Characterization of the antinociceptive effects of intrathecal DALDA peptides following bolus intrathecal delivery

2.1 Animals

Male Sprague-Dawley rats (225–300 g; Harlan, Indianapolis, IN, USA) were individually housed in standard cages and maintained on a 12-h light/dark cycle (lights on at 7 am). Testing occurred during the light cycle. Food and water were available ad libitum. Animal care was in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85-23, Bethesda, MD, USA) and as approved by the institutional Animal Care and Use Committee of the University of California, San Diego, La Jolla, CA, USA.

2.2 Surgical preparation

Each rat was implanted with a single IT catheter for drug delivery, as described previously [10]. In brief, rats were anesthetized by induction with 4% isoflurane in a room air/oxygen mixture (1:1), the anesthesia was maintained with 2% isoflurane delivery by mask, and the animal placed in a head holder. The cisternal membrane was exposed through a midline incision and a polyethylene (outer diameter 0.36 mm) catheter was passed into the IT space to the level of the L2–L3 spinal segments (8.5 cm). The catheter was externalized on the top of the head. Rats were given carprofen (5 mg/kg in lactated Ringer’s solution) subcutaneously and allowed to recover under a heat lamp. Rats exhibiting motor weakness or signs of paresis were sacrificed. Animals were allowed to recover for 5–7 days before the experiment.

2.3 Study paradigm

In the present work, opioid peptide agonists and morphine sulfate were delivered exclusively via an implanted IT catheter. Antagonists (naloxone, naltrindole) were given intraperitoneally (IP).

2.4 Behavioral testing

Before initiation of drug delivery, baseline behavioral and testing data were taken. At selected times after infusion of the test or control article, these data were again collected. All assessments were made with the observer blinded as to the drug being delivered.

2.5 Drugs and chemicals

The following drugs were synthesized as previously described [2], [14]: dDALDA (H-Dmt-D-Arg-Phe-Lys-NH_2; Dmt=2′,6′-dimethyltyrosine) (MW 981). (DMT-DALDA is abbreviated as dDAL in Figs. 2 and 4–6).

dDALc (H-Dmt-D-Cit-Phe-Lys-NH₂; Cit=citrulline) (MW 868).

dDALcn (H-Dmt-D-Cit-Phe-Nle-NH₂; Nle=norleucine) (MW 739).

TAPP (H-Tyr-D-Ala-Phe-Phe-NH₂) (MW 659).

dDAL-TICP (H-Dmt-D-Arg-Phe-Lys-NH-(CH₂)₂-NH←TICP[Ψ]; TICP[Ψ]=H-Tyr-TicΨ[CH₂-NH]Cha-Phe-OH [Tic=1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid; Cha=cyclohexylalanine]) (MW 1519).

dDAL-TIPP (H-Dmt-D-Arg-Phe-Lys(N^ε←TIPP)-NH₂; TIPP=H-Tyr-Tic-Phe-Phe-OH (MW 1604).

Morphine SO₄ (MS) (MW: 759) (MOR) was obtained from Merck Pharmaceuticals (Rahway, NJ, USA). Naloxone hydrochloride (Nx) and Naltrindole (Ntd) were obtained from Sigma Chemical (St. Louis, MO, USA). Each drug was dissolved in saline such that the total dose was delivered in 10 μL. Isoflurane was obtained from VETone (Meridian, ID, USA).

2.6 Sample size

For the thermal escape studies group size was based on a power analysis of the area under the curve (%MPE×h) observed with the appropriate control group, e.g. animals receiving intrathecal saline. For the IT saline control group, the mean±SD group thermal escape response latency with each animal assessed out to 4 h was 10.5±1.3 s (vs. 10.1±1.5 s in uninjected rats; p>0.10), which corresponded to a mean±SD AUC of 569±427 %MPE×h. An increase of approximately 50% over baseline (6 s) for 0.5 h was considered to be behaviorally meaningful and this corresponded to an AUC of 1350 %MPE×h. Assuming β=0.8 and designating p<0.05 as statistically significant, a power analysis indicated the need for 5 animals. For the formalin model, the effects on phase 2 flinching of the IT saline control, was 1458±321 flinches/phase 2 (vs. 1521±220 flinches/phase 2 in uninjected rats; p>0.10). A decrease in phase 2 flinching of approximately 50% below IT saline baseline (729 flinches/phase 2) was considered to be behaviorally meaningful. Assuming β=0.8 and designating p=0.05 as statistical significance, a power analysis indicated the need for 4 animals. Analyses were based on assumptions of normality https://www.stat.ubc.ca/∼rollin/stats/ssize/n2.html).

2.7 Statistical analysis

The data were compiled in Excel (v.14.4.9, Microsoft Corporation, USA), and statistical analyses were performed using Prism (v.6.0, GraphPad Software, Inc., La Jolla, CA, USA).

The response latencies were normalized by converting them to percentage of the maximum possible effect [(Post–Pre)/20 s – Pre]×100 and the area under the percentage max possible effect curve calculated (AUC). Treatments were compared using one-way or two-way analysis of variance as indicated, and post hoc comparisons across treatment groups accomplished with Sidak post hoc comparison. Differences reaching the p<0.05 level of rejection were considered to be statistically significant.

3.1 IT bolus delivery and thermal escape

3.1.1 Morphine

IT morphine sulfate (MS) resulted in a dose-dependent (3–30 nmol) increase in acute thermal escape latencies starting at 30 min and lasting in excess of 90 min (Fig. 1A). Pretreatment with naloxone (intraperitoneal administration; IP 1 mg/kg) but not naltrindole (IP 3 mg/kg) prevented this increase (Fig. 1B and C). Straub tail displayed a dose-dependent incidence across the range of doses which was unaltered by naloxone or naltrindole. Doses of >40 nmol resulted in severe truncal rigidity (Fig. 1D).

Fig. 1:

(A) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of morphine sulfate or saline vehicle. (B) Scattergraph of area under the curve of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists, and saline vehicle. One-way ANOVA analysis yielded p<0.0001. Sidak post hoc comparison showed **p<0.01, ***p<0.001, ****p<0.0001 compared to MTD (30 nmol) and ^##p<0.01, ^####p<0.0001 compared to saline vehicle. (C) Time effect graph of IT morphine with pretreatment (15 min) of systemic antagonists (naloxone [Nx] and naltrindole [Ntd]). (D) Scattergraph of the observance of Straub tail and motor dysfunction at any time during the time course.

3.1.2 DMT-DALDA

IT DMT-DALDA resulted in a dose-dependent (1–10 pmol) increase in acute thermal escape latencies starting by 15 min and lasting 2–4 h (Fig. 2A). Pretreatment with naloxone (IP1 mg/kg) but not naltrindole (IP 3 mg/kg) prevented this increase. IT DMT-DALDA resulted in a dose-dependent incidence of Straub tail during the first 30 min after injection at doses up to 10 pmol, which was not prevented by either naloxone or naltrindole (Fig. 2A). Strong, reversible, truncal rigidity was observed at doses of 30 pmol.

Fig. 2:

(A) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of dDALDA or saline vehicle, scattergraph of area under the curve and observance of Straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis showed statistical significance of p<0.0001 with Sidak post hoc comparisons of **p<0.01, ****p<0.0001 compared to MTD (10 pmol) and ^#p<0.05, ^##p<0.01, ^###p<0.001 compared to saline vehicle. (B) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of cDALc or saline vehicle and scattergraph of area under the curve and observance of Straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis showed statistical significance of p=0.0002 with Sidak post hoc comparisons of *p<0.05, **p<0.01, ***p<0.001 compared to MTD (10 pmol) and ^##p<0.01, ^###p<0.001 compared to saline vehicle. (C) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of cDALcn or saline vehicle and scattergraph of area under the curve and observance of Straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis showed statistical significance of p=0.0014 with Sidak post hoc comparisons of **p<0.01 compared to MTD (300 pmol) and ^#p<0.05, ^##p<0.01 compared to saline vehicle.

3.1.5 TAPP

IT TAPP was with minimal effect on thermal escape latencies at 10 nmol. Higher doses were not examined due to truncal paralysis at 46 nmol (Fig. 3A).

Fig. 3:

(A) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of TAPP or saline vehicle, scatter of area under the curve and observance of Straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis shows p=0.0906 and post hoc analysis were no significant difference compared to MTD (10 nmol). (B) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of dDAL-TICP or saline vehicle and scattergraph of area under the curve and observance of straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis showed statistical significance of p=0.0003 with Sidak post hoc comparisons of *p<0.05, **p<0.01 compared to MTD (300 pmol) and ^##p<0.01 compared to saline vehicle. (C) Dose-response curve of thermal latencies (seconds) vs. time post IT administration of dDAL-TIPP or saline vehicle and scattergraph of area under the curve and observance of Straub tail or motor dysfunction of MTD (1, 1/2 log, 1 log), MTD with pretreatment of antagonists or saline vehicle at any time during the time course. One-way ANOVA analysis showed statistical significance of p=0.0014 with Sidak post hoc comparisons of **p<0.01, ***p<0.001, ****p<0.0001 compared to MTD (30 pmol) and ^##p<0.01 compared to saline vehicle.

3.2 IT bolus DMT-DALDA: formalin flinching

Intraplantar delivery of formalin resulted in biphasic flinching of the injected hind paw over the 60 min after formalin injection. IT delivery of DMT-DALDA (10 pmol) resulted in a significant reduction in phases 1 and 2 flinching (Fig. 4A–D) as compared to IT vehicle control when administered as soon at 5–6 h in advance of formalin, but not when given 24 h before formalin, emphasizing the long duration of action (Fig. 4E and F).

Fig. 4:

(A, B, C, D). Minute by minute plot of formalin-induced flinching at 0.2, 2, 6 and 24 h after pretreatment with IT DMT-DALDA (10 pmol/10 L) or IT saline vehicle (10 μL). (E/F) Total flinch count at 15 min, 2, 6 and 24 h after pretreatment with IT DMT-DALDA or IT vehicle for phase 1 (E) and phase 2 (F). Comparisons were made with two-way ANOVA (Treatment main effect p-value shown in each graph). Post hoc comparisons were carried out between each drug treatment and vehicle using Sidak post hoc test (*p<0.05, **p<0.01, ***p<0.01).

3.3 IT bolus morphine and fentanyl: formalin flinching

For comparison with standard opiate analgesic molecules, pretreatment studies were carried out with morphine (30 nmol/10 μL) and fentanyl (1 nmol/10 μL). As indicated in Fig. 5, Morphine produced significant reductions of phase 1 and phase 2 through 6 h, but not 24 h. In contrast, fentanyl (1 nmol/10 μL) resulted in a significant elevation at 15 min pretreatment, but not at 2 h reflecting its rapid spinal clearance and short duration of action (Fig. 5).

Fig. 5:

(A) Minute by minute plot of formalin-induced flinching at 15 min and 2, 6 and 24 h after pretreatment with IT morphine (30 nmol/10 μL) or the IT vehicle (10 μL). (B) Minute by minute plot of formalin induced flinching at 15 min (0.2 h) and 2 h after pretreatment of IT Fentanyl (1 nmol/10 μL) or the IT vehicle (10 μL). (C) Total flinch count during phase 1 (0–10 min) and phase 2 (11–60 min) at (C) 15 min, 2, 6 and 24 h after pretreatment with Morphine or (D) 15 min or 2 h for IT Fentanyl. Comparisons were made with two-way ANOVA (Treatment main effect p-value shown in each graph). Post hoc comparisons were carried out between each drug treatment and vehicle using Sidak post hoc test. (*p<0.05, **p<0.01, ***p<0.01).

3.4 IT bolus DMT-DALDA: tolerance and cross-tolerance on thermal escape

3.4.1 Tolerance after repeated bolus delivery of DMT-DALDA

DMT-DALDA (10 pmol) or saline were administered IT for 3 days (Fig. 6). On day 4, both groups received DMT-DALDA. As shown, on day 4 DMT-DALDA in animals which had received DMT-DALDA on three preceding days showed significantly less analgesia than did animals receiving three preceding days of saline, indicating a loss of DMT-DALDA activity (e.g. tolerance) (Fig. 6B). On day 5, all animals received IT morphine (30 nmol). As shown in Fig. 6B, animals that received 4 days of saline and 1 day of DMT-DALDA showed a greater response to morphine than did animals with 4 preceding days of DMT-DALDA (e.g. evidence of a cross-tolerance between morphine and DMT-DALDA).

Fig. 6:

(A) Time effect graph of thermal latencies of daily bolus IT DMT-DALDA for 4 days group vs. one-time bolus IT DMT-DALDA on day 4 group. On Day 5 both groups were given morphine. (B) Area under the curve (AUC) of each group on Days 1, 4, 5 and saline vehicle given on Day 1. One-way ANOVA analysis yielded p<0.0001 with Sidak post hoc comparisons of *p<0.05, **p<0.01 compared to each group.

3.5 Tolerance after repeated bolus delivery of morphine

The same experimental paradigm was used to examine the properties of IT morphine and its interaction with DMT-DALDA. In Fig. 7, IT morphine or saline was given for 3 days. On day 4, morphine was given to both groups. As indicated (Fig. 7B) morphine given to animals receiving morphine for the three preceding days showed a significantly lesser response as compared to animals that had received saline on days 1–3, e.g. tolerance (Fig. 7B). On day 5, DMT-DALDA showed an equal degree of analgesia whether given in a rat that had 3 days of saline and 1 day of morphine as compared to 4 days of morphine (Fig. 7C).

Fig. 7:

(A) Time effect graph of thermal latencies of daily bolus IT Morphine for 4 days group vs. one-time bolus IT morphine on Day 4 group. On Day 5 both group were given DMT-DALDA. (B) Area under the curve (AUC) of each group on Days 1, 4, 5, and saline vehicle given on Day 1. One-way ANOVA analysis yielded p<0.0001 with Sidak post hoc comparisons of *p<0.05, **p<0.01, ***p<0.001 compared to each group.

4.1 Efficacy and potency

In the present work, these agents displayed a dose dependent effect on acute thermal escape. Further, with DMT-DALDA, at doses effective in blocking acute thermal escape, a significant suppression was noted in phase 1 and phase 2 formalin flinching. Phase 1 is considered to be an endpoint reflective of acute nociception, while phase 2 reflects the role of a facilitated state of spinal nociceptive processing [13].

4.2 Duration of action

The duration of action of spinal drugs depends largely upon the rate at which they are cleared into the systemic circulation after delivery and to a lesser degree by their metabolism in the CNS [1]. Early work with peptides such has β endorphin and DADLE ((D-Ala², D-Leu⁵)-enkephalin) [15], [16], [17] revealed long durations of actions after IT delivery as compared to a small polar molecule such as morphine and in particular when compared to small lipophilic molecules (such as remifentanil, fentanyl or sufentanil) [18], [19]. In this work we systematically compared the bolus effects of a number of dermorphin-derived peptides and analogues that ranged in IT potency from pmol to nmol doses. Previous work had shown that the DALDA analogues were resistant to metabolism in ex vivo blood incubation studies [20]. Importantly, this duration of activity was noted in not just an acute thermal threshold model but was confirmed by the potent effects upon the two-phased flinching observed in the formalin model. The use of increased pretreatment intervals also emphasized the extended duration of action of the potent DMT-DALDA molecule, which compared most favorably to that associated with morphine. The fentanyl-formalin data in contrast emphasizes the brevity of the behavioral action of mu opioids that are rapidly cleared.

4.3 Pharmacology

The antinociception observed after IT agents were uniformly reversed by naloxone and not by naltrindole. The doses employed were previously shown to be respectively shown to be effective in reversing the analgesia produced by the IT delivery of mu and delta-preferring ligands [21]. An interesting variant to the possible mechanisms involved in DMT-DALDA is that this tetrapeptide amide (H-Dmt-D-Arg-Phe-Lys-NH₂) is not only a potent and selective μ opioid receptor (MOR) agonist but is reported to display antioxidant activity due to its N-terminal Dmt (2′,6′-dimethyltyrosine) residue [22] and to block norepinephrine reuptake in synaptosomes [23]. Given the efficacy of naloxone in reversing these effects, the role of the antioxidant and uptake blocking properties of these molecules in the present model of acute thermal nociception and formalin evoked flinching appear minimal, though if there is a robust synergy between mu receptor activity and the other two mechanisms, then loss of mu function would result in a total collapse of effect. This possibility was not studied in the present work.

While both MOR and DOR-preferring agonists can block substance P release from C-fibers to yield analgesia after IT delivery [21], [24] and interact synergistically [25], it has been reported that the severity of tolerance development in morphine-treated animals is reduced in DOR knockout mice [26] or when using DOR antagonists [27], [28], [29]. This phenomenon has been suggested to reflect a modulatory role of DOR on MOR function [30]. In the present study IT delivery of dDAL-TICP and dDAL-TIPP (agents with significant MOR agonist and DOR antagonist activity) displayed a surprisingly minimal effect on pain behavior. The reason for the contrast with previous reports may reflect spinal vs. supraspinal systems. The interpretation of the spinal reflex as a marker of nociceptive processing may be rendered moot where there is an effect of the drug on motor function, which, in this case, results in bilateral increases in motor out flow to the tail musculature.

4.4 Motor effects

The principal motor effect first observed with escalating doses of IT morphine and peptides was the appearance of the classic Straub tail [31]. This is a marker of increased motor tone in the sacrococcygeus dorsalis muscle, mediated by increased opiate receptor-initiated activation of lumbosacral outflow. The lack of effect of spinal section on the Straub tail phenomena argues that it can be mediated at the spinal level [32]. Importantly, the appearance of Straub tail renders a spinal response such as the tail flick difficult to interpret as indices of nociception. In the present studies, IT saline had no effect upon tail appearance, whereas the agents employed resulted in an increased incidence of at least brief signs of Straub (stiffened) tail. Several studies have reported that the motor effects after systemic [33], [34], [35] or IT opiates [36] are at least partially diminished by naloxone. In the present work, naloxone at a dose completely reversing the effect on thermal escape latency had no effect upon the incidence of the Straub tail endpoint. A gradation assessment of the magnitude of the Straub tail response was not made, and we recognize that changes in the degree of motor effects may have been missed. Further, we note our studies were undertaken with a single antagonist dose (albeit one that completely blocked the intrathecal drug effects upon thermal escape) in the face of a single just maximally usable agonist dose. At this moment we speculate that these data support the possibility that a nonopiate receptor mechanism may be at play in this spinal effect in the rat, including a possible role involving toll receptors [37] or Mas-related G-protein–coupled receptors (MRGPRs) [38].

4.5 Tolerance

In the present studies repeated bolus delivery of equianalgesic doses of morphine and DMT-DALDA resulted in evidence of tolerance over a 5-day interval. Of note, animals tolerant to morphine continued to respond to DMT-DALDA, whereas animals tolerant to DMT-DALDA did not respond to morphine. Such asymmetric cross-tolerance has been previously described for IT infusions of mu agonists (morphine and sufentanil) [39], [40], [41]. The mechanisms of such asymmetry are not clear. Previous work has suggested that it may reflect the relative intrinsic activity of the two agonists, reflecting different degrees of receptor occupancy required by different agonists to yield a criterion effect, e.g. a measure of intrinsic activity. With continued drug exposure, the pharmacodynamics resemble the condition associated with a loss of functional receptors over time, [42] with the changes having a greater impact upon the agent with lower intrinsic activity [39], reflecting the magnitude of a receptor reserve [43]. In addition, as noted above, DMT-DALDA, aside from being a MOR agonist, also has other pro-analgesic actions that might account for the asymmetry in tolerance.

4.6 Mu/delta interactions

Several studies have suggested that μ-receptor activity could be modulated by δ-ligands. Several disparate conclusions have been noted. First, it has been reported that IT delivery of μ and ∂ ligands can result in a positive synergy [25], [44], [45]. Second, other observations have suggested that antagonists for the δ-receptor can enhance the analgesic potency and efficacy of μ-agonists [9]. The present examination of two such compounds, dDAL-TICP and dDAL-TIPP [9], were without evident activity in thermal escape at maximal doses without effects upon motor function. These results with these agents at these doses employed do not support a positive synergy between spinal μ agonism and δ antagonism.