A snake toxin as a theranostic agent for the type 2 vasopressin receptor

Abstract

Rationale: MQ1, a snake toxin which targets with high nanomolar affinity and absolute selectivity for the type 2 vasopressin receptor (V2R), is a drug candidate for renal diseases and a molecular probe for imaging cells or organs expressing V2R. Methods: MQ1's pharmacological properties were characterized and applied to a rat model of hyponatremia. Its PK/PD parameters were determined as well as its therapeutic index. Fluorescently and radioactively labeled MQ1 were chemically synthesized and associated with moderate loss of affinity. MQ1's dynamic biodistribution was monitored by positron emission tomography. Confocal imaging was used to observe the labeling of three cancer cell lines. Results: The inverse agonist property of MQ1 very efficiently prevented dDAVP-induced hyponatremia in rats with low nanomolar/kg doses and with a very large therapeutic index. PK (plasma MQ1 concentrations) and PD (diuresis) exhibited a parallel biphasic decrease. The dynamic biodistribution showed that MQ1 targets the kidneys and then exhibits a blood and kidney biphasic decrease. Whatever the approach used, we found a T1/2α between 0.9 and 3.8 h and a T1/2β between 25 and 46 h and demonstrated that the kidneys were able to retain MQ1. Finally, the presence of functional V2R expressed at the membrane of cancer cells was, for the first time, demonstrated with a specific fluorescent ligand. Conclusion: As the most selective V2 binder, MQ1 is a new promising drug for aquaresis-related diseases and a molecular probe to visualize in vitro and in vivo V2R expressed physiologically or under pathological conditions.

Keywords: PET imaging; PK/PD; Snake toxin; cancers; theranostic agent.

Figure 1

(A) Inverse agonist property of MQ1. cAMP concentration produced by the CHO cell line stably expressing hV2R as a function of MQ1 concentration. Representative curve, n=3, error bars are S.E.M. (B) Representative [³H]AVP competition curves by AVP (red) and MQ1 (black) on the CHO cell line stably expressing hV2R (open circle) or the COS cell line transiently expressing rV2R (closed circles). Representative curve, n=4-11, error bars are S.E.M.

Figure 2

(A) Diagram of the experimental protocol. (B) Blood sodium as a function of time. Black circle, control. Red circle, 10 µg/kg (1.55 nmol/kg) MQ1.Blue circle, 100 µg/kg (15.5 nmol/kg) MQ1. (C) Rats bodies weights normalized to their own basal body weight.

Figure 3

Rat diuresis (A) and urine osmolality (B) over a 24-hour period for various doses of MQ1. (C) Rat diuresis versus time for various doses of MQ1 administered by the i.p. route. Same color code for (A), (B) and (C). (D) Plot of diuresis (red and left axis) and MQ1 pharmacokinetics (black and right axis) for 356 nmol/kg MQ1. (E) Modeled pharmacokinetics vs pharmacodynamics relationship (black). Linear regression between 2- and 96-hour pharmacodynamics (red). PD= -5.6 + 102*PK. R²=0.98.

Figure 4

(A) Structural organization of DBO-MQ1. (B) Representative curves for the binding of MQ1 (black), 6-azidohexanoïc-MQ1 (blue) and DBO-MQ1 (red) to hV2R stably expressed in CHO cells. (C) Time activity curves of various organs within the first 60 minutes following intravenous injection in mice.

Figure 5

(A) Representative PET scans of healthy C57BL6 mice following the kinetics of tracer elimination within the seven days after intravenous injection of 89Zr-DFO-MQ1. (B) Biodistribution derived from (A). PET imaging by drawing VOI for various organs. (C) Blood and kidney activity curves over time.

Figure 6

(A) Rat diuresis over four days. Red arrows indicate 3 nmol/kg MQ1 i.p. injections. (B) Superposition of rat diuresis monitored for four days.

Figure 7

Labeling of endogenously expressed V2R in the LLC-PK1 cell line, in either fresh or PFA-fixed cells by (A) 100 nM of AFD-488-MQ1, (B) 100 nM of Cy5.5-MQ1. Specificity is determined by the last panels in the presence of either 3.4 µM MQ1 or 1 µM SR121463. (C) Cy5.5-MQ1 labeling at 100 nM in wild-type CHO cells (CHO WT), CHO cells stably expressing human V2R (CHO hV2R) and in renal cancer cell lines CAKI2, ACHN and A498 expressing hV2R. (D) Specificity is determined in the presence of 3.4 µM MQ1.