Presentación de resultados

human receptor

There are only three studies directly comparing the affinity of cannabinoid ligands to both the rCB2 and the hCB2 receptor. They are limited to testing eight different

cannabinoids57,105,125_{. Direct comparison of binding affinity is important, as it}

overcomes the experimental error associated with different techniques employed by different laboratories performing the binding assay. Comparison of the binding affinity of ligands, performed in different independent studies have been reviewed51,80 but due to the variability of these results it is difficult to draw meaningful

comparisons from the work.

§§ _{Web of Knowledge (http://wok.mimas.ac.uk) citation search conducted on 17/06/04} *** _{Author’s personal observation.}

A summary of both the direct and indirect comparative work is shown in Table 5-1. Griffin et al. analysed their affinity data for statistical significance between the rCB2

and hCB2 receptor105. Using this study it was straightforward to determine which of

the ligands tested showed significant species differences. Their analysis of seven different cannabinoids demonstrated that only three, JWH051 (classical cannabinoid), Win 55212-2 3, and anandamide 4 had significant differences between the rat and human CB2 receptors105. The differences observed for the three cannabinoid ligands

were a 9, 8, and 32 fold decreased affinity to the rCB2 receptor respectively105. The

observation that SR144528 6 exhibited little species selectivity was corroborated in a study by Rinaldi-Carmona et al. which reported that the rCB2 and hCB2 affinities

were similar57_{, however work by Iwamura et al. was able to show an 8 fold decreased}

affinity to the hCB2 receptor (Table 5-1)125. Iwamura et al. also demonstrated that

Win 55212-2 3 had a 5 fold decreased affinity towards the rCB2 receptor125, which is

in agreement with the results generated by Griffin et al.105.

The results for Win 55212-2 3 and ∆9_{-THC 1, shown in Table 5-1, highlights}

how difficult interpreting results obtained from different laboratories can be. Using Win 55212-2 3 as an example, it can clearly be seen that although two studies both examined the same ligand on the rat and human CB2 receptors, they have both

produced different Ki values for the two receptor species (rCB2 Ki = 10.4 or 1.30 nM

and hCB2 Ki = 1.19 or 0.29 nM). It was for this reason that where possible we tried to

compare direct experimental comparisons of Ki values to assess for species’

differences.

The two most significant results relating to our work are for the compounds JTE1-22 61 and JTE907 66 (Table 5-1). These Japan Tobacco compounds were shown to exhibit large species differences in affinity, between the rat and human CB2

receptor. The direct comparison conducted by Iwamura et al. of the compound JTE907 66 demonstrated that the compound had a 95 fold less favourable binding affinity to the hCB2 receptor than to the rCB2 (Ki 35.9 and 0.38 nM respectively)125.

This interesting result was attributed to the low sequence homology between the rat and human receptors and to the novel structure of JTE907 66125. The one indirect comparison included in Table 5-1, is of another Japan Tobacco compound, JTE1-22 61, which is of interest to us. The results, are extracted from the original Japan Tobacco patent126 and from a later publication by the Bristol Myers Squibb (BMS)

company who were interested in employing the novel structures as a template for their own CB2 receptor ligands197. The original patent reported affinity of JTE1-22 61 in rat

splenocyte membranes whilst workers at BMS screened the compound in the hCB2

receptor expressed in CHO cells. It is observed, from Table 5-1, that the binding of 61 at the rat and human receptors differs by a factor of 86. The low Ki observed in the

rCB2 receptor (1.4 nM) is altered to a moderate Ki value in the hCB2 receptor (120

nM). Although the BMS workers who tested the compound in human CB2 receptors

did not comment on the species difference, this second example of a Japan Tobacco compound exhibiting a large difference in affinity towards the CB2 receptor is

noteworthy. Both of the ligand comparisons available for the Japan Tobacco ligands, show large differences (86 and 95 fold) in affinity between the rat and human CB2

receptor, with a higher binding observed at the rCB2 receptor.

We will be demonstrating, in the remainder of this chapter, our own evidence that another of Japan Tobacco’s amide containing cannabinoid also displays a species selective binding, with greater affinity observed for the rCB2 receptor. We will also

extend the use of molecular modelling discussed in chapter 3 to explore the intimate nature of ligand-receptor binding to rationalize these findings.

Compound Study type rCB2 Ki (nM) hCB2 Ki (nM) hCB2/rCB2 ratio Statistical analysis Reference (s) SR144528 6 Direct 0.30 0.32 1 NS 105 Direct 0.24 1.99 8 ND 125 Direct 0.30 0.60 2 NS 57 Win 55212-2 3 Direct 10.4 1.19 0.1 P < 0.05 105 Direct 1.30 0.29 0.2 ND 125 ∆9_{-THC 1 Direct 28.3 44.9 1.6 NS 105} Direct 6.80 3.13 0.5 ND 125 JWH015 53 Direct 269 98.4 0.4 NS 105 JWH051 Direct 3.16 0.33 0.1 P <0.05 105 JTE907 66 Direct 0.38 35.9 95 ND 125 JTE1-22 61 Indirect 1.4a ₁₂₀b _{86 ND a = 197 b = 126}

Table 5-1 Comparisons of the binding affinity (Ki (nM)) of a selection of cannabinoids towards the

rCB2 and hCB2 receptor. ND not determined. NS not significant.

5.3 Pharmacology results

In-order to be able to directly compare human versus rat affinities of JTE2-3 62 it was necessary for us to calculate its Ki value at the rCB2 receptor using a radioligand

displacement assays. From the result obtained we could directly compare the rat Ki

value to the human Ki obtained in chapter 4. The Ki value is calculated using the

Cheng and Prussoff equation which uses the Kd value of the radioactive ligand ([3H]

CP55, 940) being displaced. Therefore a standard curve was first produced in order to determine the Kd value of [3H] CP55, 940 in a rat splenocyte assay. A method by

DeBlasi et al was followed whereby [3H] CP55, 940 is displaced by cold CP55, 940; which avoids the use of problematic saturation binding experiments252. Full details of the preparation of the rat splenocyte membranes and the binding experiment are given in the experimental section (section 8.2)

-13 -12 -11 -10 -9 -8 -7 -6 0 20 40 60 80 100 120 EC50 9.084e-010 log [CP55, 940] % bound [ 3H] CP55, 940

Figure 5-1 Graph used to calculate the Kd of [3H] CP 55, 940, showing the displacement of [3H] CP

55, 940 from membranes derived from rat splenocytes by cold CP 55, 940 (n=3).

The displacement graph shown in Figure 5-1 was used to calculate the EC50 of 0.91

nM. Using the calculation from DeBlasi et al.252_{, shown below, we calculated the K} d

DeBlasi et al. calculation252: Kd = EC50 – L

Kd = 0.91 – 0.32

Kd = 0.59 nM

where:

L = concentration of radioligand (nM)

EC50 = concentration of CP 55, 940 that displaces half the specific binding of [3H] CP

55, 940 (nM)

Kd = equilibrium dissociation constant for [3H] CP 55, 940 (nM)

Using the calculated Kd value of 0.59 nM for [3H] CP 55, 940 we were able to use the

Cheng and Prussoff calculations, within GraphPad® _{Prism3, to determine the K} i of

JTE2-3 62 using a competitive radioligand displacement of the non-selective cannabinoid [3_{H] CP 55, 940 from rat splenocyte membranes. The displacement of}

[3H] CP 55, 940 by JTE2-3 62 from the rCB2 receptor is shown in Figure 5-2. From

this curve the Ki value for JTE2-3 62 was calculated to be 5.02 nM (95% confidence

intervals 0.85 – 29 nM). This value is 600 fold less than the Ki of 3.11 µM calculated

for the hCB2 receptor in chapter 4.

-12 -11 -10 -9 -8 -7 -6 0 20 40 60 80 100 120 log [JTE2-3] % bound [ 3H] CP55, 940

Figure 5-2 Displacement curve of [3H] CP 55, 940 by JTE2-3 62 from membranes derived from rat

Due to the species selective nature of JTE2-3 62 (and at least two other Japan Tobacco Compounds), it became prudent for us to retest the two BODIPY Japan Tobacco fluorescent derivatives synthesised in chapters 2 and 4, against the rCB2

receptor. Thus Bodipy-JTE2-6 100 and Bodipy-JTE2-3 121 were used in competitive displacement assays against [3H] CP 55, 940 using membranes from rat splenocytes. The binding curves from these assays are shown in Figure 5-3

-12 -11 -10 -9 -8 -7 -6 -5 0 50 100 Bodipy-JTE2-6 (100) Bodipy-JTE2-3 (121) log [ligand] % bound [ 3H] CP55, 940

Figure 5-3 Displacement curve of [3H] CP 55, 940 by Bodipy-JTE2-6 100 (solid) and Bodipy-JTE2-3 121 (dashed) from membranes derived from rat splenocytes (n=4).

Although the Ki for both Bodipy-JTE2-6 100 and Bodipy-JTE2-3 121 could not be

calculated from this assay due to maximal [3H] CP 55, 940 displacement not being achieved, it could be shown that at concentrations of 1 µM, 44% and 48% of the bound [3H] CP 55, 940 could be displaced by Bodipy-JTE2-6 100 and Bodipy-JTE2-3 121 respectively. This should be compared to no detectable displacement with these compounds at concentrations ≥ 32 µM at the hCB2 receptor.