observed with VEGFR2-inhibitors. SPAR analysis indicated that fostamatinib caused a vasoconstriction in the mesenteric vascular bed, again reflecting effects typically observed with VEGFR2-inhibitors. Although entospletinib did not affect the renal, mesenteric or hindquarters VC in the conventional analysis, in the SPAR analysis this compound showed similar flow wave morphology changes to those observed with sunitinib, a known VEGFR2-inhibitor. These results indicated that entospletinib caused small changes in the compliance of blood vessels and may be associated with subtle CV effects.
Lastly, the effects of fostamatinib and entospletinib at VEGFR2 were evaluated in vitro. An intracellular nanoBRET assay in HEK293 cells was developed and employed to determine the compounds’ direct binding affinities to the intracellular site of VEGFR2. Furthermore, an nuclear factor of activated T-cells (NFAT) reporter gene assay in HEK293 cells was performed to determine the compounds’ effect on the downstream signalling of VEGFR2. These studies showed that fostamatinib bound to the receptor and inhibited its downstream signalling. Entospletinib did not directly bind to VEGFR2. Yet the latter inhibited both VEGF165a- and ionomycin-induced NFAT signalling and did so with an approximately 100x higher potency than fostamatinib indicating entospletinib exerted off-target effects beyond its Syk inhibition.
In conclusion, results from this thesis showed that SPAR is a valuable method for detection of subtle cardiovascular side effects. For fostamatinib, SPAR detected CV ADR earlier than MAP, HR or PP could. Additionally, SPAR exposed entospletinib-induced CV effects that were consistent with an in vitro inhibition of the VEGFR2 signalling that might be relevant to its safety, in particular since this compound is in development in combination therapy with certain chemotherapeutics that have been associated with CV safety issues.
| Item Type: |
Thesis (University of Nottingham only)
(PhD)
|
| Supervisors: |
Woolard, Jeanette
Hill, Stephen J. |
| Keywords: |
Safety pharmacology; adverse drug reactions; Symmetric Projection Attractor Reconstruction; Wave analysis; Cardiovascular side effects |
| Subjects: |
R Medicine > RM Therapeutics. Pharmacology |
| Faculties/Schools: |
UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences |
| Item ID: |
76890 |
| Depositing User: |
Van Daele, Marieke
|
| Date Deposited: |
16 Jul 2024 04:40 |
| Last Modified: |
16 Jul 2024 04:40 |
| URI: |
https://eprints.nottingham.ac.uk/id/eprint/76890 |
Actions (Archive Staff Only)
 |
Edit View |
Tools
Tools
