Pharmacology, pharmacokinetics, and metabolism of the DNA-decoy AYX1 for the prevention of acute and chronic post-surgical pain - PubMed (original) (raw)
Pharmacology, pharmacokinetics, and metabolism of the DNA-decoy AYX1 for the prevention of acute and chronic post-surgical pain
Julien Mamet et al. Mol Pain. 2017 Jan.
Abstract
Background AYX1 is an unmodified DNA-decoy designed to reduce acute post-surgical pain and its chronification with a single intrathecal dose at the time of surgery. AYX1 inhibits the transcription factor early growth response protein 1, which is transiently induced at the time of injury and triggers gene regulation in the dorsal root ganglia and spinal cord that leads to long-term sensitization and pain. This work characterizes the AYX1 dose-response profile in rats and the link to AYX1 pharmacokinetics and metabolism in the cerebrospinal fluid, dorsal root ganglia, and spinal cord. Results The effects of ascending dose-levels of AYX1 on mechanical hypersensitivity were measured in the spared nerve injury model of chronic pain and in a plantar incision model of acute post-surgical pain. AYX1 dose-response profile shows that efficacy rapidly increases from a minimum effective dose of ∼ 0.5 mg to a peak maximum effective dose of ∼ 1 mg. With further dose escalation, the efficacy paradoxically appears to decrease by ∼ 30% and then returns to full efficacy at the maximum feasible dose of ∼ 4 mg. The reduction of efficacy is associated to doses triggering a near-saturation of AYX1 metabolism by nucleases in the cerebrospinal fluid and a paradoxical reduction of AYX1 exposure during the period of early growth response protein 1 induction. This effect is overcome at higher doses that compensate for the effect of metabolism. Discussion AYX1 is a competitive antagonist of early growth response protein 1, which is consistent with the overall increased efficacy observed as dose-levels initially escalate. Chemically, AYX1 is unprotected against degradation by nucleases. The sensitivity to nucleases is reflected in a paradoxical reduction of efficacy in the dose-response curve. Conclusions These findings point to the importance of the nuclease environment of the cerebrospinal fluid to the research and development of AYX1 and other intrathecal nucleotide-based therapeutics.
Keywords: Chronic pain; cerebrospinal fluid; early growth response protein 1; nucleases; oligonucleotide.
Figures
Figure 1.
(a) Effect of ascending AYX1 dose-levels on the development of pain measured as mechanical hypersensitivity in the spared nerve injury model of chronic neuropathic pain in the ADY-SNI2 study. Total responses (number of paw withdrawals) to repetitive von Frey stimulation in animals treated with vehicle (black circles), 0.7 mg (black squares), 1.05 mg (white squares), 1.4 mg (white circles), or 2.8 mg (white lozenges) AYX1 are presented; _T_-test, different from vehicle at a given time-point: *p < 0.05, data distribution over the testing period: p < 0.001 for all tested doses. Vehicle or AYX1 were administered once IT at the time of surgery, n = 4 to 5 per group; values are presented as mean + SEM. (b) Effect of ascending AYX1 dose-levels on the development of pain measured as mechanical hypersensitivity in the plantar incisional model of acute pain in the ADY-INC5 study. The effect of AYX1 0.56, 0.84, 1.12, 1.40, 2.80, and 4.20 mg was tested against vehicle. For clarity in light of the amount of tested groups, the vehicle (black circles), 1.12 (black triangles), 2.8 (white circles), and 4.2 mg (white squares) of AYX1 groups are displayed as representative responses observed during the study. The magnitude of effects of all tested doses is presented in Table 1. _T_-test followed by a T-Welsh analysis: *p = 0.07, **p = 0.02, data distribution over the testing period, vehicle versus 4.2 mg: p = 0.02; values are presented as mean + SEM. Vehicle or AYX1 were administered once IT at the time of surgery, n = 6 per group. (c) and (d) Dose-response patterns observed in the ADY-SNI2 and ADY-INC5 studies, respectively. The magnitude of effect for each dose-level normalized on the maximum efficacy measured within each study is presented (black triangles). Connecting curves are presented as dotted lines. Data are fitted with an exponential association fit (dashed line), excluding the outlier dose (circled). For the ADY-SNI2 study, excluding the outlier dose shifts the coefficient of regression _R_2 of the exponential fit from 0.75 to 0.97 and from 0.44 to 0.85 in the ADY-INC5 study.
Figure 2.
(a) Meta-analysis of AYX1 dose-response profile including results from the ADY-SNI2, INC5, SN1,2-4 (Mamet et al., 2014), -INC25 (Mamet et al., 2014), and -DOD1 (Mamet et al., 2014) studies testing AYX1 efficacy in either the spared nerve injury of neuropathic pain or in the incisional model of post-surgical pain. Data are normalized internally for each study against the maximum observed efficacy relative to vehicle-treated animals and presented as mean ± SEM. The connecting curve is presented with a dotted line, and data fitted with a polynomial equation of the fourth order are presented as a dashed line. A systematic series of potential dose-response fits were tested. The highest coefficients of regression were _R_2 ∼ 0.35 for the polynomial fit above and equivalent or lower for various association equations, including one-phase exponential or hyperbolic fits. In absence of a robust coefficient of regression and considering that the polynomial curve fits the meta-analysis and the results of the majority of individual studies, it was selected as the closest representation of AYX1 dose-response over a hyperbolic or exponential plateauing curve. (b) Concentration of AYX1 shortmers in the LCSF 30 min following injection of 1.1, 2.2, or 3.85 mg of AYX1; n = 5 per dose-level; results are presented as mean ± SEM for each dose-level. Data are fitted with a sigmoidal function (dotted line).
Figure 3.
(a) Rate of AYX1 metabolism as a function of AYX1 concentration in fresh spinal cord homogenates. The rate of AYX1 metabolism is presented as the amount of full-length AYX1 degraded per minute as a function of AYX1 concentration introduced in the homogenates. Data are presented for each measured time-point: 5 (circle), 30 (square), and 60 (triangle) min. Linear regression for each data set are presented; coefficient of linear regression _R_2 is ∼0.99 for 5, 30, and 60 min; data are presented as mean + SEM. (b) Metabolic patterns of full-length AYX1 and individual N-1 to N-6 metabolites measured with the CGHE assay. For each analyzed sample, the relative amount of AYX1 or individual metabolite species was normalized on the species found in largest amount. Each curve represents the metabolite pattern observed for a dose-level, compartment, and time, and the corresponding concentration of total AYX1 is listed in the legend. The amounts of N-4 to N-6 metabolites were pooled together due to the frequent fusion of their corresponding peaks in the CGHE assay. Values are presented as mean + SEM, n = 2 to 4 per condition, FL = full-length AYX1, N-x = metabolite species. (c) Illustration of electropherograms from the CGEH analytical method from the LCSF at 60 and 240 min following injection. Right arrows show full-length AYX1 and the left arrows the extremity of the analyzable area. Peaks in between the two arrows represent AYX1 shortmers from N-1 to N-6. LCSF: cerebrospinal fluid; DRG: dorsal root ganglia.
Figure 4.
AYX1 presence in lumbar DRG (a) and spinal cord (b) cells was observed 30 min following an IT injection of 1.7 mg of AYX1 conjugated to an ALEX488 tag (green). Tissue auto-fluorescence was controlled using non-injected rats (c). Areas identified in the top panels with white rectangles are magnified in the bottom panels. One example of AYX1-positive cell nuclei is pointed out for the DRG and spinal cord with a vertical arrow. A few cells show AYX1 present in the cytoplasm, and one example is pointed out in both tissues by a horizontal arrow. This observation is consistent with independent experiments showing the presence of decoys in the nucleus and/or cytoplasm of cells following various administration methods–; Top panel scale bar = 100 µm, bottom panel scale bar = 25 µm, and n = 2 rats per condition.
References
- Apfelbaum JL, Chen C, Mehta SS, et al. Postoperative pain experience: results from a national survey suggest postoperative pain continues to be undermanaged. Anesth Analg 2003; 97: 534–540. -PubMed
- Johansen A, Romundstad L, Nielsen CS, et al. Persistent postsurgical pain in a general population: prevalence and predictors in the Tromso study. Pain 2012; 153: 1390–1396. -PubMed
- Kehlet H, Jensen TS, Woolf CJ. Persistent postsurgical pain: risk factors and prevention. Lancet 2006; 367: 1618–1625. -PubMed
- Lavand'homme P. The progression from acute to chronic pain. Curr Opin Anaesthesiol 2011; 24: 545–550. -PubMed
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