Immunologic Effects of Morphine in Rodents, Rabbits, Monkeys, and Cats1)

z. Z. Immun.-Forsch.

vol. 151, pp. 126-137 (1976)

Biological Sciences Division, Midwest Research Institute, Kansas City, Missouri, USA

Immunologic Effects of Morphine in Rodents, Rabbits, Cats!) Monkeys, and Cats-) DAVID A. RINGLE and BETTY L. HERNDON 1975 . Accepted September 4, 1975 With 2 Figures . Received June 19, 1975

Abstract The effects of prolonged morphine administration on immunologic reactivity against morphine was studied in a number of animal species: rabbit, monkey, guinea pig, rat, and cat. Some evidence for increased serum binding oP'C-labeled morphine was noted after morphine treatment in all test species, with the rabbit no response. In addition to the best responder and the cat showing little or no HC·labeled morphine, other methods (meas(meas· measurements on serum binding of HC-labeled HC·labeled codeine and methadone, competitive urement of serum binding of HC-labeled inhibition tests, radial immunodiffusion, and passive hemagglutination) were used for one or more of the species. Overall, results with these test methods have shown that prolonged morphine administration can result in immunologic responsiveness to morphine in animals.

Introduction

Studies carried out by a number of investigators have indicated that morphine administration is capable of evoking immunologic responsiveness against morphine, for example NADEAU and SOBOLEWSKI (1960), RYAN et al. (1972), BERANEK (1974), and our own studies cited later. There is also evidence that tolerance to morphine may be, at least in part, immunologic in nature (COCHIN 1971). We have previously reported on the occurrence of an increase in morphine binding by whole serum and serum Ig-containing fractions from rabbit bleedings taken after prolonged administration of morphine with either morphine sulfate (MS) or morphine alkaloid pellet (MP) pretreatments (RINGLE and HERNDON 1972, 1975). In addition to the serum morphine-binding response, we found that morphine administration in rabbits also showed other evidence for immunogenicity in this species, including positive reactions in in vitro and in vivo immunologic tests employing morphine-carrier conjugates, as well as a reduction in the serum morphinebinding response by cyclophosphamide administration. The study reported here was carried out to extend our earlier findings on immunologic responsiveness to morphine in morphine-treated rab1) This work was supported by United States Public Health Service Grant No. 1) DA 00125.

Immunologic Effectss of Morphine in R odents odents,, Rabbits, Monkeys, Immunol ogi c Effect R abbit s , Monkey s , and a n d Cats . 127

bits species.. W eefound ev evidence bi t s to other species ide nce for immunologic responsiveness responsiven ess against agains t morphine after morphine treatmen treatmentt in 4 of of the t he species ttested ested (rabbit,, monkey monkey,, guinea pig, and rat) and an indication indi cation of a po possible ssible (rabbit serum response on se in the cat. ser um morphine-binding resp Materials and Methods Animals morphi 1. An ima ls and and m or p h i ne n e dosage Adolescent young Ado lescen t or you ng adult a dult virgin females femal es of of the following foll ow ing species species and a n d strains used:: rrabbits, New Zealand River COBS were used abbits, N ew Zeal a nd aalbino; lb ino ; rats, rat s , Charles R iv er CO BS outbred albino Sprague-Dawley erived (L-E); guinea a lbino Sprague -D a wl ey d eriv ed (S-D), (S -D ), and hhooded ooded Long-Evans (L -E) ; guin ea pigs, a lb ino; cats, sho short-hair rt-h ai r domesti domestic; c ; and mon monkeys, k eys, cynamolgus pi gs , Camm-Hartley Ca m m -H a rtley albino; fascicul aris (Macaca fasci cul ar is ). Unless otherwise indicat in di cated ed,, m orphine orphine was given gi v en for ddosage osa ge courses 10 days in length, with courses separat se parated ed by drug-free periods la lasting stin g 4-6 weeks. Moradministered either -filtered phine was administer ed eit he r as MS (Merck, USP) in sterile -filter ed isotonic saline solutions or as MP (Grnsox (GIBSON and TINGSTAD TINGSTAD 1970) m made a d e with wi t h purified morphine product a lk aloid (Mallinckrodt prod uct No. 1616), except for one exp ex p eriment erim en t with p h ine alkaloid rats which were given m morphine orphin e hydrochloride h ydrochlorid e (Mer (Merck, ck, USP) instead in st ead of MS. Mo Morphine rphi ne in solution so lution was administer administered ed to rrats a t s at ddaily a ily increasing ddose ose levels ily bbeginning mg/d g/d ose/rat ggiven iv en twice d aaily eginn in g with 2 m ose/ra t and ending with 32 mg/dose/rat for each 10-d lO-day compounded contain a y course. MP were co m pou n ded to contai n morphine morph ine a lkaloid lkaloid at eith ellet (forr use in pigs, ellet eit h er 75 mg/p rug /p ell et (fo in rats ra t s and a n d guinea pi gs , and for t he first fir st 2 ppell et monkeys. Placebo courses in monkeys) m onkey s) or 300 300 m gg/p /p ellet ell et for fo r rabbits, rabb it s , cats, and monk eys. Pla ceb o pellets ne solutions samee way except p ellets (PP) (P P) and a nd control sali sa line so lutions were prepared prep a red in in the t h e sam omission orphine.. P Pellets each for tthe h e om issio n of of m orphine ellets were we re surgically su rgically rremov em ov ed ed at the end en d of of each implantation course. impl ant ation cours e. bindi n g measurements m ea s u r eme nts 2. Morphine M orphin e bindin Measu Measurement rement of m morphine orphin e binding b in d in g was car carried r ied out eit either her by equilibrium eq u ilib r iu m di diaalysis modification a rr ammonium sulfate echnique HC_ odifica t io n of the t h e F arr a m moniu m su lfate t ech niqu e with 14C_ ly sis or bby y a m labeled narcotics: (N -rn -methyl-l4C) drochloride codeine la b eled narcot ics : morphine (N othyl-t' D) hhy ydrochl or id e and co d eine (Nt.h y l-HC) I-PC) H Cl , specifi of 54-58 mCi/mMol (Amers harn /Searle} HCI, specificc activities activit ies of (Amersham/ Searle);: m ce th DL-methadone-2C) , specific activity 7 m Ci/mMol Ci/m Mol (Applied Science a nd Dls-m ot.hadone- z -t14-C}, Sc ie nce LaboL a b oEquilibrium simila r to tthe h e one ddescribed ratories). Equilib r iu m dialysis was done bbyy a mmethod ethod similar escri b ed by EISEN (1964): -ml quantities of serum (1964 ): I -rnl serum diluted dilu t ed I : 2 with phosphate p hosp hate buffer ed ed saline saline (P (PBS) B S) w were er e di diaallyzed y zed against 11 ml of pH 7. 7.22 PBS contain containing ing 2.17 nMol Radioactivity of HC-morphine. 14C-morphine. R adioa ct iv it y was measured by b y liquid liqu id scintillation scintilla ti on spectrom etry, and counts p er min minute transformed ute values were transfor m ed to t o disintegrations d isintegrations pper er m inute (DPM) values fo in g serum morphin minute forr calculat calculating morphinee binding (sa (sacc DPM minus D DPM P M measured for an an equal equ a l vo volume lu me of external di dialysate al y sate).). Farr tests t ests were done method by a m ethod used bby y uuss previously (RINGLE and H ERNDON 1975) 197 5) and a n d similar to MINDEN WEKSLER et al. (1973). the ones ddescribed escribed by MI NDEN and FARR (1967) and by 'VEKSLER et al, of so solution containF or these Farr ttests, ests, 0.2 0 .2 ml m l of serum ser um was mixed with 0.2 0 .2 m l of lu t io n conta ining 0.0 54 nMol of 14C-morphin l4C-morphinee a nnd, d, aft after er incubation inc ubatio n at 4°C overnig overnight, h t, 0.4 ml in g 0.054 saturated sulfate of cold co ld satu rated ammonium ammon ium su lfate was added to pprecipitate reci p it a te t hhee globulins glo b uli ns.. easurem en tss were carried Radioactivity m ea surement car r ied out, as a s d escribed escr ib ed for equilibrium eq u ilib riu m dialy lysis sis sa samples, m p les, on 0.5 0. 5 ml supernatant su pernatant a liq liquots uots after a fter packing protein precipitates precipitat es calculated experiby centrifugation. centrifugatio n . Serum Seru m binding values were wer e calc u lated for control con t rol and a n d ex p erimental sera by by subtracting su btracting their t h eir DPM values from se r u m -fr ee stan dard ttube ube serum-free standard D P M values (standard tubes DPM t ubes co contained n t a in ed an a n equivalent equ ivalent volume v olume of PBS substituted su bs t ituted fo forr ser serum). um) . Co Control ntrol se sera, ra, for comparison com p a rison with sera se ra from fr om mor morphine-treated p hine -t reated animals, a n imals, were from bleedin bleedings gs taken taken pprior rior to the t he animals a n imals receivi receiving n g any morp morphine h ine adm admii-

B. L. 128 . D. A. RINGLE and B. L. HERNDON nistration or from animals either given morphine-free control treatments or untreated, as identified in the Results section. Serum samples were run in triplicate, except for a small number of tests run in duplicate because of limited serum. 3. Competitive inhibition ofuC-morphine Inhibition of 14C-morphine binding by various analgesic compounds was tested by a modification of our Farr test described above. For these tests, 0.15 ml of isotonic saline containing the competitive inhibitor drug (0.054, 0.108, 0.216, or 0.432 X 10-9 MoljO.15 ml) was mixed with 0.2 0.2 ml of serum and incubated at room temperature for 1 hour, followed by the addition of 0.216 nMol of 14C_ in 0.05 ml of PBS, as morphine contained in as initial steps in the Farr method. ComHCI, codeine (S. B. pounds tested for competitive inhibition were: morphine HCl, HCI (donated by Eli Lilly); levorphanol tartrate (donated PENICK); methadone HCl HCI (donated by Endo Laboratories); and by Hoffmann-LaRoche); naloxone HCl HCI (provided by Dr. E. J. WOODHOUSE, Midwest Research Institute). heroin HCl 4. Morphine-carrier conjugates Two different morphine-carrier conjugates were prepared for use in various tests: 3-0-carboxymethylmorphine (CMM) conjugated to protein with l-ethyl-3HCI (ECDI) by the method of SPECTOR (3-dimethylaminopropyl) carbodiimide HCl and PARKER (1970); and morphine-6-hemisuccinate (M6HS) conjugated to carrier protein by a mixed anhydride (MA) reaction (WAINER et al. 1972). Protein carriers used were crystallized rabbit serum albumin (RSA) and bovine serum albumin (BSA) (Pentex, Miles Laboratories). For control use, proteins MA procedures but without added morphine were carried through ECDI or MA derivatives. 5. Radial immunodiffusion MANCINI et al. Radial immunodiffusion was carried out by the method of MANCINI (1965) using plates prepared with 1.5% SeaKem agarose (Marine Colloids) diluted 1: 2 with serum. Four ,ul of test solutions (antigen concentrations 0.5-1.25 mgj ml) were added to the wells. Test antigens employed were CMM-RSA, CMMBSA, M6HS-RSA, and M6HS-BSA; control antigens were ECDI-RSA andMARSA. A number of tests were also carried out with commercial immunodiffusion plates (Meloy Laboratories) to measure IgG and IgM levels in monkey serum (6 ftl It1 serum added per well). After immunodiffusion, plates were washed, dried, and stained with amido black (URIEL 1971) to aid in revealing weak antigenantibody precipitates. 6. Hemagglutination (HA) tests M6HS-BSA (BSA for control cells) was conjugated to goat RBC by the glutaraldehyde method (AVRAMEAS et al. 1969). Sera were heat-inactivated and plateR. HA tests were carried out with 50 ,ul Microtiter equipment and plastic plates.

Results 1. 14C-morphine binding by rabbit serum

Increased morphine binding measured in one group of 7 rabbits following course-III MP MP implantation was similar to increased morphine binding reported earlier for MP-treated rabbits (RINGLE and HERNDON 1975b, HERNDON 1974). Serum morphine-binding values per

Immunologic Effects of Morphine in Rodents, Rabbits, Monkeys, and Cast . 129

ml of serum (represented by DPM, mean ± S.E.), measured in equilibriumdialysis tests with 14C-morphine, were 21,600 ± 3,800 for bleedings taken prior to any morphine administration, in comparison with 20,600 ± 2,200 and 56,800 ± 8,400 for course-III bleedings on day 0 (pre-morphine) and day 8, respectively. Increased morphine-binding for day-8 serum was statistically significant in comparisons with binding for either the initial or course-III, day-O morphine bleedings (p < 0.01, Student t-test). In our other studies, individual control sera from PP-implanted rabbits never gave 14C-morphine binding values of over 27,400 DPM/ml serum. 2. 14C-morphine binding by monkey serum One of 3 MP-treated cynamolgus monkeys showed a significant increase in morphine binding by its serum after MP implantation beginning with the second morphine course (Tab. 1). Course-VI sera tested by the Farr method (see final column of Tab. 1) gave binding results similar to ones found for courses II-V by equilibrium dialysis, namely a marked increase in morphine binding by the serum of monkey B. 3. 14C-morphine binding by guinea pig serum Increased binding of morphine following MP administration was found in 3 of 5 experimental guinea pigs (Tab. 2). A prominent increase in morphine binding measured by equilibrium dialysis was shown by one guinea pig (MP-7) whose serum, after 4 courses of morphine, bound approximately 10 times as much morphine as did control sera. Farr test results on course-IV, day-5 sera confirmed the high morphine-binding ability of serum from guinea pig MP-7 and also MP-l and showed higher morphine binding by sera from guinea pigs MP-I MP-6. Differences in animals showing serum morphine binding found for the 2 test methods could have been due to variations in non-specific binding of morphine by albumin in equilibrium dialysis. 4. 14C-morphine binding by rat serum Some evidence for increased morphine binding by serum following prolonged morphine treatment was shown by sera from both strains of rats studied, although the increases in binding were less consistent and were lower in magnitude than the increases found for rabbit sera. Modest increases in morphine binding were noted in equilibrium dialysis tests for pooled serum (6 rats/serum pool) from 12 S-D rats after 3 courses of morphine HCI in comparison with binding for pooled serum (5 "and 6 rats/serum pool) from 11 control rats. Morphine bind2 /ml ing (DPM X 101O-2/ ml serum) averaged 226 before morphine treatment and 301 after morphine (course III, day 12) for morphine-treated rats,

192

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152

148

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108

156 156

104 104

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116

568

56

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184

738 738

208 208

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170 170

530

166

66118 8

1 ,7 68 1,768

2,576

12,744 12,7 44

2,032

140

190 190

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individua l monkey se sera epresen t ed by by DPM DPM valu es (ser a eeit er pre-drug p er -drug or for day dayss ind icated HC-m orphine bbinding * HC-morphine inding for individual ra r epresented it hher ica ted aafter fter em oval).. ellet et r emoval) p ell

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Treatment Course Course group I III*** and animal Pre-drug Pre-drug Day-5 no. Morphine pellet MP-l 140 124 MP-3 124 MP-4 266 220 MP-6 220 MP-7 144 144 Control c.i C-l C-2 C-3 C·3 C-4 C-5

152 88 72 90 40

200 200 160 232 220 220 248

152 200 200 232 220 1,192

DPMjml serum**

Day-12

200 244 244 276 276 204 1,332

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426 316 316 272 296 1,344

8,168 3,048 2,272 5,088 15,560

182 152 124 104 120

1,920 2,984 1,816 1,112 1,720

* HC-morphine binding represented by DPM values for individual guinea pig or for days insera measured by equilibrium dialysis (sera either pre-drug or dicated after pellet removal). * * Morphine binding measured by the Farr method. *** Comparison of post-morphine course-III and -IV results for MP-animals with combined pre-morphine, course-I course-L results for MP- and control animals showed significantly higher binding associated with morphine treatment (p < 0.002, Mann-Whitney U test).

in comparison with 208 und 215 for concurrently bled control rats (p < 0.02, Mann-Whitney U test). In later tests using some of these same rats (after injection with a control antigen, ECDI-RSA, for another study), 14C-morphine binding by serum was measured by the Farr method using individual serum samples (5 rats/group). 14C-morphine binding results were 3,200 ± 280 and 2,432 ± 312 DPM (mean ± S.E.) per ml serum for morphine-treated and control rats, respectively (p < 0.05, Mann-Whitney U test), after 4 s.c. courses of morhine or saline. S-D rats, no significant eviIn contrast to findings for serum from SoD dence for increased binding of morphine was shown for L-E rats in either equilibrium dialysis or Farr tests on pooled serum samples (3 rats/ serum pool) throughout 5 courses of MS or MP (6 rats/treatment group), in comparison with results for pooled serum (2 and 3 rats/serum pool) from 5 PP-treated rats. However, in other experiments involving additional courses of morphine, some individual L-E rat sera showed unusually high 14C-morphine binding values in Farr tests (e.g., 5,344

132 . D. A.

RINGLE

and B. L.

HERNDON

DPM/ml serum) in comparison with 2,352 ± 232 DPM (mean and 4,288 DPMjml ± S.E.) per ml serum from PP-treated rats. 5. 14C-morphine binding by cat serum Neither of the 2 young cats given morphine showed a marked increase in serum binding of morphine after MP administration, although some indication of increased morphine binding was shown by the serum of cat No.2. In Farr tests, control (pre-morphine) serum HC-morphine binding DPM values of 1,872 and 1,944 were measured for cats Nos. 1 and 2, respectively. Maximum 14C-morphine binding values achieved for sera after final MP courses were 2,184 DPM (cat No.1, course IV, day 5) and 3,888 DPM (cat No.2, course III, day 12). Similar results were obtained for morphine binding by equilibrium dialysis. 6. Serum binding of HC-codeine and 14C-methadone l4C-Iabeled morphine, codeine, and methadone, employed Binding of 14C-labeled at molar concentrations identical to the one standardly used for 14C_ morphine binding, were carried out by the Farr method with various monkey, guinea pig, and rat sera. Results of these tests showed an in14C-codeine, but not of 14C-methadone, by crease in serum binding of 14C-codeine, sera of animals that showed increased serum binding of l4C-morphine HC-morphine (rat sera not tested for methadone binding). 7. Competitive inhibition Results of competitive inhibition tests on monkey and guinea pig sera are shown in Figure 1. These results indicate that the increased 4C-morphine found for the 2 test sera from responding oP4C-morphine serum binding of1 morphine-treated animals was relatively specific for the morphine configuration. These results also agree with the findings described above 14C-Iabeled narcotics in showing essentially no inhibition for binding of 14C-labeled by methadone for either the monkey or guinea pig as well as in the

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Fig. 1. Competitive inhibition of 14C-morphine binding by various drugs: M, L, levorphanol; N, N, naloxone; and M, methamorphine; C, codeine; H, heroin; L, done. Part A: course-VI, day-5 serum from MP-treated monkey B. Part B: course-IV, day-5 serum from MP-treated guinea pig MP-7.

Immunologic Effects of Morphine in Rodents, Rabbits, Monkeys, and Cats . 133

slight differences in inhibition of 14C-morphine binding by codeine and morphine for the monkey and guinea pig sera. 8. Radial immunodiffusion tests Radial immunodiffusion tests were carried out with course-VIII, course-IV, day-5 guinea day-5 monkey sera (monkeys A and B) and course-TV, pig sera (morphine-treated guinea pigs MP-l, MP-4, and MP-7, and control guinea pigs C-l and C-4. Only sera showing increased binding 4C-morphine gave positive radial immunodiffusion reactions against oP4C-morphine of1 one or more of the morphine hapten-carrier conjugates. Sera from monkey B and guinea pig MP-7 reacted with both CMM-RSA and CMM-BSA; guinea pig MP-l reacted only with CMM-RSA. These sera failed to react against any of the other morphine conjugates or control antigens. Immunodiffusion tests carried out on monkey sera with anti-IgG and anti-IgM plates prepared for use with human serum, but which (NEOH et al. 1973), also cross react with monkey immunoglobulins (NEOR showed an increase in IgM and a decrease in IgG levels after long-term morphine administration only for the serum of monkey B (Fig. 2). Sera from monkeys A and C, which showed little or no increase in 14C_ morphine binding after prolonged morphine administration, showed decreased IgM levels and no consistent changes in IgG levels associated with morphine treatment. IgG

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134 . D D.. A. Rr"'GLE RINGLE a and B. L. nd B.

HERNDON HERND O'"

Passive 9. Pas sive HA tests t e sts The presence of antibody ant ibody against morphine in morphine-binding sera from morphine-treated monkey and guinea guine a pigs was also shown by HA tests M6HS-BSA-tagged goat RBC (cat and rat ser seraa were no nott test s using M6HS-B SA-tagged goat from monkeyss A, B B,, and C respectively, om monkey respectively , gave titers of ttested). est ed). Sera fr bleedings 1 :: 1, 1 : 4, and 0, for bleedin gs taken before befor e any morphine administrapost-morphine ttion, ion , and titers t iters of 1 : 1, 1 :: 16, and 1 : 1, for po st-morphine sera (MP course IV, MPI V, day 5). Tests Te st s employing employ ing course-IV, day-5 sera from 5 1\IPtreated higher t reate d guinea pigs gave high er HA titers than t han sera from 5 control guinea pigs, differences averaged (1 : 4 to guine a pig s, although tthe he differen ces av er aged only one titer (1 1 : 8 for MP-treated guinea pig sera versus 1 :2 to t o 1 : 4 for control guinea pig sera). In tests with sera from MP- and PP-treated rabbits (7 rabbits/group) involving diluting these sera 1 : 2 with rabbit antiserum against CMM-RSA, dayday-8S sera from MP courses III and V lowered HA titers by one dilution tube in comparison with titers ti t ers for antiCMM-RSA serum combined with sera from PP-treated rabbits. rabbits. These sera from MP-treated MP- treated rabbits were previously shown to possess weak but measM6HS-BSA-tagged (RINGLE urable HA activity ac tiv ity against M6HS-BSA-t agged RBC (R INGLE and HERNDON 1975). Discussion results extended other The re sults of this t his study have extende d ttoo ot her species our earlier increased findings on the t he occurrence occurre nce of in creased serum ser um morphine binding after prolonged morphine ad administration (RINGLE HERNDON prolonged ministration in rabbits (R INGLE and HERNDON 1972, 1975) and have hav e further demonstrated that t hat there t he re are marked differences differen ces in tthe he magnitude magni tude for the t he serum seru m morphine-binding response tested. incidence occurring among the t he species teste d. In contrast to 100% 100 % in cidence of increased serum im-in creased ser um morphine binding occurring in rabbits rabbit s after afte r MP im plantation, he other species tested teste d did all of the t he morphineplan t ation , in none of tthe serum Differences ttreated reated animals show tthis his ser um response. Differ en ces were also noted significant in in-between the 2 rat r at strains, although the failure to t o detect det ect significant serum creases in pooled L-E rat ser um in equilibrium dialysis ttesting esting was probably incidence ab ly due both to a low in cidence of occurrence of the serum morphinese when it did occur. binding response as well as to a low level of respon response Our previous findings on rabbits were consistent with wit h the idea that morphine administration is capable of evoking an immune response morphine configuration, directed against the mo rp hine configur at ion, and the results reported here her e on other species indicate that tha t a humoral immunologic response rat,, guinea pig pig,, against morphine can aalso lso be elicited elicite d in tthe he monkey, monkey , rat he cat. Relative Relati ve specificity of the evoked serum and perhaps, in tthe component morphine-binding compone nt for the morphine configuration was shown both sho wn bo th by binding tests t ests using 14C-labeled HC-Iab eled narcotics narcoti cs (morphine, methadone) inhibition one) and by competitive competi ti ve inhibiti on tests t ests with codeine, and methad various narcotics va rious narcoti cs (Fig. 1).

Immunologic Effects of Morphine in Rodents, Rabbits, Monkeys, and Cats . 135

Some serum binding of drugs such as morphine and codeine is due to non-specific binding by albumin (OLSEN 1973), but the increased morphine binding noted in our studies on morphine-treated rabbits was not due to increased binding by serum albumin (RINGLE and HERNDON 1975). The evidence from our earlier studies on rabbits indicated that part, and perhaps all, of the increased serum morphine binding associated with morphine administration was in the serum's gamma globulin component, although we have not yet determined which specific type of globulin was involved. A possible role for IgM in the humoral response against morphine is suggested by our findings reported here for monkey serum which showed an increase in IgM level for the one monkey (B) which showed a serum morphine-binding response (Fig. 2). Also, a number of other investigators (NICKERSON et al. 1970, KILOOYNE et al. 1972, CUSHMAN and GRIEOO 1973, RABIN and D'AMANDA 1973, and WEKSLER et al. 1973) have shown the occurrence of elevated IgM levels in human recovering from opioids, but it is not known if IgM is the component responsible for the increased morphine binding found for human addict sera by RYAN et al. (1972). Evidence against IgM as the morphine-binding antibody is offered by the low HA activity of these sera in tests with M6HS-BSA tagged goat RBC, although higher HA titers might have been obtained using RBC tagged with M6HS-RSA or with CMM-albumin conjugates. The poor HA activity of these sera was also shown by their inhibition of HA activity effected by antiCMM-RSA serum described in the Results section (9. Passive HA tests), since these HA results indicate that the test sera contained a morphine-binding component poor in HA capability relative to antiCMM-RSA antibody. Positive precipitin reactions against CMM-protein conjugates by morphine-binding sera, like binding of 14C-morphine l4C-morphine and positive HA activity, indicate the presence of antimorphine antibody. Also indicative of the presence of antibody in animals showing the morphineCMM -protein binding serum response were positive dermal responses to CMM-protein conjugates in rabbits (RINGLE and HERNDON 1975) and in the one responder monkey, B (unreported results). In other unreported experiments, we have isolated morphine-binding protein from MP-rabbit serum with an immunoadsorbent column prepared from CMM covalently linked to aminopropyl agarose, although we have not yet identified the nature of the eluted protein. Further evidence for the antibody nature of the morphine-binding serum component from morphinetreated animals is provided by other studies in which we have shown that pretreatment of rats with serum from the MP-treated rabbit is capable of attenuating the analgesic effects of morphine in a way similar to that shown by pretreatment with antiserum made against an immunogenic morphine-carrier conjugate (HERNDON and RINGLE 1975).

A.. RINGLE RINGLE and L.. HERNDON HERNDON 136 . D. A a nd B . L Acknowledgement wish Mrs. V.. CROUCH CROUCH and SHELLEY D. LARLARThe authors wi sh to t o thank Mr s . JJUDY UDY V a n d Miss SHELLEY SON for their hhelpful elpful t echnical assistance. a ssist ance. SON

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