Alpha-1 Acid Glycoprotein as a Biomarker for Subclinical Illness and Altered Drug Binding in Rats.

Alpha-1 acid glycoprotein (AGP) is a significant drug binding acute phase protein that is present in rats. AGP levels are known to increase during tissue injury, cancer and infection. Accordingly, when determining effective drug ranges and toxicity limits, consideration of drug binding to AGP is essential. However, AGP levels have not been well established during subclinical infections. The goal of this study was to establish a subclinical infection model in rats using AGP as a biomarker. This information could enhance health surveillance, aid in outlier identification, and provide more informed characterization of drug candidates. An initial study (n = 57) was conducted to evaluate AGP in response to various concentrations of Staphylococcus aureus (S. aureus) in Sprague-Dawley rats with or without implants of catheter material. A model validation study (n = 16) was then conducted using propranolol. Rats received vehicle control or S. aureus and when indicated, received oral propranolol (10 mg/kg). Health assessment and blood collection for measurement of plasma AGP or propranolol were performed over time (days). A dose response study showed that plasma AGP was elevated on day 2 in rats inoculated with S. aureus at 106, 107 or, 108 CFU regardless of implant status. Furthermore, AGP levels remained elevated on day 4 in rats inoculated with 107 or 108 CFUs of S. aureus. In contrast, significant increases in AGP were not detected in rats treated with vehicle or 10³ CFU S. aureus. In the validation study, robust elevations in plasma AGP were detected on days 2 and 4 in S. aureus infected rats with or without propranolol. The AUC levels for propranolol on days 2 and 4 were 493 ± 44 h × ng/mL and 334 ± 54 h × ng/mL, respectively), whereas in noninfected rats that received only propranolol, levels were 38 ± 11 h × ng/mL and 76 ± 16. h × ng/mL, respectively. The high correlation between plasma propranolol and AGP demonstrated a direct impact of AGP on drug pharmacokinetics and pharmacodynamics. The results indicate that AGP is a reliable biomarker in this model of subclinical infection and should be considered for accurate data interpretation.

Factor XIIa inhibition by Infestin-4: in vitro mode of action and in vivo antithrombotic benefit.

Coagulation factor XII (FXII) plays a central role in initiating the intrinsic cascade of blood coagulation. Purified recombinant Human Albumin-tagged Infestin-4 (rHA-Infestin-4) is a recently described FXIIa inhibitor that displayed strong anticoagulant activity without compromising haemostasis in several animal models. We pursued detailed in vitro characterisation of rHA-Infestin-4 and demonstrated that it is a competitive inhibitor of FXIIa with slow on and off rate constants for binding (kon=5x105 M⁻¹s⁻¹, koff=6x10⁻4 s⁻¹), it can block FXIIa activation of its physiological substrates (plasma prekallikrein and FXI), and it can inhibit ellagic acid-triggered thrombin generation in plasma. Potency and selectivity profiling in enzyme assays suggest that rHA-Infestin-4 is indeed highly potent on FXIIa (IC50=0.3 ± 0.06, 1.5 ± 0.06, 1.2 ± 0.09 nM, for human, rat, and rabbit FXIIa, respectively) with at least >100-fold selectivity against factors IIa, Xa, IXa, XIa, VIIa, and plasma kallikrein in all three species. rHA-Infestin-4 dose-dependently and markedly reduced clot weight in the arteriovenous shunt thrombosis model in rats and rabbits, accompanied with minimal increase in cuticle bleeding times in either species. rHA-Infestin-4 treatment at 5 mg/kg in rabbit resulted in a 13% reduction in ex vivo FXa activity, demonstrating a modest off-target effect. In summary, our findings confirmed and extended previous reports that inhibition of FXIIa by rHA-Infestin-4 can produce strong antithrombotic efficacy while preserving haemostasis. Our comprehensive selectivity profiling, mode of action, and kinetic studies of rHA-Infestin-4 reveal limitations of this molecule and offer new perspectives on any potential effort of discovering novel FXIIa inhibitors.

Discovery of 3-arylpropionic acids as potent agonists of sphingosine-1-phosphate receptor-1 (S1P1) with high selectivity against all other known S1P receptor subtypes.

A series of 3-arylpropionic acids were synthesized as S1P1 receptor agonists. Structure-activity relationship studies on the pendant phenyl ring revealed several structural features offering selectivity of S1P1 binding against S1P2-5. These highly selective S1P1 agonists induced peripheral blood lymphocyte lowering in mice and one of them was found to be efficacious in a rat skin transplantation model, supporting that S1P1 agonism is primarily responsible for the immunosuppressive efficacy observed in preclinical animal models.