In human CB2-expressing CHO cells, AM630 enhanced forskolin-stimulated cAMP and reversed inhibition by CP55940, consistent with inverse agonist/antagonist behavior. Micromolar AM630 also reduced stimulated cAMP in CB1-expressing cells. The reported absence of effects without forskolin and in untransfected cells limits interpretation to the tested receptor and assay context. [1]
AM630 responses depend on pretreatment and assay context. In human CB2-expressing CHO cells, 24 h AM630 pretreatment followed by washing abolished its subsequent cAMP enhancement, whereas SR144528 pretreatment enabled cAMP inhibition at 25–50 µM AM630. In membranes prepared after AM630 pretreatment, inhibition of basal GTPγS binding persisted. The cause of this whole-cell versus membrane difference remains unresolved. [2]
Human CB1/CB2 radioligand displacement
Biochemical assay · Membranes from CHO cells stably expressing human CB1 or CB2 receptors [1]
AM630 preferentially displaced [3H]-CP55940 from human CB2 receptors. The reported selectivity ratio is based on affinity, not a treatment dose or functional selectivity guarantee.
Literature-Reported Treatment Conditions
- Treatment concentration
- Concentration-response displacement; 0.5 nM [3H]-CP55940
- Exposure duration
- 90 min at 30°C
- Method
- Filtration radioligand-binding assay; Ki calculated by Cheng–Prusoff equation
- Vehicle
- Ethanol stock diluted in assay buffer; vehicle alone had no detectable effect at highest tested level (data not shown).
- Reported limits
- Exact AM630 displacement concentration grid and final ethanol percentage are not specified in the read Table 1/Methods.
Methods and source locations
Compound reported in the paper: AM630; Compound label in the paper: 6-iodopravadoline
Evidence for the named compound in the stated model; no salt conversion, clinical inference or Adooq lot validation.
Table 1 (image viewed); Methods Binding experiments, CHO cells and Drugs; Figure 1
Displacement of [3H]-CP55940 from human CB2 receptor membranes: 0.5 nM radioligand; nonspecific binding defined with 1 µM unlabelled cannabinoid — Table 1 AM630 row, [3H]-CP55940; Methods Analysis of data
Displacement of [3H]-CP55940 from human CB1 receptor membranes: Same assay framework; CB1 and CB2 membrane preparations differ in receptor expression — Table 1 AM630 row, [3H]-CP55940; Methods CHO cells
Relative affinity at human CB1 and CB2 receptors: Ratio reported by authors; not an independent experiment — Results Cannabinoid binding experiments; Discussion paragraph beginning AM630 bound to both
Receptor-dependent cAMP responses and CP55940 reversal
Cell-based study · CHO cells expressing human CB1 or human CB2 receptors; untransfected CHO control [1]
AM630 enhanced forskolin-stimulated cAMP in CB2-expressing cells and reversed CP55940 inhibition. In CB1-expressing cells it reduced stimulated cAMP at micromolar concentrations; attempted reversal of CP55940 inhibition was not significant. Neither AM630 alone without forskolin nor AM630 in untransfected cells changed cAMP in the reported controls.
Literature-Reported Treatment Conditions
- Treatment concentration
- AM630 1 nM–10 µM in Figures 3 and 7; Figure 4 uses 1 µM AM630
- Exposure duration
- 30 min AM630/IBMX preincubation, then 30 min with 2 µM forskolin at 37°C
- Method
- Radioimmunoassay for cellular cAMP; 50 µM IBMX
- Vehicle
- Ethanol stock diluted in assay buffer; vehicle alone had no detectable effect (data not shown).
- Co-treatment
- CP55940 concentration response in Figure 4 or fixed 10 nM in Figure 7; AM630-alone comparisons in Figure 3.
- Reported limits
- No Adooq lot tested. Numerical vehicle percentage not supplied in the read methods.
Methods and source locations
Compound reported in the paper: AM630; Compound label in the paper: 6-iodopravadoline
Evidence for the named compound in the stated model; no salt conversion, clinical inference or Adooq lot validation.
Figures 3, 4 and 7 (images and captions); Results Effects on cyclic AMP and Effect of AM630; Methods Cyclic AMP assay, Drugs and Analysis
Enhancement of forskolin-stimulated cAMP in CB2-expressing cells: Nonlinear regression; normalization to forskolin-only response — Figure 3 caption
Forskolin-stimulated cAMP remaining in CB1-expressing cells at 1 and 10 µM AM630: One-sample t-test; control normalized to 100% — Results paragraph beginning AM630 produced a significant inhibitory effect; Figure 3
Reversal of inhibition by 10 nM CP55940 in CB2-expressing cells: Half reversal between CP55940-inhibited and agonist-free stimulated cAMP levels; not binding Ki — Results paragraph beginning A further set of experiments; Figure 7
Specificity and negative controls: Untransfected-cell and forskolin-omission controls; CP55940-only comparison — Results Effects on cyclic AMP, paragraph beginning AM630 produced; final paragraph before Discussion; Figures 4 and 7
Pretreatment-dependent AM630 cAMP phenotype
Cell-based study · Human CB2-transfected CHO whole cells, compared after vehicle, AM630 or SR144528 pretreatment [2]
AM630 enhanced stimulated cAMP in vehicle-pretreated cells. Its enhancement was abolished after AM630 pretreatment and washing. After SR144528 pretreatment and washing, AM630 instead inhibited stimulated cAMP at 25 and 50 µM, but not at 2.5 µM or less. These data support condition-dependent functional behavior in this recombinant model.
Literature-Reported Treatment Conditions
- Treatment concentration
- Pretreatment: 10 µM AM630 or 10 µM SR144528; subsequent AM630 concentration response, including 25 and 50 µM
- Exposure duration
- 24 h pretreatment, extensive washing; assay drug incubation 30 min at 37°C
- Method
- HitHunter cellular cAMP assay with forskolin and 10 µM rolipram
- Vehicle
- Dimethyl sulphoxide vehicle pretreatment
- Co-treatment
- SR144528 is an alternative pretreatment, not simultaneous AM630 combination treatment.
- Reported limits
- Assay Methods names forskolin without fixing its concentration for every panel; 10 µM forskolin is specified in the Results comparison of pretreatment-dependent baseline stimulation. No uniform panel concentration inferred.
Methods and source locations
Compound reported in the paper: AM630; Compound label in the paper: 6-iodopravadoline
Evidence for the named compound in the stated model; no salt conversion, clinical inference or Adooq lot validation.
Figures 1 and 6 (images/captions); Results Effects of pre-incubation; Methods cAMP assay and Materials
AM630 enhancement of forskolin-stimulated cAMP in untreated (vehicle-preincubated) cells: Nonlinear regression; enhancement is above forskolin-stimulated response, not percent viability — Figure 1 caption; Methods Analysis of data
Effect of 10 µM AM630 pretreatment on subsequent AM630 response: Matched assay comparison with untreated cells; no fitted pretreatment-group EC50 invented — Figure 1 and Results first paragraph
AM630 after 10 µM SR144528 pretreatment for 24 h: Alternative pretreatment followed by washing and subsequent AM630 cAMP assay — Figure 6 caption and preceding Results paragraph
Membrane GTPγS inverse agonism persists after AM630 pretreatment
Biochemical assay · Membranes prepared from human CB2-transfected CHO cells preincubated with vehicle or AM630 [2]
AM630 inhibited basal [35S]-GTPγS binding in both membrane preparations. Pretreatment did not significantly change its potency or efficacy, unlike the loss of cAMP enhancement in intact cells. The reason for the assay difference was not established.
Literature-Reported Treatment Conditions
- Treatment concentration
- 10 µM AM630 pretreatment; subsequent AM630 concentration response; 25 µM AM630 in CP55940 antagonism comparison
- Exposure duration
- 24 h cell pretreatment and extensive washing; membrane assay 60 min at 30°C
- Method
- [35S]-GTPγS binding with 0.1 nM tracer and 30 µM GDP
- Vehicle
- Dimethyl sulphoxide vehicle pretreatment
- Co-treatment
- CP55940 concentration response ±25 µM AM630 in Figure 5B
- Reported limits
- Whole-cell and membrane endpoints are distinct assays; no mechanism for the difference is established.
Methods and source locations
Compound reported in the paper: AM630; Compound label in the paper: 6-iodopravadoline
Evidence for the named compound in the stated model; no salt conversion, clinical inference or Adooq lot validation.
Figure 5 (image/caption); Results Effect of pre-incubation on GTPγS assay; Methods Membrane preparation and GTPγS assay
AM630 inhibition of basal [35S]-GTPγS binding: Nonlinear regression; overlapping confidence limits used by authors to assess lack of significant difference — Figure 5A caption and preceding Results paragraph
Change in basal [35S]-GTPγS binding: Signed percent change relative to basal binding; nonspecific binding measured with 30 µM unlabelled GTPγS — Figure 5A caption; Methods GTPγS binding assay
CP55940 stimulation of membrane GTPγS binding after AM630 pretreatment: Concurrent AM630 versus vehicle during CP55940 assay — Figure 5B and caption
