For more information on the protocols used in the experiments, please refer to here. Knowledge of abbreviations stated in the experimental protocols will be assumed in this page. Other abbreviations will be defined.
We have created our desired aptamer to solve the main focus of our project: to design a sclerostin inhibitor without sacrificing the cardiovascular properties of sclerostin. Hence, we are to first evaluate the affinity between the Ostamer and human sclerostin to ensure that the effects of the Ostamer will be exerted.
Affinity evaluation of the Ostamer has already been completed here.
As normal aptamers have a very short half-life when present in human serum[1], we have performed additional chemical modifications to O to increase serum stability.
Affinity comparisons between modified ostamer (O') and O has already been completed here.
Efficacy evaluation of the Ostamer has already been completed here.
We have determined that the Ostamer is able to promote bone anabolism effectively and reverse the effects brought about by osteogenesis imperfecta (OI). However, we must also determine the cardiovascular risks that this promotion of bone anabolism will bring.
We have used mice affected by OI and Apolipoprotein E deficiency (ApoE-/-).
Fig R.1: Graphs comparing the serum levels of inflammatory cytokines and chemokine with different reagents in OI-ApoE-/- mice. [unpublished data]
Angiotensin II (AngII) is a naturally-occuring hormone that acts on the central nervous system to increase vasopressin production, and also acts on venous and arterial smooth muscle to cause vasoconstriction[2]. The presence of this hormone will lead to a higher blood pressure due to vasoconstriction, and this feature is used in medications that treats hypotension resulting from septic shock or other distributive shock[3]. Synthetic AngII (identical to its natural counterpart) is used to allow the cardiovascular effects of the reagents used to be exaggerated, allowing for better comparisons against the conditions present in normal blood.
Examining the effects of O' against the antibody, from these three graphs, it can be determines that cytokine and chemokine activity induced by the introduction of the antibody is at around 2.5 to 3.2 times that amount induced by the introduction of O', which is about the same level as in normal blood.
As control, loop2 mutant sclerostin (loop2m, does not suppress cardiovascular effects, but still binds to the antibody, thereby inhibiting the antibody) was also introduced with and without the sclerostin antibody (in separate trials) and the data (along with data provided by literature[4]) above suggests that the reason behind the increased cytokine and chemokine activity was due to the interactions between the antibody and sclerostin at loop 3, re-confirming that loop 3 of sclerostin is responsible for cardiovascular risk regulation.
Fig R.2: Graphs and images displaying the progression of aortic aneurysms (AA) in OI-ApoE-/- mice under different infusions. [unpublished data]
As seen from R.5, compared to the baseline (AngII+veh), the introduction of the sclerostin antibody has nearly doubled AA incidence, and that the introduction of O' decreased AA incidence by roughly 15%.
On the left, it can be seen that the size of the aortic aneurysm in the aorta with the introduction of O' is much smaller than that of the baseline. On the right, the maximum diameter of the aneurysms in the aortas when O' was introduced was roughly similar (slightly smaller) to that of the baseline, but that when the antibody was introduced were nearly double the size for the thoracic aorta, and 1.2 times the size for the suprarenal aorta.
This shows that O' slightly decreases the risk of developing aortic aneurysms in mice, while the antibody increases that risk by a huge amount.
Fig R.3: Graphs and images displaying the progression of atherosclerosis in mice under different infusions. [unpublished data]
Oil Red O stain is a lysochrome (fat-soluble dye) diazo dye used for staining of neutral triglycerides and lipids on frozen sections and some lipoproteins on paraffin sections, and can make fat more visible in various cuts in pathology[5]. The staining techniques used are non-destructive (i.e. does not destroy the exhibit and does not prevent the use of other techniques) and mainly targets fat deposits on the surface of porous exhibits[6]. This will allow atheromas deposited on the aortas to be stained for easy identification.
The graphs on the right show that the amount of positive stain deposited into the atheroma tissue was double the baseline when the sclerostin antibody was introduced, while that when O' was introduced did not change.
The maximum diameter of the aortic arch in mice was slightly smaller when O' was introduced, but had increased in size when the sclerostin antibody was introduced. The images of aortic arches agree with this, with significantly less Oil Red O stain deposited on the aortic arches for when O' was introduced than that when the sclerostin antibody was introduced. From the images of cross-sections of the aortas, it can be clearly seen that when O' was introduced, the diameter of the aortic root was much wider than that when the sclerostin antibody was introduced.
This shows that O' slightly decreases the risk of developing atherosclerosis in mice, while the antibody increases that risk by a huge amount.
Combining the results from above, it can be concluded that the induced cardiovascular effects by the addition of the ostamer does not affect the risk of contracting cardiovascular diseases in OI-ApoE-/- mice, and the risk is maintained at a significantly lower level than that induced by the addition of the sclerostin antibody.
In summary of the results and conclusions above, we have successfully created an aptamer-based sclerostin inhibitor that does not impose severe cardiovascular risk on mice, and our experiments have demonstrated that our ostamer is technically superior to the available sclerostin antibody.
Fig R.7: US FDA Orphan Drug Designation of our aptamer-based drug.
We have received US FDA Orphan Drug Designation, and our team is now in the process of applying for clinical trials for human testing to ensure that the bone anabolic and cardiovascular effects described above are sustained in humans.
[1] Thiviyanathan, V., & Gorenstein, D. G. (2012). Aptamers and the next generation of diagnostic reagents. Proteomics. Clinical applications, 6(11-12), 563–573. [2] Chawla LS, Busse L, Brasha-Mitchell E, Davison D, Honiq J, Alotaibi Z, Seneff MG (October 2014). "Intravenous angiotensin II for the treatment of high-output shock (ATHOS trial): a pilot study". Critical Care. 18 (5): 534. [3] Kaufman MB (March 2018). "Pharmaceutical Approval Update". P & T. 43 (3): 141–170. [4] Veverka V et al. (2009). Characterization of the structural features and interactions of Sclerostin: molecular insight into a key regulator of Wnt-mediated bone formation. The Journal of biological chemistry. 284. 10890-900. [5]"Forensic Pathology". [6] Beaudoin, A. New technique for revealing latent fingerprints on wet, porous surfaces: Oil Red O. Journal of Forensic Identification, 2004, 54 (4), 413-421.
