the alkaloid morphine has been used in the treatment of pain , cough , and diarrhea . unfortunately , unpleasant and/or potentially dangerous side effects such as respiratory depression , nausea , vomiting , and constipation can accompany its use . psychological and physiological processes such as abuse , tolerance , and physical dependence have been associated with chronic use and limit the utility of morphine and other mu - opioid agonists in the treatment of chronic pain . in a continuing search for potent analgesics free of these undesirable side effects , the evolution of this search was summarized in a succinct review of the exciting but vain quest for the holy grail of opioid research . results of an investigation of the effects of leucine and methionine enkephalin on morphine - induced analgesia suggested an interaction between mu- and delta - opioid receptors . over a decade later , investigators found that naltrindole , a selective delta - opioid receptor antagonist , blocked the morphine tolerance without diminishing its antinociceptive potency . these reports and the finding that g - protein - coupled opioid receptors existed as heterodimers : mu - kappa , mu - delta [ 5 , 6 ] , and kappa - delta   led to a hypothesis - driven synthesis   of a series of conjugates containing the mu - opioid receptor agonist ( derived from oxymorphone ) linked with delta - opioid receptor antagonist ( related to naltrindole ) with spacers ranging from 16 to 21 atoms . studies in mice indicated that 7-{2-[(7-{2-[({(5 , 6)-4,5-epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonyl)-metoxy]-acetylamino}-heptylaminocarbonyl)-methoxy]-acetylamino}-naltrindole or mdan-21 , ( see figure 1 ) , with a 25.4   spacer , ( 21-atoms ) , was the most potent analgesic of the series . significantly , naloxone , an opioid receptor antagonist , failed to precipitate withdrawal signs after chronic administration . these properties suggested that mdan-21 may , to a significant extent , fulfill the long quest for a strong opioid analgesic with greatly reduced side effects . we decided to determine mdan-21 's effects on morphine - induced physical dependence and explore its analgesic profile in nonhuman primates . the rhesus monkey study models were selected because of their excellent relationship to the behavioral expressions of opioid effects in humans and their predictive value regarding pharmacotherapy   especially regarding opioids . all monkeys had prior exposure to drugs and to the behavioral procedures in which they were tested . the subjects in the substitution of morphine assay were housed in groups , whereas those in the thermal nociception and capsaicin - induced thermal allodynia assays were individually housed . a 12  h light/12  h dark cycle was in effect ( lights on from 7am7pm ) . animal maintenance and research were conducted in accordance with the guidelines provided by the nih committee on laboratory animal resources . the facility was licensed by the united states department of agriculture and accredited by the association for the assessment and accreditation of laboratory animal care . monkeys also had access to puzzle feeders , mirrors , and chew toys to provide environmental enrichment . nature videotapes or music twenty - four monkeys in the weight range 3.57.5  kg comprised the group tested . morphine was given subcutaneously ( s.c . ) daily at 6 am , 12 noon , and 6 pm . all the animals had received morphine for at least 3 months and were maximally dependent on morphine . at least 4 monkeys / treatment were used and a minimal two - week wash - out period was allowed between testing . it was initiated by the injection ( s.c . ) of the test drug or control substances ( morphine and vehicle ) into animals in a group that had not received morphine for 14 - 15  hr and showed definite signs of withdrawal . each animal was randomly chosen to receive one of the following treatments : ( a ) a dose mdan-21 ; ( b ) morphine sulfate control ( 4.0  mg / kg ) ; ( c ) vehicle control ( 1.0  ml / kg ) . withdrawal signs were scored , absent , or present , once during each of five consecutive 30-min observation periods . withdrawal signs included slowing of movement , drowsiness ( sitting with eyed closed and lethargic or being indifferent to surroundings ) , fighting , vocalizing , rigidity of abdominal muscles , vocalization during palpation of abdominal muscles , restlessness ( pacing ) , tremors , coughing , retching , vomiting , wet - dog shakes , and masturbation . the cumulative number of withdrawal signs was analyzed using the kruskal - wallis analysis of variance ( anova ) and post hoc mann - whitney u tests . the stat view statistical package ( brainpower , agoura hills , ca ) was utilized for these analyses . in all cases , significance ( p ) they were seated in acrylic restraint chairs so that their tails hung down freely . during tail - withdrawal measurements , the bottom 15  cm of each monkey 's shaved tail was immersed in a thermal container of warm water . if the subject did not withdraw its tail within 20  s , the tail was removed from the water by the experimenter , and a latency of 20  s was assigned to that measurement . each test session consisted of multiple cycles . before mdan-21 administration , baseline tail - withdrawal latencies from 38 and 50c water were determined . testing continued only if tail withdrawal from 38c water did not occur before the 20  s cutoff , and if tail withdrawal occurred in 2  s from 50c water . the effects of mdan-21 intramuscularly ( i.m . ) were evaluated using a time - course procedure , in which a single dose of mdan-21 ( 0.00320.32  mg / kg ) was administered at the start of the session , and tail withdrawal latencies from 50c water were redetermined 3 , 6 , 10 , 18 , 32 , 56 , and 100  min after the injection . accordingly , a follow - up experiment was conducted in these two monkeys to determine whether mdan-21 might function as an antagonist of the high - efficacy mu - agonist methadone . for these studies , monkeys were pretreated with vehicle or 0.32  mg / kg mdan-21 15  min before treatment with 0.32  mg / kg methadone . tail - withdrawal latencies were then determined 10 , 20 , and 30  min after methadone administration . drug effects were expressed as percent maximum possible effect ( % mpe ) using the following equation :  % mpe = ( test latency  baseline latency)/(20  baseline latency)100  , where  test latency  was the tail - withdrawal latency from 50c water obtained during each cycle after drug administration ,  baseline latency  was the latency from 50c water during the baseline determinations before drug injection , and  20  was the cutoff latency in s. three different male rhesus monkeys 512  kg were studied . monkeys were seated in acrylic restraint chairs as described above . to determine tail - withdrawal latencies , the lower 15  cm of each monkeys shaved tail was immersed into a thermal container of warm water heated to the designated temperature ( see below for temperatures ) . the latency in seconds for the monkey to remove its tail from the water was measured using a hand - held stopwatch . if the subject did not withdraw its tail within 20  s , the tail was removed from the water by the experimenter , and a latency of 20  s was assigned to that measurement . experimental sessions were conducted once per week . at the beginning of each session , tail withdrawal latencies were determined for each monkey from water heated to 38 , 42 , 46 , and 50c , and the order of temperature presentations was randomized across sessions . by this procedure , baseline temperature - effect curves were determined in each monkey at the beginning of each session , and the highest temperature to produce a tail withdrawal latency > 15  s was identified . water heated to this  threshold  temperature then served as the thermal stimulus for subsequent studies of allodynia during that session . the threshold stimulus intensity was 42c for two monkeys and 46c for the third monkey throughout the study . allodynia was elicited by topical application of capsaicin as described previously [ 15 , 16 ] . following baseline tail withdrawal latency determinations , a topical patch treated with capsaicin solution or vehicle was prepared as described below ( see drugs ) , and the patch was applied to a region approximately 7  cm from the bottom of the tail for 5  min . tail withdrawal latencies were then redetermined 15 , 30 , 45 , and 60  min after patch removal using the thermal stimulus identified from the baseline temperature - effect curve in each monkey ( i.e. , 42c in two monkeys , 46c in the third monkey ) . morphine ( 1.0  mg / kg , i.m . , 15  min pretreatment ) was tested for comparison . raw tail withdrawal latencies obtained 15 , 30 , 45 , and 60  min after removal of the capsaicin patch were converted to percent maximum possible effect ( % mpe ) using the equation % mpe = [ ( test latency  capsaicin alone latency)/(20  capsaicin alone latency)100 ) ] , where  test latency  was the tail withdrawal latency obtained at each time point after drug pretreatment + capsaicin patch treatment , and capsaicin alone latency  was the latency obtained at the corresponding time point after treatment with the capsaicin patch alone . all drugs were dissolved in sterile water for injection ( hospira , inc . , forest hills , il ) . louis , mo ) was dissolved in vehicle composed of 70% alcohol and 30% sterile water and was delivered transdermally ( topical patch ) as described previously [ 15 , 16 ] . the concentration of capsaicin in the solution was individually determined for each monkey as the lowest concentration to produce sustained decreases in tail - withdrawal latencies from the threshold temperature to 5  s throughout the 1  hr testing period ( 0.61  mg / ml ( 2  mm ) ) for all three monkeys in the study . within 30 s of preparing the capsaicin patch , it was secured onto the monkey 's tail with elastic tape and left on for 5  min . it is evident that mdan-21 had a short onset and long duration of action , that is , it effectively suppressed withdrawal as soon as 30  min and was still effective at 150  min . mdan-21 and morphine suppressed the full spectrum of withdrawal signs exhibited by the vehicle controls . the effective dose range was 0.0060.03  mg / kg . the kruskal - wallis anova p values are as follows : 30  min-0.01 ; 60  min-0.0002 ; 90  min-0.0002 ; 120  min-0.0001 ; 150  min-0.0001 . post hoc mann - whitney comparisons with probability values of 0.05 or less are indicated in figure legends and with appropriate superscript letters in figure 2 . the mean  s.e.m baseline tail withdrawal latency from 50c was 0.75  0.18  s.   figure 3 shows the time course of antinociceptive effects produced by doses of 0.00320.32  mg / kg mdan-21 in three individual monkeys . in monkey m1470 ( one of the two males ) , mdan-21 doses of 0.0032 and 0.032  mg / kg produced a dose - dependent increase in tail withdrawal latencies that peaked after 18 and 32  min and then dissipated after 56  min . however , in the other two monkeys , mdan-21 doses of 0.032 and 0.32  mg / kg were largely ineffective , producing only small increases in tail - withdrawal latencies after 6  min in monkey m1472 . higher doses were also tested in m1472 ( 2.6  mg / kg ) and m1475 ( 1.0  mg / kg ) and these doses were also ineffective ( maximum effect of 15% mpe at any time in either monkey ; data not shown ) . figure 4 shows the effects of pretreatment with vehicle or mdan-21 on the antinociceptive effects of 0.32  mg / kg methadone in monkeys m1472 and m1475 . after vehicle pretreatment , pretreatment with 0.32  mg / kg mdan-21 had no effect on methadone antinociception in either monkey . the mean  s.e.m baseline tail withdrawal latency at the threshold temperature was 19.59  0.41  s , and treatment with capsaicin alone reduced tail withdrawal latencies at the threshold temperature to mean  s.e.m . 66  0.23 , 1.65  0.41 , and 1.49  0.10  s at times 15 , 30 , 45 , and 60  min after capsaicin patch removal , respectively . figure 5 shows that pretreatment with mdan-21 ( 0.00320.32  mg / kg ) produced a dose- and time - dependent prevention of capsaicin - induced allodynia . the highest dose of 0.32  mg / kg mdan-21 produced peak antiallodynic effects after 15  min , and these effects dissipated after 45 to 60  min . for comparison , the antiallodynic effect of 1.0  mg / kg morphine peaked after 30  min and remained at levels greater than 50% mpe in all three monkeys at 45  min and in two of three monkeys at 60  min . it had a remarkably long duration of action compared to that in the capsaicin - thermal assay in nondependent monkeys . perhaps , mdan-21 is much more effective in situations involving physical dependence . increased pain sensitivity in chronic pain patients and opioid abusers mdan-21 failed to produce a reliable antinociceptive effect in the assay of thermal antinociception in nondependent monkeys . in one monkey , a dose of 0.032 , mdan-21 was fully effective , but neither this dose nor a 10-fold higher dose of 0.32  mg / kg was active in the other two monkeys . conversely , mdan-21 was as effective as morphine in all monkeys tested in the assay of capsaicin - induced thermal allodynia . this profile of results is consistent with the possibility that mdan-21 does not readily cross the blood - brain barrier in rhesus monkeys . on the other hand , suppression of withdrawal signs in morphine - dependent monkeys by mdan-21 it is known that quaternary compounds , do not readily cross the blood - brain barrier . indeed , quaternary morphine compounds were reported virtually inactive in morphine - dependent rhesus monkeys and practically devoid of antinociceptive activity in mice . the variability in effects produced by mdan-21 in the assay of thermal nociception did not appear to result from low efficacy of mdan-21 at mu - opioid receptors . if mdan-21 had failed to produce antinociception in 2 of 3 monkeys due to low efficacy , then it would be expected to antagonize the effects of a higher efficacy mu - agonist in these two monkeys . however , mdan-21 failed to antagonize the antinociceptive effects of methadone in either monkey . nevertheless , it is possible that higher doses might have achieved a robust antinociceptive effect . finally , species differences in mu - delta interactions on neural substrates that mediate thermal antinociception may be involved [ 2123 ] . nevertheless , mdan-21was potently active in the capsaicin - induced thermal allodynia assay in the dose range of 0.0320.32  mg / kg . it also attenuated withdrawal signs in morphine - dependent monkeys in spontaneous withdrawal for a longer time period . the results are in accord with the findings of other investigators that pain sensitivity is increased in opioid - treated animals , chronic pain patients and opioid abusers ( see references above ) . the results also suggest that mdan-21 may be useful in the pharmacotherapy of chronic pain and chronic opioid use .
mdan-21 , 7-{2-[(7-{2-[({(5 , 6)-4,5-epoxy-3,14-dihydroxy-17-methylmorphin-6-yl}-aminocarbonyl)metoxy]-acetylamino}-heptylaminocarbonyl)-methoxy]-acetylamino}-naltrindole , a bivalent opioid ligand containing a mu - opioid receptor agonist ( derived from oxymorphone ) linked to the delta - opioid receptor antagonist ( related to naltrindole ) by a spacer of 21 atoms , was reported to have potent analgesic properties in mice . 
 tolerance , physical dependence , and conditioned place preference were not evident in that species . 
 the finding that bivalent ligands in this series , with spacers 19 atoms or greater , were devoid of tolerance and dependence led to the proposal that mdan-21 targets heteromeric mu - delta - opioid receptors . 
 the present study focused on its effects in nonhuman primates ( macaca mulatta ) , a species with a physiology and behavioral repertoire not unlike humans . with regard to opioids , 
 this species usually better predicts clinical outcomes . 
 mdan-21 substituted for morphine in morphine - dependent monkeys in the remarkably low dose range 0.0060.032  mg / kg , subcutaneously . 
 although mdan-21 failed to produce reliable thermal analgesia in the dose range 0.00320.032  mg / kg , intramuscularly , it was active in the same dose range and by the same route of administration , in the capsaicin - induced thermal allodynia assay . 
 the results suggest that mdan-21 may be useful in the treatment of opioid dependence and allodynia . 
 the data provide additional evidence that opioid withdrawal is associated with sensitized pain .