Sigmaplot 4.0 for windows

Assays of binding to CB₂ receptors were performed at Eurofins Discovery France (https://www.eurofinsdiscoveryservices.com/) under the study number 100057099 (accessed on 3 June 2021) using the protocol previously published by Munro, Thomas, and Abu-Shaar [46 (link)]. Human recombinant CB₂ receptors were expressed in CHO cells, and binding was performed with [³H]-WIN 55212-2, which is a nonselective agonist radioligand. The analysis was performed using software developed at Cerep (Hill software, Santa Barbara, CA, USA) and SigmaPlot 4.0 for Windows (^©1997 by SPSS Inc., Chicago, IL, USA).
Data obtained from duplicate experiments were fit to concentration-response curves by non-linear regression of the Hill equation. Curve-fit parameters and standard errors were calculated using GraphPad Prism 6.0 (Hercules, CA, USA).

Sample preparation: Several concentrations were tested in triplicate. For the preparation of the different concentrations, the original formulation was diluted with vehicle. Vehicle composition contained 0.41 mg of glacial acetic acid and 0.10 mg of sodium acetate per 1 mL of water, adjusted to pH = 4 when necessary, with NaOH 0.1 M or HCl 0.1 M.
Method: The PTH1 Human Parathyroid Hormone GPCR Cell-Based Agonist cAMP Assay (Item 2260, Eurofins Cerep) was used to compare the PTH1 agonist response of Teriparatide BGW and RMP samples in SaOS-2 cells endogenously expressing PTH1R and determined by HTRF technology. The EC₅₀ values (concentration producing a half-maximal response) were determined by non-linear regression analysis of the concentration–response curves generated with mean replicate values. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot^® 4.0 for Windows^® (©1997 by SPSS Inc., Chicago, IL, USA). After qualification with PTH (1-34) standard reference agonist, the assay acceptance range was set as mean EC₅₀ ± ½ log.

Binding affinities for neurokinin receptors, NK2 and NK3, were carried out through Eurofins’ service according to standard procedures, in which affinities were determined by displacing [¹²⁵I]-labelled NKA and [³H]-labeled SR 142801 (Table 4) [41 (link),42 (link)].
The IC₅₀ values (concentration causing a half-maximal inhibition of control specific binding) and Hill coefficients (nH) were determined by non-linear regression analysis of the competition curves generated with mean replicate values using Hill equation curve fitting: $Y=D+\left(\frac{A-D}{1+{\left(\frac{C}{C_{50}}\right)}^{nH}}\right)$
where Y = specific binding; A = left asymptote of the curve; D = right asymptote of the curve; C = compound concentration; C₅₀ = IC₅₀; and nH = slope factor. This analysis was performed using software developed at Cerep (Hill software) and validated by comparison with data generated by the commercial software SigmaPlot^® 4.0 for Windows^® (© 2021 by SPSS Inc., Chicago, IL, USA).
The inhibition constants (K_i) were calculated using the Cheng Prusoff equation
$K_{\mathrm{i}}=\frac{I{C}_{50}}{\left(1+\frac{L}{K_D}\right)}$
where L = concentration of radioligand in the assay; and K_D = affinity of the radioligand for the receptor. A Scatchard plot is used to determine the K_D.